Stem Cells and its use in Dentistry

INDEX

1. INTRODUCTION & DEFINITIONS

2. HISTORY

3. PROPERTIES OF STEM CELLS

4. HUMAN STEM CELL NICHES

5. TYPES OF STEM CELLS

6. TYPES OF DENTAL STEM CELLS

7. TOOTH ELIGIBILITY CRITERIA

8. STEM CELL ISOLATION, CHARACTERIZATION & CULTURE

9. STEM CELLS-APPLICATION

10. ROLE OF STEM CELLS IN MEDICAL THERAPIES

11. CANCER STEM CELLS

12. STEM CELL BANKING

13. ETHICS

14. CONCLUSION

15. GLOSSARY

16. REFERENCES

For centuries, scientists are trying to known how certain animals can regenerate missing parts of their bodies. Humans actually share this ability with animals like the starfish and the newt (aquatic amphibian). Our bodies are constantly regenerating blood, skin, and other tissues. The identity of the powerful cells that allow us to regenerate some tissues was first revealed in 1950s1 when experiments with bone marrow established the existence of stem cells in our bodies and led to the development of bone marrow transplantation, a therapy now widely used in medicine. This discovery raised hope in the medical potential of regeneration. For the first time in history, it became possible for physicians to regenerate a damaged tissue with a new supply of healthy cells by drawing on the unique ability of stem cells to create many of the body’s specialized cell types. Once they had recognized the medical potential of regeneration through the success of bone marrow transplants, scientists sought to identify similar cells within the embryo. Early studies of human development had demonstrated that the cells of the embryo were capable of producing every cell type in the human body. Scientists were able to extract embryonic stem cells from mice in the 1980s, but it wasn’t until 19981,2 that a team of scientists from the University of Wisconsin–Madison1 became the first group to isolate human embryonic stem cells and keep them alive in the laboratory.¹

“Stem cells are human cells that have the potential to develop into other cell types within the body and thereby repair injured cells.”³

“A stem cell is a cell with the ability to divide indefinitely in culture and with the potential to give rise to mature specialized cell types.”⁴

“Stem cells are undifferentiated embryonic or adult cells that continuously divide.”⁵

“Stem cells are immature, unspecialized cells that have the potential to develop into many different cell lineages via differentiation. These cells can renew themselves indefinitely through “self renewal”, and they vary in terms of their location in the body and the type of cells that they can produce.”⁶

Stem cells have remarkable potential to develop into much different cell type in the body during early life and growth. In many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the creature is alive. Stem cells are primitive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, red blood cell, or a brain cell.²

One of the fundamental properties of stem cell is that it does not have any tissue specific structure that allows it to perform specialized function. Unspecialized stem cells can give rise to specialized cells including heart muscle cells, blood cells, or nerve cells. This process is called differentiation.²

Recently, scientists are working with two kinds of stem cells from humans:embryonic stem cellsand non-embryonic"somatic" or "adult" stem cells.²

Embryonic stem cells, as their name suggests, are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilizedin-vitro in anin-vitrofertilizationclinic and then donated for research purposes with informed consent of the donors. They arenotderived from eggs fertilized in a woman's body.²

An adult stem cell is thought to be anundifferentiatedcell, found among differentiated cells in a tissue or organ that can renew itself and can differentiate to yield some or all of the major specialized cell types of the tissue or organ. Adult stem cells typically generate the cell types of the tissue in which they reside.²

Several types of stem cell have been discovered from germ cells, the embryo, fetus and adult. Each of these has promised to revolutionize the future of regenerative medicine through the provision of cell-replacement therapies to treat a variety of debilitating diseases. Stem cell research opens-up the new field of ‘cell-based therapies’ and, as such, several safety measures have also to be evaluated. Regenerative medicine is based on stem cells. The dental pulp contains the progenitor/stem cells which have the self renewal capability, multi-lineage differentiation capacity and clonogenic efficiency. Dental stem cells can generate many types of tissues including bone, nerve, cartilage, teeth and fat. However, the biology of these mysterious cells have yet to be understood and a lot more basic research is needed before new therapies using stem-cell-differentiated derivatives can be applied. Recently stem cells banks are present, and even some of these banks do not only freeze cord stem cells but also dental stem cells of baby teeth. Hence this review attempts to study the history, current concepts, evolution of stem cells in dentistry, dental stem cell banking in India, characterization of the stem cells in dentin regeneration and stem cell therapy in dentistry.

The term stem cell was proposed for scientific use by Russian histologist Alexander Maksimov in 1908. While research on stem cells grew out of findings by Canadian scientists in the 1960s.⁷

In 1961, Till & McCulloh establish the foundation for stem cell science. Toronto scientists Dr. James Till, a biophysicist, and Ernest McCulloch, a hematologist published accidental findings in “Radiation research” that proved the existence of stem cells- cells that can self-renew repeatedly for various uses. Both worked for the Ontario cancer institute (OCI) at that time.⁸

In the late 1970s, scientists developed a method whereby embryos are created in cell research: in-vitro (in a laboratory) through the process of in-vitro fertilization (I.V.F) for several years thereafter, research studies involving embryos and fetal tissue focused on fertility testing for IVF.³

In 1981, the first embryonic stem cells were isolated from mouse embryos, opening the door to embryonic stem cell research. Stem cell research then faced considerable opposition from both President Reagan and President Bush, Sr. during the 1980s. However, after his election in 1994, President Clinton lifted the long-standing ban on federal funding of fetal tissue research, but the National Institute of Health ("NIH") was still unable to gain the approval of Congress for federal funding of human embryo research.³

In 1998, James Thompson (University of Wisconsin - Madison) isolated cells from the inner cell mass of early embryos, and developed the first embryonic stem cell lines. Human embryonic stem cells (hESC) have the ability to reproduce limitlessly and can differentiate into many organs. On the other hand considerable controversy has arisen regarding this type of research, because derivation of hESC requires destruction of early human embryos.⁹

In 1999, Pittenger et al. characterized human MSCs from the bone marrow of the iliac crest as multipotent stem cells by demonstrating their isolation, expansion in culture and directed differentiation to osteogenic, adipogenic and chondrogenic lineages.⁶

In 1999, an adult epithelial stem cell niche in teeth was first demonstrated via organ culture of the apical end of the mouse incisor. The niche is located in the cervical loop of the tooth apex and possibly contains dental epithelial stem cells, which can notably differentiate into enamel-producing ameloblasts.⁶

Dental pulp stem cells (DPSCs) were isolated at first in 2000 by Gronthos et al. These cells can differentiate in pulp-like cells. DPSCs can also differentiate in adipocytes and neural-like cells. Gronthos et al. in 2002 have established the ability of self-duplication in-vivo. The same group in 2003, showed that bone marrow mesenchymal stem cell (BMMSC) and DPSCs can be traced in perivascular tissue of their respective origin tissue assumpting that this discovery could have implications for the tissue stem cells populations identification in other districts.¹⁰

In the year 2003, Songtao Shi, a pediatric dentist discovered baby tooth stem cells by using the deciduous teeth of his six year old daughter, he was luckily able to isolate, grow, preserve these stem cells and their regenerative ability, and he named them as SHED (Stem cells from Human Exfoliated Deciduous tooth.⁷

In 2006, Dr. Shinya Yamanaka discovered that normal mouse adult skin fibroblasts can be reprogrammed to an embryonic state by introducing four genetic factors (Oct3/4, Sox2, Klf4 and c-Myc), and the resulting cells were termed iPS cells. Just a year after the mouse study was reported, the findings were replicated in human skin cells, which opened the door to generate a patient-specific ES cell equivalent from autologous somatic cells.⁶

In 2006, Kishi et al. isolated salivary gland stem/progenitor cells from rat submandibular glands and found that the cells are highly proliferative and express acinar, ductal and myoepithelial cell lineage markers.⁶

In 2008, Ikeda et al. identified distinctive stem cells in the dental mesenchyme of the third molar tooth germ at the late bell stage (tooth germ progenitor cells: TGPCs) with high proliferation activity and the capability to differentiate in vitro into lineages of the three germ layers including osteoblasts, neural cells and hepatocytes. Stem cells from the apical papilla (SCAP) were found in the papilla tissue in the apical part of the roots of developing teeth. Compared with DPSCs, SCAP demonstrate better proliferation in vitro and better regeneration of the dentin matrix when transplanted in immunocompromised mice. These findings suggest that ‘‘developing’’ dental tissues may provide a better source for immature stem cells than ‘‘developed’’ dental tissues.⁶

In 2009, Zhang et al. first characterized human gingiva-derived MSCs (GMSCs), which exhibited clonogenicity, self-renewal and a multipotent differentiation capacity similar to that of BMSCs.⁶

In 2010, Geron Corporation announced the enrollment of the first patient in the company’s clinical trial of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells.⁸

In 2010, Regenerative Medicine Company© advanced cell technology received federal approval from the US FDA to begin a multi-center clinical trial that tests human embryonic stem cell treatment on patient with Stargardt’s Macular dystrophy, a disease that causes blindness.⁸

In 2012, Chinese scientist from Guangzhou institute of biomedical and health have converted cells found in urine into pluripotent stem cells that can be used to create neurons and brain cells. The researchers say their find holds huge potential for the rapid testing and development of new treatments for neurodegenerative disorders.⁸

Stem cells have remarkable potential to develop into much different cell type in the body during early life and growth. Stem cells have two different characteristics. (1) They are unspecialized cells capable of renewing themselves through cell division. (2) They can be induced to become tissue or organ specific cells with special function. Two type of stem cell were discovered, embryonic stem cell and non embryonic ‘somatic’ or adult stem cell. In 2006, researchers made another breakthrough by identifying condition that would allow some specialized adult stem cell to be “reprogrammed” genetically to assume a stem cell like state. This new type of stem cell called induced pluripotent stem cells.²

According to Caroline PT, 2007, all stem cells have three general properties. First, they can divide and renew themselves indefinitely, unlike muscle, blood, or nerve cells. Second, stem cells are unspecialized, meaning that they do not belong to any specific tissue structure which would allow them to perform a specialized function, such as pumping blood through the body. Finally, although stem cells are unspecialized, they may develop into specialized cells through a process called differentiation. The specialized cells produced by differentiation may then perform specific functions.³

According to Ariff B, 2008, when a stem cell divides, the daughter cells can either enter a path leading to the formation of a differentiated specialized cell or self-renew to remain a stem cell, thereby ensuring that a pool of stem cells is constantly replenished in the adult organ. This mode of cell division characteristic of stem cells is asymmetric and is a necessary physiological mechanism for the maintenance of the cellular composition of tissues and organs in the body. Other attributes of stem cells include the ability to differentiate into cell types beyond the tissues in which they normally reside. Stem cells are also believed to be slow cycling but highly clonogenic and generally represent a small percentage of the total cellular make-up of a particular organ.⁴

According to Christine MS, 2012, fundamental properties of stem cells is self-renewal or the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.⁵

DIFFERENTIATION

Differentiation is the process whereby an unspecialized early embryonic cell acquires the features of a specialized cell such as a heart, liver or muscle. Differentiation in-vitro can be spontaneous or controlled. From a teleological perspective there appears to be no limit to the types of cell that can be formed from hESC differentiation.⁴

TYPES OF DIFFERENTIATION

1: Direct differentiation: A specific type of cell in a special niche developed in a multistep unidirectional pathway (e.g., MSCs differentiating into osteoblasts/fibroblast).¹¹
2: Transdifferentiation: Direct conversion of one cell type to another different cell type (e.g., blood cells into brain cells and vice versa).¹¹
3: Dedifferentiation: A unipotent stem cell becoming a multipotent one.¹¹
4: Cell fusion: A stem cell fusing with a somatic cell resulting in another lineage (e.g., ESCs fuse in-vitro with HSCs and neuronal cells).¹¹
5: Self-renewal: They can divide without differentiation and create everlasting supply.¹¹
6: Plasticity: MSCs have plasticity and can undergo differentiation. The trigger for plasticity is stress or tissue injury which up regulates the stem cells and releases chemo attractants and growth factors.¹¹

Homing of HSC from other definitive hematopoiesis to fetal bone marrow is thought to involve some signaling factors such as stromal derived factor-1 (SDF-1 or CXCL12)/chemokine C-X-C receptor 4 (CXCR4).12 Soluble factors are not only mediated in fetal bone marrow but also in adult bone marrow to maintain HSC in undifferentiated state and regulate HSC in proliferative and differentiated states within the specific microenvironments termed “niche” throughout the life. Stem cell niche was first proposed by Schofield, with the later identification in Drosophila melanogester’s ovary to confirm the existence of HSC niche. Germ line stem cells resided in the Drosophila ovary that is surrounded by differentiated somatic cells have been shown to be essential for maintaining stem cells survival and division. Thus, HSC niche is the special local environments of HSCs that maintains and controls HSCs function by regulating survival, self-renewal ability, and cell fate decision. Such molecules have been identified to be associated with HSC homing to bone marrow, for example, SDF1-α, β1-integrins, metalloproteinase (MMP), and serine-threonine protein phosphatase (PP)2A.13 By using real-time imaging, it is possible to explore the localization of HSCs with their function. HSCs lodge in the endosteal surface, osteoblasts, and blood vessels, particularly in trabecular regions, in the mouse calvaria.¹⁴

On the contrary, more mature cells reside away from the endosteum. Similarly, mice show the homing and lodgment of transplantable HSCs in the endosteal region of the trabecular bone area where they respond to bone marrow damage by rapidly dividing. HSCs niches are suggested to be mediated in two main microenvironments within bone marrow endosteal niche and vascular niche (Figure1). First, endosteal niche: osteoblasts derived from mesenchymal precursors are localized in the endosteal regions which are well vascularized.¹⁴

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Figure-1: Candidate cellular niches mediated in maintenance and regulation of HSCs in bone marrow; endosteal and vascular niches. ¹⁴

The activation of osteoblastic differentiation is in part mediated by HSC-derived bone morphogenic protein-2 (BMP-2) and BMP-6. Osteoblasts are suggested as the niche due to the finding that the number of osteoblasts is increased from parathyroid hormone activation and results in an increase HSCs number in-vivo. This signal was found to be activated through Jagged1, a serrate family of Notch ligand, on osteoblasts. Study by Chitteti and colleagues supports this evidence and shows that enhancing hematopoiesis promoted by osteoblast via Notch signaling not only through Jagged1 up-regulation, but also Notch2, Jagged2, Delta1 and 4, Hes1 and 5, and Deltex ligands.15 Soluble factors produced from osteoblasts function in regulating HSC quiescence, HSC pool and fate such as angiopoietin-1 (Ang-1),16 SDF-1 (CXCL12),17 and osteopontin.18 Recently, osteoblasts secreted cysteine protease cathepsin-x have been found to catalyze the chemokine CXCL-12, a potent chemo-attractive cytokine for HSCs, and ablate the attachment of CD34+ cells with the osteoblasts. This result suggests the role of osteoblasts in regulate HSCs trafficking in the bone marrow.¹⁴

A group of de Borros supports this hypothesis by showing that the 3D spheroid of non-induced and one week osteo-induce bone marrow stromal cell (active osteoblasts) formed an informative microenvironment that control migration, lodgment, and proliferation of HSCs. Bone marrow endosteal cells, particularly, osteoblast-enriched ALCAM+Sca-1 cells promoted LT-reconstitution activity of HSCs via the up-regulation of genes related in homing and cell adhesion. In addition, HSCs were found to adhere with spindle-shaped N-cadherin+ osteoblastic (SNO) cells which are a subpopulation of osteoblasts. BMP receptor type IA mutant mice have been shown to increase in the number of SNO cells that correlated to an increase in HSC number.¹⁴

Green fluorescent protein (GFP+) HSCs derived from Col2.3-GFP+ transgenic mouse were found to attach to SNO cells but not all GFP+ HSCs were in contact with SNO cells showing that N-cadherin component might be the other niche for HSCs. Cumulatively, osteoblasts and SNO cells are suggested as the niche for hematopoietic stem and progenitor cells where this microenvironment termed “Endosteal niche.”¹⁴

Vascular niche, might involve in HSC maintenance within the bone marrow. Studies in osteoblast depletion demonstrated that there was a loss of B lymphopoiesis but not immediately loss of HSC number and few bone-marrow HSCs (CD150+CD48−CD41−lineage) were localized to the endosteum.¹⁴ Table-I: TYPE OF STEM CELL ON THE BASIS OF DEVELOPMENTAL POTENTIAL:¹¹

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TOTIPOTENT STEM CELLS

Stem cells that can be differentiate into the widest variety of cells. They are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells in the human body in addition to the placenta, which nourishes the human embryo in the womb. Human cells are only totipotent during the first few divisions of a fertilized egg. After three to four divisions of the totipotent cells, the cells start to specialize. At this point, the cells become pluripotent.²

PLURIPOTENT STEM CELLS

Pluripotent stem cells can give rise to all the different cell types in the human body but do not contain the genetic information to make a placenta. These stem cells are primarily found in the human embryo during its earliest stages. Pluripotent stem cells are typically what people are referring to when they generically refer to stem cell research.²

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Figure-2: Pluripotent stem cell can give rise to all of the different cells found in the human body.²

MULTIPOTENT STEM CELLS

The cells that can divide and grow into several differentiated cell types within a specific type of tissue or organ. For example, a multipotent skin stem cell could divide and grow into a hair follicle cell or a sweat gland cell; however, it would not be able to grow into a nerve cell or heart cell or any other kind of cell. A multipotent skin stem cell could only divide and grow into the different types of cells found in the skin tissue (Figure 3). These multipotent stem cells can be found in many places in the adult human body, including the skin and bone marrow.²

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Figure-3: Multipotent stem cell found in the bone marrow which makes the different cells found in the blood and lymphatic system.²

Table-II: TYPES OF STEM CELL WITH THEIR SOURCES OF ORIGIN:¹¹

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EMBRYONIC STEM CELLS

ES cells are produced by culturing cells collected from the undifferentiated inner cell mass of the blastocyst, which represents an early stage of embryonic development after fertilization. This embryonic origin is the major reason that ethical and moral questions are associated with the use of human ES cells. Nonetheless, ES cells are of great interest to scientists and clinicians because of their developmental capacity to differentiate in-vitro into cells of all somatic cell lineages as well as into male and female germ cells. In the field of dentistry, ES cells are expected to provide an in-vitro model system and transplantation substrate for animal models to study the controlled differentiation of pluripotent stem cells into specific lineages of oral tissues and organs, such as mucosa, alveolar bone, periodontal tissues and teeth. These approaches can be useful to obtain a better understanding of oral developmental biology and may lead to future strategies in regenerative dentistry that meet clinical needs. However, in addition to the ethical issues, the tissue engineering applications of ES cells are limited because the cells are allogenic and thus may be immunologically incompatible between donors and recipients. To overcome this issue, the creation of human ES cell banks with human leukocyte antigen (HLA) matching and the generation of customized, patient-specific ES cells via nuclear transplantation from the patient’s own cells have been proposed to enable combined gene and cell therapy. However, these strategies rely on inefficient and expensive techniques and are tedious and ethically cumbersome, especially for dentists, unless a cooperative team can be organized with experts who routinely deal with patients’ embryos.⁶

ADULT STEM CELLS

They are also called somatic stem cells or postnatal stem cells, and they are found in many tissues and organs. Although very few of these cells are present in adult tissues, they undergo self-renewal and differentiation to maintain healthy tissues and repair injured tissues. Recent stem cell studies in the dental field have identified many adult stem cell sources in the oral and maxillofacial region. These cells are believed to reside in a specific area of each tissue, i.e., a ‘‘stem cell niche’’. Many types of adult stem cells reside in several mesenchymal tissues, and these cells are collectively referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs).⁶

INDUCED PLURIPOTENT STEM CELLS

In 2006, Dr. Shinya Yamanaka discovered that normal mouse adult skin fibroblasts can be reprogrammed to an embryonic state by introducing four genetic factors (Oct3/4, Sox2, Klf4 and c-Myc), and the resulting cells were termed iPS cells.¹⁹ Just a year after the mouse study was reported, the findings were replicated in human skin cells, which opened the door to generate a patient-specific ES cell equivalent from autologous somatic cells. This technology is expected to revolutionize medicine because of the capacity of iPS cells to develop into all tissues/organs and thereby support the emerging field of ‘‘personalized medicine’’, which uses a patient’s own cells to provide biologically compatible therapies and individually tailored treatments.⁶

MESENCHYMAL STEM CELLS

In cell culture, MSCs can be identified and isolated based on their adherence to tissue-culture-treated plastic. MSCs are among the most promising adult stem cells for clinical applications; they were originally found in the bone marrow, but similar subsets of MSCs have also been isolated from many other adult tissues, including skin, adipose tissue and various dental tissues. The concept of using adherent fibroblastic cells isolated from the bone marrow was originally reported in 1970 by Friedenstein et al.²⁰ Those cells were referred to as colony forming units-fibroblasts, and their capability to differentiate to various mesenchymal tissues gave rise to the concept of MSCs. In 1999, Pittenger et al.²¹ characterized human MSCs from the bone marrow of the iliac crest as multipotent stem cells by demonstrating their isolation, expansion in culture and directed differentiation to osteogenic, adipogenic and chondrogenic lineages. Since then, extensive studies on MSCs have demonstrated their robust multipotency and even ‘‘stem cell plasticity’’, as exemplified by the capacity of MSCs to differentiate into lineages that are not typical mesenchymal derivatives.⁶

BONE MARROW-DERIVED MSCs (BMSCs)

Adult bone marrow contains rare multipotent progenitor cells that are generally termed BMSCs. Despite their heterogeneity, BMSCs possess a high replicative capacity and have the capacity to differentiate into various connective tissues cell types. In addition, BMSCs robustly form bone in-vivo, which makes them an appropriate stem cell source for bone regeneration therapy.⁶

UMBILICAL CORD BLOOD STEM CELLS

Umbilical cord blood contains a highly heterogeneous mixture of cells. This mixture includes hematopoietic cells including erythrocytes and leukocytes. Moreover, umbilical cord blood contains at least three types of stem cells including a population of hematopoietic stem cells (HSCs) and a population of Mesenchymal stem cells (MSCs), which are multipotent stem cells highly similar to Mesenchymal stem cells (MSCs) of the bone marrow. In addition, umbilical cord blood contains a relatively low concentration of non-hematopoietic multipotent stem cells expressing SSEA-4 protein,22 a surface marker expressed by embryonic stem cells, and the transcription factors OCT4, SOX2 and NANOG normally expressed by pluripotent stem cells.²³ The potential use of this non-hematopoietic stem cell population in a range of applications underpins the efforts to further characterize and analyze the properties of this unique cell population. Clinical applications of umbilical cord blood date back to the early 1970s, where it was used to treat lymphoblastic leukemia patients.²⁴ Since then, it was used regularly in hematology transplantations as a replacement for bone marrow following hematological malignancy or bone marrow failure.²⁵ The discovery of the unique non-hematopoietic multipotent stem cells in cord blood and the ability to differentiate these cells into many different cell types highlighted the potential use of umbilical cord blood as a therapeutic tool for a wider range of diseases and disorders . ²⁶

AMNEOTIC FLUID STEM CELL (AFDSCs)

AFDSCs belong to the group of stem cells present in extra embryonic tissues; all sharing the feature of belonging to material that is discarded after birth or that can be collected during amniocentesis. Besides the amniotic fluid, the amnion, umbilical cord and placenta have shown to contain stem cells that can be isolated at birth Bailo M et al.²⁷ The first studies of AFDSCs were completed using mesenchymal amniocytes isolated from sheep. These cells showed the ability to expand in-vitro and to integrate into a scaffold (Kaviani A et al., 2001).²⁸ In the following years, the identification of cells expressing the marker Oct4, or co-expressing Oct4, CD44 and CD105 (Tsai et al., 2004)²⁹ were discovered in amniotic fluid. More recently a clonal population of AFDSCs derived from human and mouse were isolated and characterized (De Coppi P et al., 2007).³⁰ These cells named AFSCs, were isolated through positive selection for the marker CD117 (or c-Kit), and represented 1% of cells derived from amniocentesis. AFSCs express the marker of “stemness”, Oct4, and the embryonic stem cell (ESC) marker SSEA-4. Furthermore AFSCs express markers characteristic of mesenchymal and neural stem cells such as CD29, CD44, CD73, CD90, and CD105. Interestingly, these cells are negative for markers of hematopoietic stem cells such as CD34 and CD133.³¹

ADIPOSE TISSUE-DERIVED STEM CELLS (ASCs)

Adipose tissue is an abundant source of MSCs and has been extensively studied in the field of regenerative medicine as a stem cell source. Adipose-derived MSCs can be readily harvested via lipectomy or from lipoaspirate from areas such as the chin, upper arms, abdomen, hips, buttocks and thighs in large numbers with low donor-site morbidity, as liposuction is one of the most common cosmetic procedures. The intrinsic characteristics of ASCs appear to be different from those of BMSCs, ASCs exhibit robust osteogenesis and are thus expected to be an alternative source of MSCs for bone regeneration in dentistry. Indeed, the feasibility of using autologous ASCs for orofacial bone regeneration and implant placement has been demonstrated. The transplantation of autologous ASCs with an inorganic bovine bone scaffold enhanced new bone formation and implant osseo-integration following vertical bone augmentation of the calvarial bone of rabbits, which suggests that ASCs may be useful for vertical alveolar bone augmentation for implant treatment.⁶

DENTAL TISSUE-DERIVED STEM CELLS

To date, two types of adult stem cells have been characterized in dental tissues, i.e., epithelial stem cells and MSC like cells. An adult epithelial stem cell niche in teeth was first demonstrated in 1999 via organ culture of the apical end of the mouse incisor. The niche is located in the cervical loop of the tooth apex and possibly contains dental epithelial stem cells, which can notably differentiate into enamel- producing ameloblasts. Although the epithelial stem cell niche is useful for analyses of the fate decision of stem cells in tooth development, no information is available for dental epithelial stem cells in humans.⁶

HEMATOPOIETIC STEM CELLS (HSC)

HSC are adult stem cells that contain the potentiality in self-renew and differentiation into specialized blood cells that function in some biological activities: control homeostasis balance, immune function, and response to microorganisms and inflammation. HSCs can also differentiate into other specialized cell or so called plasticity such as adipocytes, cardiyomyocytes, endothelial cells, fibroblasts/myofibroblasts, liver cells, osteo-chondrocytes, and pancreatic cells.¹⁴

Most HSCs are in quiescent state within the niches that maintain HSC pool and will respond to the signals after the balance of blood cells or HSC pool is disturbed from either intrinsic or extrinsic stimuli. HSCs have been studied extensively, especially, for the therapeutic purposes in the treatment of blood diseases, inherited blood disorders, and autoimmune diseases. Nonetheless, advanced development in this field needs knowledge in the biological studies as a background in performing strategy and maintaining of HSCs. Thus, HSC source, origin, niches for HSC pool, and signaling pathways, essential for the regulation of HSCs.¹⁴

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Figue-4: Source of blood cells during gestation through after birth. Intraembryonic yolk sac is the first site of blood cells observation at around E7.0–E7.5. The de novo hematopoiesis in the placenta and aorta-gonad mesonephros (AGM) occurs at nearly similar wave of gestation (around E8.5– E10.5) before it circulates into fetal liver where there is the large HSC pool during gestation. At around E16.5, the HSCs migrate and reside within the bone marrow which finally becomes the source of HSC in adult life. ¹⁴

HIERARCHY OF HUMAN HEMATOPOIESIS

Abbildung in dieser Leseprobe nicht enthalten Figure-5: Hierarchy of hematopoiesis. The phenotypic cell surface marker of each population of mouse and human blood system is shown (modified from [13]). In the mouse hematopoiesis system, MPPs omit CMPs which directly give rise to MEPs unlink in the human system (dash line). CLP, common lymphoid progenitor; CMP, common myeloid progenitor; DC, dendritic cell; EP, erythrocyte progenitor; GMP, granulocyte/macrophage progenitor; GP, granulocyte progenitor; HSC, hematopoietic stem cell; MacP, macrophage progenitor; MEP, megakaryocyte/erythrocyte progenitor; MkP, megakaryocyte progenitor; NK, natural killer; Lin, lineage markers. ¹⁴

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Figure-6: Sources of adult stem cells in the oral and maxillofacial region. BMSCs: bone marrow-derived MSCs from orofacial bone; DPSCs: dental pulp stem cells; SHED: stem cells from human exfoliated deciduous teeth; PDLSCs: periodontal ligament stem cells; DFSCs: dental follicle stem cells; TGPCs: tooth germ progenitor cells; SCAP: stem cells from the apical papilla; OESCs: oral epithelial progenitor/stem cells; GMSCs: gingiva-derived MSCs, PSCs: Periostium-derived stem cells; SGSCs: salivary gland-derived stem cells.⁶

Two types of adult stem cells have been characterized in dental tissues, i.e., epithelial stem cells and mesenchymal stem cell (MSC) like cells. An adult epithelial stem cell niche in teeth was first demonstrated in 199932 via organ culture of the apical end of the mouse incisor. The niche is located in the cervical loop of the tooth apex and possibly contains dental epithelial stem cells, which can notably differentiate into enamel-producing ameloblasts. Although the epithelial stem cell niche is useful for analyses of the fate decision of stem cells in tooth development, no information is available for dental epithelial stem cells in humans.⁶

Niches have been identified in the dental pulp of permanent teeth (Dental Pulp Stem Cells - DPSCs), in naturally exfoliated deciduous teeth (Stem cells from Human Exfoliated Deciduous teeth - SHED), in the periodontal ligament (Periodontal Ligament Stem Cells - PDLSC), in the apical papilla (Stem Cells from Apical Papilla), in the dental follicle (Dental Follicular FCs), and in the Periostium of the maxillary tuberosity (Oral Periostium Stem Cells). In each of these studies a multipotent mesenchymal stem cell was isolated, capable of differentiating in-vitro into at least three lines: osteo/odontogenic, adipogenic and neurogenic. For the evaluation of the potentiality of stem cells from oral niches, Bone Marrow Mesenchymal Stem Cells (BMMSCs) was used as a gold standard, as they are the most tested stem cells and the point of reference for each new line of stem cells.¹⁰

1. DENTAL PULP STEM CELLS (DPSCs)

DPSCs were first isolated from human permanent third molars in 2000. The human adult tooth pulp contains a population of neural crest-derived embryonic mesenchymal stem cell (EMSCs) that can be isolated by enzymatic digestion and which forms adherent clonogenic colonies of fibroblast like cells when cultured in-vitro. These EMSCs from dental pulp, termed DPSCs, display a great variability in growth rate and may exhibit a wide range of cell morphologies and tissue marker expression, which appears to reflect their high multilineage differentiation potential to both mesenchymal and nonmesenchymal lineages, characteristic of neural crest stem cell. Notably, DPSCs in-vivo appear to reside at the perivascular and periodontoblastic compartments within the adult tooth pulp, and their cellular phenotype corresponds to pericyte-like smooth-muscle-actin-(SMA-) expressing cells. DPSCs proliferate faster than bone marrow MSCs (BMMSCs)³³ Finally, and similarly to other MSC types, protocols have been devised that permit long-term cryopreservation of DPSCs without affecting their stemness potential.³⁴

DPSCs have been reported to in-vitro differentiate to multiple cell lineages, including odontoblasts, chondroblasts, adipocytes, muscle cells, and neurons. Differentiation of DPSC to dentinogenic cell lineages specifically seems to be favored after serial in-vitro culture passaging. When DPSCs are xenotypically transplanted in immunocompromised mice, combined with mineralized biocompatible hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolding materials, they can generate a complete dentin-pulp complex containing odontoblastic cells. Conversely, BMMSCs in the same conditions give rise to highly vascularized bone-like tissue, containing adipocytes and lamellar bone trabeculae. These different outcomes after in-vivo transplantation seem to be at least partly related with a differential secretion of paracrine signals by the grafted stem cells, which act upon surrounding host cells to generate specific tissue phenotypes.³³

It is now recognized that DSCs can play an important role in the balance of inflammation and repair/dentinogenesis during invasive caries lesions or pulp exposures. Following odontoblast damage after caries or trauma, markers of inflammation and regeneration within the pulp tissue are differentially expressed, with cross talk between the inflammatory and regenerative processes considered to determine the outcome. This notion is supported by in-vitro observations of DPSCs migrating from the perivasculature toward the dentin surface following injury to the dentin matrix and differentiating into functional odontoblasts in response to EphB/ephrin³⁵ signaling. DPSCs have also been shown to express the bacterial recognition toll-like receptors (TLR), TLR4 and TLR2, and vascular endothelial growth factor in response to lipopolysaccharide, a product of gram-negative bacteria. When compared with normal pulps, DPSCs in inflamed pulp tissues have reduced dentinogenesis activity, and an in-vitro investigation has shown reduced dentinogenic potential of DPSCs exposed to a high bacterial load that can be recovered after the inhibition of the bacterial recognition toll-like receptor TLR2. Taken together, these studies support the existence of interactions between DSCs and immune cells in pulps affected by dental caries, a better understanding of which has significant implications for the future management of teeth affected by dental caries. In an effort to determine the fate of DPSCs exposed to root canal irrigants used in regenerative endodontic therapy procedures, dentin disks were preconditioned with different irrigants (5.25% sodium hypochlorite [NaOCl] or 17% ethylene diamine tetra acetic acid [EDTA]), seeded with DPSCs, and implanted subcutaneously into immunodeficient mice. After 6 weeks, the differentiation of DPSCs into odontoblast like cells was facilitated by the use of EDTA. In contrast, the use of NaOCl resulted in resorption lacunae at the cell-dentin interface.⁵

2. DENTAL PULP PLURIPOTENT STEM CELLS (DPPSCs)

In 2006, Dr. Shinya Yamanaka¹⁹ discovered that normal mouse adult skin fibroblasts can be reprogrammed to an embryonic state by introducing four genetic factors (Oct3/4, Sox2, Klf4 and c-Myc), and the resulting cells were termed iPS cells. Just a year after the mouse study was reported, the findings were replicated in human skin cells, which opened the door to generate a patient-specific ES cell equivalent from autologous somatic cells. This technology is expected to revolutionize medicine because of the capacity of iPS cells to develop into all tissues/organs and thereby support the emerging field of ‘‘personalized medicine’’, which uses a patient’s own cells to provide biologically compatible therapies and individually tailored treatments. For dental applications, iPS cells that can be efficiently generated from tissues that are easily accessed by dentists have great potential, and iPS cells have been generated from various oral mesenchymal cells, such as SCAP, DPSCs and SHED, TGPCs, buccal mucosa fibroblasts, gingival fibroblasts and periodontal ligament fibroblasts. Most of these cells have a higher reprogramming efficiency than the conventionally used skin fibroblasts, possibly because of their high expression of endogenous reprogramming factors and/or ES cell-associated genes as well as their high proliferation rate. Therefore, cells of oral origin are expected to provide an ideal iPS cell source, especially for dentists and dental researchers. These iPS cells may be of particular importance for developing innovative technologies to regenerate missing jaw bones, periodontal tissues, salivary glands and lost teeth. In a mouse model, iPS cells combined with enamel matrix derivatives provided greatly enhanced periodontal regeneration by promoting the formation of cementum, alveolar bone and periodontal ligament. In-vitro studies demonstrated that differentiation of mouse iPS cells into ameloblasts and odontogenic mesenchymal cells, which may be useful approach for tooth bioengineering strategies. However, the scientific understanding of iPS cells and how to control their differentiate fate is still limited. Despite the similarities between iPS cells and ES cells, it remains unclear whether these pluripotent stem cells are exactly equal. It is indicated that not all iPS cells are equal and that iPS cells retain an epigenetic memory of their former phenotype that can limit their differentiation potential. Therefore, strategies that bypass the epigenetic memory to create more ES-like iPS cells or that can identify iPS cell sources that are amenable to efficient guided differentiation to the target lineage may be necessary. To achieve this goal, the generation of more stringent markers of pluripotency and assays to determine the abilities of a given iPS cell line is critical. In addition, the prevention of tumor formation upon in-vivo implantation of iPS cells is critical for their clinical application. The original protocol to generate iPS cells uses the c-Myc oncogene as one of the reprogramming factors and a retroviral vector for gene transfer, which raises concern about possible carcinogenic properties. Recent rapid progress in iPS cell research has virtually resolved these problems, e.g., by using L-Myc as a replacement for c-Myc or via the application of small molecules rather than viral gene delivery, the generation of reprogramming protocols to enhance the reprogramming efficiency without requiring c-Myc and the use of non-viral components such as protein, micro-RNA, synthetic mRNA or episomal plasmids for reprogramming. However, serious clinical problems can still arise when residual undifferentiated iPS cells remaining among the differentiated target cells uncontrollably proliferate to form teratomas in the transplanted site. To address this critical issue, several approaches are being investigated, such as a selective ablation method to remove teratomas via suicide genes and chemotherapy, as well as a cell sorting method to remove teratoma-forming cells using specific antibodies.⁶

3. STEM CELLS FROM HUMAN EXFOLIATED TEETH (SHED)

SHED cells are highly proliferative stem cells isolated from exfoliated deciduous teeth capable of differentiating into a variety of cell types, including osteoblasts, neural cells, adipocytes, and odontoblasts, and inducing dentin and bone formation. Like DPSCs, SHED cells can generate dentin-pulp like tissues with distinct odontoblast like cells lining the mineralized dentin-matrix generated in HA/TCP scaffolds implanted in immunodeficient mice. However, SHED cells have a higher proliferation rate than DPSCs and BMMSCs, suggesting that they represent a more immature population of multipotent stem cells. SHED cells have shown different gene expression profiles from DPSCs and BMMSCs; genes related to cell proliferation and extracellular matrix formation, such as transforming growth factor (TGF)-b, fibroblast growth factor (FGF), TGF-b2, collagen (Col) I, and Col III, are more highly expressed in SHED cells compared with DPSCs.³⁶ In tissue engineering studies, odontoblastic and endothelial differentiation occurred when SHED cells were seeded in tooth slices/scaffold and implanted subcutaneously into immunodeficient mice. The resultant tissues closely resembled those of human dental pulp, and tubular dentin mediated by dentin-derived BMP-2 protein was secreted. These findings, together with those of other studies, suggest that SHED cells from exfoliated deciduous teeth may be an excellent resource for stem cell therapies, including autologous stem cell transplantation and tissue engineering. Regenerative endodontic therapy procedures should avoid compromising the attachment of stem cells to dentin. An in-vitro study showed that the root canal irrigants 6% NaOCl and 2% chlorhexidine (CHX) were cytotoxic to SHED cells. In addition, the attachment of SHED cells to root canal dentin pretreated with NaOCl or CHX was reduced compared with negative controls (saline pretreatment).⁵

4. PERIODONTAL LIGAMENT STEM CELLS (PDLSCs)

McCulloch³⁷ reported the presence of progenitor/stem cells in the periodontal ligament of mice in 1985. Subsequently, the isolation and identification of multipotent MSCs in human periodontal ligaments were first reported in 2004.³⁸ Seo and colleagues demonstrated the presence of clonogenic stem cells in enzymatically digested PDL and further showed that human PDLSCs transplanted into immunodeficient rodents generated a cementum/PDL-like structure that contributed to periodontal tissue repair. Later work showed that PDLSCs differentiation was promoted by Hertwig’s epithelial root sheath cells in-vitro. PDLSCs have the capability to differentiate into cementoblast like cells, adipocytes, and fibroblasts that secrete collagen type I. As with BMMSCs, PDLSCs can undergo osteogenic, adipogenic, and chondrogenic differentiation. PDLSCs have also been shown to differentiate into neuronal precursors. The therapeutic potential of autologous periodontal ligament progenitor cells obtained from third molar teeth implanted on bone grafting material into intrabony defects in 2 patients. After 32 to 70 months, a marked improvement was found in all sites. The progenitor cells behaved like PDLSCs, although they did not express the same markers.⁵

Enzyme mediated digestion of the PDL also yields a MSC population with clonogenic potential, and similar proliferation rates to adult DPSCs, and PDLSCs also display a multilineage differentiation potential. When transplanted in-vivo in the presence of scaffolds, these PDLSCs are able to generate properly arranged cementum and PDL like structures, including Sharpey’s fiber bundles providing cementum attachment. These properties make PDLSCs a good choice for their use in periodontal tissue engineering therapies, possibly in combination with other adjuvant factors such as platelet-derived plasma rich in growth factors, or PRGFs. Moreover, PDLSCs also maintain their stem cell properties after cryopreservation.³⁴

5. STEM CELLS FROM APICAL PAPILLA (SCAP)

The apical papilla is essential for root development. SCAP cells were first isolated in human root apical papilla collected from extracted human third molars. The apical papilla is the tissue which surrounds the apices of developing teeth near Hertwig’s epithelial root sheath and which takes part in tooth root formation. An EMSC population presenting a high proliferative capacity can be isolated from there. The apical papilla may also be present in some pre erupting little developed wisdom teeth. SCAP can be induced to differentiate to multiple cell lineages and possess a large potential for dental and periodontal repair therapies. SCAP can also be cryopreserved without losing stem cell activity.³⁴

The cells are clonogenic and can undergo odontoblastic/ osteogenic, adipogenic, or neurogenic differentiation. Compared with DPSCs, SCAP cells show higher proliferation rates and greater expression of CD24, which is lost as SCAP cells differentiate and increase alkaline phosphate expression. SCAP cells seeded onto synthetic scaffolds consisting of poly-D,L-lactide/glycolide inserted into tooth fragments, and transplanted into immunodeficient mice, induced a pulp like tissue with well-established vascularity, and a continuous layer of dentin like tissue was deposited onto the canal dentinal wall.⁵

In a mini pig, a bio-root was created by using autologous human SCAP cells seeded in an HA/TCP root-shaped carrier coated with Gel foam carrying PDLSCs that were implanted in the alveolar socket of a recent extracted anterior tooth. After 4 months, the resulting bio-root was capable of supporting a porcelain crown and participating in normal tooth function. Root canal irritants’ used in regenerative endodontic therapy procedures should ideally support cell survival, or at least not compromise survival. An in-vitro study showed that 17% EDTA used alone supported SCAP cell survival better (89% survival) than when used with either 6% NaOCl (74% survival) or 2% CHX (0% survival).⁵

6. STEM CELLS FROM DENTAL FOLLICLE (SCDF)

The dental sac or follicle is the ectomesenchymal embryonic tissue surrounding the tooth germ. It is a loose vascular connective tissue that contains the developing tooth germ, and progenitors for periodontal ligament cells, cementoblasts, and osteoblasts. DFPCs were first isolated from the dental follicle of human third molars. Because DFPCs come from developing tissue, it is considered that they might exhibit a greater plasticity than other DSCs. Indeed, different cloned DFPC lines have demonstrated great heterogeneity. In addition, after transplantation in immunodeficient rodents, DFPCs differentiated into cementoblast like and osteogenic like cells, and surface markers compatible with those of fibroblasts were identified in human dental follicle tissues, suggesting the presence of immature PDL fibroblasts. DFPCs were able to differentiate into odontoblasts in-vitro, and four weeks after combining rat DFPCs with treated dentin matrix the root-like tissues stained positive for markers of dental pulp. Both DFPCs and SHED cells can differentiate into neural cells; however, these are differentially expressed when the cells are grown under the same culture conditions.⁵

7. TOOTH GERM PROGENITOR CELLS (TGPCs)

Distinctive stem cells are found in the dental mesenchyme of the third molar tooth germ at the late bell stage (tooth germ progenitor cells: TGPCs) with high proliferation activity and the capability to differentiate in-vitro into lineages of the three germ layers including osteoblasts, neural cells and hepatocyte. TGPCs showed high proliferation activity and capability to differentiate in-vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate amino-transferase and alanine amino-transferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.⁶

8. GINGIVA DERIVED MESENCHYMAL STEM CELL (GMSCs)

In 2009, Zhang et al.³⁹ first characterized human gingiva-derived MSCs (GMSCs), which exhibited clonogenicity, self-renewal and a multipotent differentiation capacity similar to that of bone marrow stem cell (BMSCs). GMSCs proliferate faster than BMSCs, display a stable morphology and do not lose their MSC characteristics with extended passaging. A multipotent neural crest stem cell-like population, termed oral mucosa stem cells (OMSCs), can be reproducibly generated from the lamina propria of the adult human gingiva and can differentiate in-vitro into lineages of the three germ layers. The inherent stemness of gingival cells may therefore partly explain the high reprogramming efficiency of gingiva-derived fibroblastic cell populations during iPS cell generation. The multipotency of GMSCs/OMSCs and their ease of isolation, clinical abundance and rapid ex vivo expansion provide a great advantage as a stem cell source for potential clinical applications.⁶

9. ORAL MUCOSA-DERIVED STEM CELLS (OMSCs)

The oral mucosa is composed of stratified squamous epithelium and underlying connective tissue consisting of the lamina propria, which is a zone of well vascularized tissue, and the submucosa, which may contain minor salivary glands, adipose tissue, neurovascular bundles and lymphatic tissues depending on the site. To date, two different types of human adult stem cells have been identified in the oral mucosa. One is the oral epithelial progenitor/stem cells, which are a subpopulation of small oral keratinocytes (smaller than 40 mm). Although these cells seem to be unipotential stem cells, i.e., they can only develop into epithelial cells, they possess clonogenicity and the ability to regenerate a highly stratified and well-organized oral mucosal graft ex-vivo, which suggests that they may be useful for intra-oral grafting. Other stem cells in the oral mucosa have been identified in the lamina propria of the gingiva, which attaches directly to the periosteum of the underlying bone with no intervening submucosa. The gingiva overlying the alveolar ridges and retromolar region is frequently resected during general dental treatments and can often be obtained as a discarded biological sample. In 2009, Zhang et al.39 first characterized human gingiva-derived MSCs (GMSCs), which exhibited clonogenicity, self-renewal and a multipotent differentiation capacity similar to that of BMSCs. GMSCs proliferate faster than BMSCs, display a stable morphology and do not lose their MSC characteristics with extended passaging. Recently, Marynka-Kalmani et al.40 reported that a multipotent neural crest stem cell-like population, termed oral mucosa stem cells (OMSCs), can be reproducibly generated from the lamina propria of the adult human gingiva and can differentiate in-vitro into lineages of the three germ layers. The inherent stemness of gingival cells may therefore partly explain the high reprogramming efficiency of gingiva-derived fibroblastic cell populations during iPS cell generation. The multipotency of GMSCs/OMSCs and their ease of isolation, clinical abundance and rapid ex vivo expansion provide a great advantage as a stem cell source for potential clinical applications.⁶

10. PERIOSTEUM-DERIVED STEM/PROGENITOR CELLS

The periosteum is a specialized connective tissue that covers the outer surface of bone tissue. The osteogenic capacity of the periosteum of long bones was reported in 1932,⁴¹ and the periosteum membrane was found to form a mineralized extracellular matrix under the appropriate in-vitro conditions. Several subsequent studies have addressed other aspects of periosteal osteogenesis, including long bone development and the periosteum, the relationship between the vasculature and the periosteum and the periosteal osteogenic capacity. Histologically, the periosteum is composed of two distinct layers and up to five distinctly different functional regions when it is dissociated enzymatically and cultured. The outer area contains mainly fibroblasts and elastic fibers, and the inner area contains MSCs, osteogenic progenitor cells, osteoblasts and fibroblasts, as well as micro vessels and sympathetic nerves. Although the heterogeneous cell population isolated from the periosteum seems to preferentially undergo osteogenic differentiation, these cells are capable of differentiating into osteoblasts, adipocytes and chondrocytes and expressing the typical MSC markers. In addition, De Bari et al.⁴² demonstrated that single-cell-derived clonal populations of adult human periosteal cells possess mesenchymal multipotency, as they differentiate to osteoblast, chondrocyte, adipocyte and skeletal myocyte lineages in-vitro and in-vivo. Therefore expanded periosteum-derived cells could be useful for functional tissue engineering, especially for bone regeneration. A comparative analysis of canine MSCs/progenitor cells showed that the in-vivo potential of periosteum cells to form bone was higher than that of ilium-derived BMSCs and alveolar bone cells. The phenotypic profiles of human maxillary/ mandibular periosteum cells were comparable to those of maxillary tuberosity-derived BMSCs, and both cell populations formed ectopic bone after subcutaneous implantation in mice. Agata et al.⁴³ reported that human periosteal cells proliferated faster than marrow stromal cells, and subcutaneous transplants of periosteal cells treated with a combination of recombinant growth factors formed more new bone than BMSCs in mice. Periosteal grafts have been shown to induce cortical bone formation, whereas bone marrow grafting induced cancellous bone formation with a bone marrow-like structure in a rat calvarial defect model, which implies that the source of the transplanted cells can influence the structural properties of the regenerated bone.⁶

11. SALIVARY GLAND-DERIVED STEM CELLS

Patients afflicted with head and neck cancer who receive radiotherapy suffer from an irreversible impairment of salivary gland function those results in xerostomia and a compromised quality of life. Therefore, stem cells in the adult salivary gland are expected to be useful for autologous transplantation therapy in the context of tissue engineered-salivary glands or direct cell therapy. The salivary glands originate from the endoderm and consist of acinar and ductal epithelial cells with exocrine function. After ligation of the salivary gland duct, the acinar cells undergo apoptosis, and the duct epithelium subsequently proliferates. A single stem cell that gives rise to all epithelial cell types within the gland has not yet been identified. Thus far, the isolation of stem cells in the salivary glands has been attempted through the cell culture of dissociated tissue. Kishi et al.44 isolated salivary gland stem/progenitor cells from rat submandibular glands and found that the cells are highly proliferative and express acinar, ductal and myoepithelial cell lineage markers. In-vitro floating sphere culture method could be used to isolate a specific population of cells expressing stem cell markers from dissociated mouse submandibular glands. These cell populations could differentiate into salivary gland duct cells as well as mucin and amylase-producing acinar cells in-vitro. Progenitor/stem cells were also isolated from swine and human salivary glands. In addition, the intra-glandular transplantation of cells isolated from mouse submandibular glands successfully rescued the salivary function of irradiated salivary glands and Neumann et al.⁴⁵ reported the long-term cryopreservation of integrin-a6b1 expressing cells as a sub-population of rat salivary gland progenitor cells. These reports suggest that the salivary gland is a promising stem cell source for future therapies targeting irradiated head and neck cancer patients. However, primary cultures of dispersed cells will always contain a number of cells with different origins, such as parenchymal cells, stromal cells and blood vessel cells, which makes it difficult to select salivary gland stem cells. Indeed, Gorjup et al.46 isolated primitive MSC-like cells from the human salivary gland, but possibly from stromal tissue, which expressed embryonic and adult stem cell markers and could be guided to differentiate into adipogenic, osteogenic and chondrogenic cells. To obtain a genuine stem cell population that can be considered to be a true stem cell for the salivary gland, it is necessary to select cells carrying a specific marker or labeled with induced reporter proteins.⁶

STEM SAVE® has given tooth eligible criteria for stem cell isolation from different teeth. A healthy pulp contains viable stem cells. For a pulp to be considered healthy, the tooth must have an intact blood supply, be free of infection, deep caries and other pathologies. Stem cells are not concentrated within any particular area of a healthy pulp, but are diffusely spread throughout the cellular zone adjacent to the nerve and blood vessels within the pulp. Specific criteria must be met in order for a tooth to be eligible for stem cell recovery. Stem save has broken this down into three distinct tooth groups in which patients have the opportunity to recover their stem cells. It is best to recover stem cells when a patient is young and healthy and the stem cells are at their most proliferative. Stem cells can also be recovered from the permanent teeth of middle-aged individuals. Benefits are realized at this age when compared to current life expectancy statistics, coupled with the almost certain need for their use in future regenerative therapies.⁴⁷

DECIDUOUS TEETH

The healthy pulps of deciduous teeth are a rich source of viable stem cells. Scientific data supports that stem cells isolated from healthy pulp of deciduous teeth are highly proliferative, even when the pulp is recovered in small quantities. Certain factors will determine whether viable stem cells can be recovered from deciduous teeth.⁴⁷

The ideal deciduous tooth for stem cell recovery is a canine or incisor that has just started to loosen, has more than a third of the root structure left intact, and is not extracted for reasons such as infection or associations with pathology. ⁴⁷

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(A) (B) (C)

Figure-7: Figure A & B showing more than 1/3rd of root left intact for stem cell saving, while Figure C indicates less than 1/3rd of root left. ⁴⁷

Supernumerary or mesodens are another ideal source for dental stem cells. In most cases when these teeth are removed, they still have a complete root, intact blood supply and healthy pulp. ⁴⁷

HARVEST ZONE

The harvest zone for stem cells is from the deciduous canine to canine. Deciduous molars may have their pulp chambers obliterated by the erupting permanent bicuspids by the time they become loose. In most cases, the remaining pulpal tissue may not be adequate for dental stem cell recovery. Over-retained molars and molars extracted for orthodontic reasons may also be considered. ⁴⁷

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Figure-8: Showing harvested zone of deciduous anterior. ⁴⁷

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Figure-9: Showing retained molar can be a source of stem cell. 47

The pulps of naturally exfoliated teeth or teeth that have fallen out on their own are most likely necrotic, as they have been separated from their blood supply. A patient bringing a tooth in hand to the office is not a good candidate for recovery.⁴⁷

An excessively loose tooth or one that is “hanging on by a thread” is not a candidate for stem cell recovery. Even though the tooth is still attached to gingiva, the pulp most likely is necrotic.⁴⁷

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Figure-10: Showing loose or hanging tooth is not good source of stem cell. ⁴⁷

PERMANENT TEETH

All permanent teeth with healthy pulp are potential sources of stem cells. Permanent teeth to avoid include endodontically-treated or nonviable teeth, teeth with active infections, teeth with severe periodontal disease and excessive mobility, teeth with deep caries or large restorations, and teeth with sclerosing or calcified pulp chambers.⁴⁷

THIRD MOLARS

The healthy pulp from wisdom teeth is another excellent source for viable stem Cells. Whole or sectioned portions of third molars containing healthy pulp can be recovered at the time of their removal. When an impacted third molar needs to be sectioned for removal, the pulp is often exposed. ⁴⁷

Developing third molars have a larger volume of pulpal tissue than teeth that are mature with their roots completely formed. ⁴⁷

It is best to recover these teeth during the developmental stage (between 16-20 years of age), when the stem cells are very active in the formation of the root and supporting root structures. ⁴⁷

Third molars with healthy pulp can also be recovered later in life and are always considered a source for viable stem cells.⁴⁷

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Figure-11: Showing stem cell can be saved from third molars. ⁴⁷

AGE IS IMPORTANT

The stem cells from within the pulp become less proliferative as individuals age, so it is best to recover stems cells at the earliest opportunity.⁴⁷

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( A ) Healthy pulp molar of 18 year old ( B) Sclerosed pulp molar of 70 year old. ⁴⁷

Figure-12: (a) and (b) showing Healthy pulp molar of 18 year old pulp molar of 70 year old.⁴⁷

A fundamental approach to isolate MSCs in tissue samples involves the enzymatic digestion of tissue followed by the growth of isolated cells (expansion) in medium rich in growth factors.⁴⁸ The isolation of more immature stem cells involves a multistep explants approach whereby pieces of tissue are first cultured until progenitor cells grow after which enzymatic digestion and expansion in media proceed. The identification of MSCs uses a series of in-vitro tests. Colony-forming assays are used to confirm clonogenicity (the ability to generate identical stem cells with the appropriate cell morphology), which is a consistent characteristic of MSCs. Phenotypic assays evaluate cell morphology or shape (fibroblastic when flat and elongated) and cell behavior (secretory). The possession of one or several cell surface markers found on cells in representative tissues is evaluated by flow cytometry, which sorts cells with specific surface protein, such as bone marrow surface protein STRO-1, found on stem cells that can differentiate into multiple mesenchymal lineages, including dental pulp cells. DSCs can also express specific proteins associated with endothelium (CD106, CD146), perivascular tissues (a-smooth muscle actin, CD146, 3G5), bone, dentin and cementum (bone morphogenic protein [BMP], alkaline phosphatase, osteonectin, osteopontin, and bone sialoprotein), and fibroblasts (type I and III collagen).49 In-vitro functional assays test putative MSCs for multipotency by confirming that differentiated cells demonstrate the appropriate phenotypic characteristics. Accordingly, the in-vitro confirmation of the multipotency of dental pulp stem cells (DPSCs) can be demonstrated by the evidence of odontoblast like differentiation (verified by the deposition of mineralized matrix and positive staining for dentin sialophosphoprotein), adipogenic differentiation (by the accumulation of lipid vacuoles), chondrogenic differentiation (by the production of collagen type II), and neurogenic differentiation (by neuronal-cell morphologies and markers).50 In-vivo functional assays are used to confirm that stem cells implanted into a new environment (immunodeficient mice) successfully integrate with adjacent cells, survive, and function as differentiated cells.51 Several studies have demonstrated the formation of new pulp and dentin like tissues following the insertion of DSCs seeded onto scaffolds in emptied human root canals or dentin disks embedded into immunocompromised mice; the resulting dentinogenesis is accomplished by odontoblast like cells derived from MSCs.⁵

STORAGE OF STEM CELLS

Adult stem cells can be obtained from individuals at any stage in life and, therefore, can provide a source of cells for autologous transplants.⁵² Such procedures invariably require stem cell storage, which is achieved by cryopreservation in liquid nitrogen (-1960C). Stem cells can survive these low temperatures as long as they are dispersed in cryprotectants.53 Human periodontal ligament stem cells (PDLSCs) have been successfully recovered after cryopreservation for 6 months; although the number of colonies was less than for fresh PDLSCs, the proliferation rate was similar.54 Similarly, stem cells isolated from human third molar teeth and cryopreserved for at least 1 month retained STRO-1 marker expression and the potential to proliferate into neurogenic, adipogenic, osteogenic/odontogenic, myogenic, and chondrogenic pathways in inductive media. Cryopreservation of intact teeth provides another potential storage method that can allow later extraction of stem cells demonstrating similar behavior as stem cells extracted from fresh teeth.⁵

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Figure-13: Techniques for generating embryonic stem cell cultures.⁵⁵

ISOLATION AND CULTURE hESCs

The human blastocyst in-vitro consists of 200 to 250 cells. It is the source of stem cells, at this stage it is composed of 30 to 34 cells. To derive stem cells from a blastocyst, the trophoblast is removed, either by microsurgery or immunosurgery. The isolated cells is then plated onto a tissue culture dish precoated with mitotically inactivated mouse embryonic fibroblasts (mEF) or human embryonic fibroblasts (hEF) in hESC culture medium.56 Since the ES cells are not able to attach and grow on non-coated culture dishes, the presence of feeder layer is essential for their attachment and growth. After the cells have been isolated from the inner cell mass (ICM), they divide and spread over the surface of the dish. The outgrowth is dissociated into small pieces mechanically, using a Stem Cell Cutting Tool shaped as a micropipette. The cells are then re-plated on new mEF or hEF layers in new hESC medium. The small pieces attach to the new feeder layer and grow as individual colonies with undifferentiated morphology (Figure 14). The same procedure is being repeated every 4 to 5 days. This culture method is known as a feeder-dependent culture of hESCs.⁵⁵

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Figue-14: Human blastocyst showing inner cell mass, trophoblast and blastocoele.⁵⁵

DPSCs ISOLATION

Dental pulp (DP) was isolated under the sterile conditions using different procedures, for DP extraction third molars, premolars and exfoliated teeth taken. To avoid thermic damage, diamond burr for cooling turbine is used following tooth splitting; DP is isolated using excavator. The tooth and the pulp are then transported in Hank’s balanced salted solution (HBSS) to the laboratory. Another DP harvesting procedure is completely done in tissue cultures laboratory. Intact tooth with the pulp is transported in HBSS into laboratory. Luer’s forceps is also used to break the roots in order to extract the pulp through the root canals. If the roots are not developed and apical foramen is widely open, sharp needle can be used to release DP from the pulp chamber. If the roots are not wide enough, extirpation needle are used. Both, the dental pulp and tooth, are enzymatically treated with collagenase and dispase for 70 minutes. Cell pellet of two fractions are obtained by centrifugation: a) cell fraction from subodontoblastic compartment (SOc) and b) cell fraction from perivascular compartment (PVc).⁵⁷

ISOLATION OF hMSC

In another experiment aspirated bone marrow and was diluted and cooled (4°C) in HBSS with Heparine and transported to laboratory. Bone marrow mononuclear cells are obtained by optimized Ficoll-Paque density gradient centrifugation .⁵⁷

The bone marrow sample obtained after filtering is diluted (1:3) with phosphate-buffer saline (PBS), and then transferred to a 50mL conical tube containing 20mL of Ficoll/Hypaque and centrifuged for 30 minutes at 500g, 22oC. After centrifugation the cells are transferred to another tube and centrifuged again for 5 minutes at 500g, 22oC; the supernatant is discarded and cells are resuspended with DMEM-LG supplemented with 10% FBS in order to achieve a concentration of 1x105cells/mL. Next, the cells are cultivated in 25cm2 flasks maintained in humidified 5% CO2 incubators at 37oc to favor the attachment of the hMSC to the flask bottom.⁵⁸

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Figure-15: Showing bone marrow aspiration.⁵⁸

ISOLATION OF SHED

The pulp is separated from a remnant crown and then digested in a solution of 3mg/ml collagenase type I and 4 mg/ml dispase for 1 h at 37°C. Single-cell suspensions are cultured in a regular medium. These techniques resulted in populations that are termed stem cells from human exfoliated deciduous teeth (SHED). Conditions for the induction of calcium accumulation, recombinant human BMP-4 is used to induce osteogenic differentiation. Calcium accumulation is detected by 2% Alizarin red S (pH 4.2) staining. The calcium concentration is measured by using a commercially available kit (calcium kit 587-A, Sigma). The induction of adipogenesis is performed for neural differentiation, Neurobasal A, B27 supplement, 1% penicillin, 20ng/ml epidermal growth factor and 40ng/ml fibroblast growth factor (FGF) are used to culture cells attached to 0.1% gelatin-coated dishes. For sphere like cell-cluster formation, 3% rat serum and B27 are added.⁵⁰

ANTIBODIES

Rabbit antibodies including anti-HSP90 and basic FGF (bFGF) anti-core-binding factor, runt domain, subunit 1 (CBFA1); anti-endostatin, human specific mitochondria, and glutamic acid decarboxylase (GAD) and anti-alkaline phosphatase (ALP) (LF-47), bone sialoprotein (LF-120), matrix extracellular phosphaglycoprotein (MEPE) (LF-155), and dentin sialophosphoprotein (DSPP) (LF-151) (National Institute of Dental and Craniofacial Research[1]National Institutes of Health). Goat antibodies include anti-MAP2 and Tau. Mouse antibodies include anti-STRO-1 and CD146 (CC9); glial fibrillary acidic protein (GFAP), nestin, neurofilament M (NFM), neuronal nuclei (NeuN), and 2,3-cyclic nucleotide-3-phosphodiesterase (CNPase) and anti-III-tubulin. Rabbit and murine isotype-matched negative control antibodies are also used.⁵⁰

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Figure-16: (A) The exfoliated primary incisor containing dental pulp as shown (black triangles). The dashed line shows the occlusion edge of the incisor. (B and C) Hematoxylin-eosin staining indicated dentin (D) and pulp of exfoliated deciduous teeth. The pulp contain odontoblasts (arrows), blood vessels (open arrows), and connective tissues. The straight and curved dashed lines in B represent the occlusion and resorbed root surfaces, respectively. (D) Single colonies are formed after SHED is plated at low density and culture for 2 weeks. (E) SHED is capable of forming sphere-like clusters. (F) The sphere-like clusters could be dissociated by passage through needles and subsequently grew on 0.1% gelatin-coated dishes.⁵⁰

ISOLATION OF NEURAL STEM CELL (NSCs)

NSCs are isolated from brain tissues by cell-surface markers (such as CD133) or green fluorescent protein (GFP) expression driven by NSC-specific promoters. These promoters include Sox1, Sox2, Nestin, and fibroblast growth factor 1 (FGF1). Isolated NSCs are cultured in the presence of growth factors and examined to determine whether they could expand to form neurospheres. The capacity to form neurospheres is defined as self-renewal. The potential for neural differentiation of these isolated cells upon withdrawal of growth factors or administration of inducing factors is used to determine multipotency.59 FGF1 is expressed in ventral cochlear neurons, olfactory bulbs, and hippocampal neurons, but not in glial cells. The brain-specific FGF-1B promoter is active only in the brain. Interestingly, it has been shown that FGF-1B mRNA is up-regulated for the maintenance of NSCs in hippocampus dentate gyrus in response to activity-induced neurogenesis. Furthermore, FGF-1B promoter (–540 to +31)-driven GFP reporter (F1B-GFP) can be used to isolate NSCs with self-renewal and multipotent capacities from human glioblastoma tissues and developing neonatal or adult mouse brains. NSCs could be isolated as GFP-positive cells when adult mouse brain cells are transferred with F1B-GFP plasmid. This F1B-GFP plasmid comprises the GFP coding sequences driven by the human FGF1 promoter. Notably, F1B-GFP-selected NSCs from mouse brains are able to repair the damaged sciatic nerve of paraplegic rats. Micro patterned nerve conduits, together with NSCs and FGF1, could repair PNI in animals. Recently, a novel material has been developed named as ultra nanocrystalline diamond (UNCD) and demonstrated that UNCD can enhance the differentiation of NSCs (Figure 17) and (Figure 18) and can be used as the standardized differentiation method of NSCs for clinical application in PNIs.⁵⁹

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Figure-17: (A) F1B-GFP permits the isolation os F1B(+) cells from adult mouse brain, Schematic illustration of the isolation of F1B-GFP(+) neural stem cells from adult mouse bain (NSA, Neurosphere assay). (B) KT98/F1B-GFP(+) mouse brain cells showed strong fluoesccnce intensity. GFP- positive cells formed 2500 neurosperes out of 8 x 104 cells plated, while GFP-negative cells formed 26 spheres. A 100-fold enhancement for the neurosphere forming efficiency was demonstrated for the F1B-GFP(+) cells. (C) differentiation potential between F1B-GFP(+) mouse brain cells.⁵⁹

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Figure-18: Erk1/2 and F1K signaling pathways are activating during neuronal differentiation that is induced by H-UNCD films. The left panel show the poshphorylated Erk ½ and phosphorylated Fak (A,C,E) cultred on polysteren, while the right panels (B,D,F) show the NSCs culture on H-UNCD films (H). Immunofluorescence triple labeling shows staining patteren of pErk (red), pFak (green), and DAPI (blue) on NSCs at three different time points : 4h (a,b), 8 h (C,D) and 12 h(E,F).⁵⁹

ISOLATION OF MONONUCLEAR CORD BLOOD CELLS

Cord blood is aspirated and drawn directly into a 50 mL tube containing 5 mL of buffer and stored at 4 oc prior to separation.60

Dilution of anticoagulated cord blood with 3 times the volume of buffer and then carefully layer 35 mL of diluted cell suspension over 15 mL of Ficoll-Paque in a 50 mL conical tube. Centrifugation is done at 400x for 35 minutes at 20 oc in a swinging- bucket rotor without break.⁶⁰

Aspirate the upper layer leaving the mononuclear cell layer (Lymphocytes, Monocytes, and Thrombocytes) undisturbed at the interphase. Then carefully transfer the mononuclear cell layer to a new 50 mL conical tube which is filled with buffer, then mixed and centrifuged at 300x for 10 minutes at 20 oc. After removal of supernatant completely MNCs can be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous serum.⁶⁰

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Figure-19: Schematic figure showing density gradient centrifugation.⁶⁰

CHARACTERIZATION OF UNDIFFERENTIATED HESCs

There is no agreed standard test that demonstrates that stem cells are in their undifferentiated stage, but there are several kinds of criteria that a hESCs shall fulfill to be considered undifferentiated. The cell morphology is one of those criteria. The undifferentiated ESCs have epithelial-like cell morphology and grow in monolayer colonies. The other criterion is the proof of pluripotency and the ability to generate all three embryonic germ layers (mesoderm, endoderm and ectoderm). This can be demonstrated in-vitro using a three dimensional culture system which triggers spontaneous differentiation of the cells. This system is denoted embryoid body formation (EB). The corresponding in-vivo system is the test of teratoma formation, i.e. injecting hESCs into immunocompromised mice. Expressing pluripotency markers in monolayer culture is also good evidence. Some of those widely used markers are Octamer Transcription Factor-4 (Oct-4), stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-rejection antigen (TRA)-1-60, TRA-1-81 as well as the marker of early differentiation SSEA-1. Finally, retaining a normal karyotype after long-term growth and self-renewal is also a prerequisite for their future clinical use.⁵⁵

IN -VIVO/EMBRYONIC DIFFERENTIATION

Differentiation is the process by which a less specialized cell such as a stem cell becomes a more specialized cell type. Embryonic development is a three dimensional spontaneous process in which the cells gradually differentiate into more specialized cells. The zygote divides and forms an embryo which differentiates to derivate of the three major embryonic germ layers; endoderm, mesoderm, and ectoderm. These three germ layers subsequently give rise to all cell types and tissues in an organism’s body. The ectoderm (external layer) gives rise to skin, neural cells and pigment cells; the mesoderm (middle layer) gives rise to muscle, cartilage, bone and blood cells; the endoderm (internal layer) gives rise to the internal organs (Figure 20). However, the differentiation process is also common in a fully grown organism, as an adult stem cell divides and creates differentiated daughter cell during tissue repair. Differentiation dramatically changes the morphology, size, metabolic activity, and gene and protein expression of a cell. A cell differentiates by internal gene signaling and external signals such as factors secreted by other cells, physical contact with other cells and exchanging materials and molecules in the microenvironment. The internal gene signaling has a central role in cell differentiation, and is a combination of several different and specific genes which are turned on or off at the right time point to transform a cell into a specific cell type. For instance, the SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and Oct4 are all expressed by pluripotent cells in the ICM. An early differentiation marker is SSEA-1 as well as III tubulin, a primitive ectodermal marker. Hepatocyte nuclear factor 3 (HNF3) is an endodermal marker and smooth muscle actin (ASMA) a mesodermal marker. However, cellular proliferation and differentiation processes are controlled by external signals as well. Such signal molecules, called growth factors, are for example, fibroblast growth factor (FGF), bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), insulin-like growth factor (IGF) and transforming growth factor beta (TGF-β) are some of the most well known growth factors.⁵⁵

IN-VITRO DIFFERENTIATION

Differentiation of hESCs may be directed by controlling cell culture condition to desired cell lineages in unlimited numbers. There are several reports describing in-vitro differentiation of hESCs into neural,⁶¹ cardiomyogenic,⁶² hematopoietic,⁶³ pancreatic⁶⁴ and osteogenic lineages.⁶⁵ Similar to the in-vivo differentiation, the In-vitro differentiation requires internal gene signaling and the impact from environmental factors. One can stimulate differentiation of the cells with the assistance of factors such as three dimensional culturing methods, different biomaterials as well as the culture medium and its components such as serum, different chemicals and growth factors and its concentration in the culture medium. Other factors, such as cell-cell contact, co-culture with other cell types, as well as using conditioned medium i.e. factors secreted in the medium by other cells are also effective methods to induce cellular differentiation. Using conditioned medium as an indirect co-culture model can also lead to a better understanding of several molecular pathways leading to specification and terminal differentiation of embryonic cells.⁵⁵

There are several studies investigating the impact and the importance of such factors. For instance, the ability of conditioned media to drive specific differentiation of ESCs from mouse and humans towards e.g. early primitive ectoderm-like (EPL) cells or hepatocyte-like cells has been demonstrated. Directing hepatic differentiation of hESCs has also been demonstrated by co-culture. The potential of the culture micro-environment to influence cellular differentiation has been demonstrated by co-culture to drive stem cells towards required lineages. For instance, differentiation toward distal lung epithelium by co-culturing EBs with distal embryonic lung mesenchyme or toward hematopoietic cells by co-culture with human fetal liver cells and inducing cardio-myocyte differentiation controlled by co-culture as a differentiation model system initiating differentiation to beating muscle. In fact, using cell-cell interaction as a differentiation model system is central in all of these studies and has not been well investigated in developmental biology. Hence, the effect of direct co-culture is essential to understanding the role of cell-cell interaction.⁵⁵

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Figure-20: Differentiation to all three embryonic germ layers and subsequently to different tissues. Microphotographs showing morphology of stem cells in culture: DPSCs (A), SHED cells (D), and SCAP cells (G) (phase contrast, original magnification-200). Microphotographs for cytoskeleton labeled for actin-F (green fluorescent dye), nuclei with DAPI (blue fluorescent dye), and STRO-1 positive cells (red fluorescent dye) for DPSCs (B, C), SHED cells (E, F), and SCAP cells (H, I) ([B, E, H] immunofluorescence, original magnification-200; [C, F, I] STRO-1 positive, original magnification-400). DPSC, dental pulp stem cells; SCAP, stem cells from apical papilla; SHED, stem cells from human exfoliated deciduous teeth .⁵⁵

CHONDROGENIC AND MYOGENIC DIFFERENTIATION

IDPSCs are chosen to demonstrate their differentiation capacity. At day 21 after induction of chondrogenic differentiation, IDPSCs demonstrate the formation of an extracellular cartilage matrix which is intensively stained by Masson`s trichrome (Figure 21A, Inset). Toluidine blue staining was used to detect essential cartilage matrix proteins such as proteoglycans (Figure 21B). IDPSCs maintain in basal culture medium (control) do not form any cell pellet. Additionally, chondrogenic differentiation is confirmed by the expression of COMP (Cartilage Oligomeric Matrix Protein) gene, which encodes a pentameric non-collagenous matrix protein that is mainly expressed in articular cartilage. The expression of COMP is observed in both EP and LP of IDPSCs (Figure 21C). It is important to highlight that chondrogenic differentiation of IDPSCs is uniform even in the absence of TGF-b, which is known to be a strong inductor of chondrogenesis in bone marrow derived MSCs. Following myogenic differentiation, IDPSCs showed typical cells elongation and fusion leading to small myotubes formation at day 7 (Figure 21D). At day 21, this cell fusion is obvious and most of the cells formed small myofibers (Figure 21E). MyoD transcription factor, which is a master regulatory gene of skeletal muscle differentiation, as expected, is expressed in IDPSC-derived myoblasts in nucleus or in perinuclear space following immunostaing using anti-MyoD1 antibody (Figure 21F). These myoblasts further form myosacs and MyoD1 protein is observed in the cytoplasm of these more mature cells (Figure 21G). Titin is the third most abundant skeletal muscle filamentous protein that forms a separate myofilament system in both skeletal and cardiac muscle.⁶⁶

It is expressed in IDPSC-derived muscle cells at more advanced stages of differentiation (Figure 21H). Some titin negative cells are also observed (Figure 21I). Troponin I is a protein responsible for immobilizing the actin tropomyosin complex in place. The expression of this protein is visualized in more mature myofibers derived from IDPSCs (Figure 21J). Human specific anti-actinin and anti-myosin antibodies reacted positively with differentiated IDPSCs (Figure 21K–N). Myosin positive immunostaining is observed in myofibers (Figure 21K) and also in differentiated small cells, which presented spot-like immunolabeling (Figure 21L). Singular binuclear differentiated IDPSCs are alpha-actinin positive (Figure 21M). This marker showed differential expression pattern within myosacs: some cells are strongly positive, while others presenting only shadow like immunostaining (Figure 21N). RT-PCR is used to verify the expression of MyoD1 and ACTB (Beta cytoskeletal actin) genes during IDPSCs myogenic differentiation. Both genes are found to be expressed in EP and LP (Figure 21O). EP and LP of IDPSCs showed similar chondrogenic and myogenic differentiation before and after cryopreservation. Control culture of IDPSCs do not present any signals of myogenic differentiation.⁶⁶

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Figure-21: Chondrogenic differentiation. A) Pellet culture: collagen fibers intensively stained by Masson` s trichrome. same as in (A) high magnification. B) The proteoglycans presence is revealing by Toloudine blue staining. C) RT-PCR shows the expression of COMP gene in EP and LP of IDPSCs. Housekeeping gene GAPDH is used as control. D–O) myogenic differentiation. D, E) morphological aspect showing stages of muscle fibers formation. F) Nuclear expression of MyoD1 protein in LP of IDPSCs-derived myocyte-like cells. G) Myosac composed by MyoD1 positive cells. H, I) Titin protein expression in LP of IDPSCs-derived myotubes. J) Expression of troponin I in Z-bands of myofibers. K) Myosin protein expression. L) Very small, satellite-like cells, showing positive myosin immunostaining. M) Binuclear cell positive for alpha-actinin (spot-like labeling). N) Fuse myotubes, which deferentially express alpha-actinin protein. O). ⁶⁶

CHARACTERIZATION

Magnetically separate cells are sub cultured in four-chamber slides (5000 cells per slide) to determine the characteristics of the stem cells. After being fixed with 4% paraformaldehyde, the slides are labeled with the different primary antibodies, followed by Alexa Fluor-568-conjugated secondary antibodies. The following antibodies produced in mouse are used: anti-STRO-1, anti-CD133, antic-met, and anti-CD117. The samples are washed three times with phosphate-buffered saline (PBS) after each antibody layer, and the stained cells are observed under a confocal scanning laser fluorescence microscope.⁶⁷

HEPATOGENIC DIFFERENTIATION

After the cells had attached to the surface, the serum containing medium is replaced with serum-free DMEM (SFM) supplemented with 1% insulin-transferrin selenium-x (ITS-x) (Invitrogen) and 100 μgml−1 of embryotrophic factor (ETF) produced according to the method described by Ishiwata et al 2000.68 After the cells reached 70% confluence, recombinant human hepatocyte growth factor (HGF) (20ng/ml) is added to the SFM for five days to induce endodermal specialization of the cultures. A mixture of 10ng/ml oncostatinM and 10nM dexamethasone is added for another 15 days. All the media are changed every second or third day.⁶⁷

CELL DIFFERENTIATION

The flask containing the cells is verified by optical microscopy every day in order to confirm the adherence of hMSC colonies. After the establishment of hMSC cultures on the fourth passage, the cells differentiated in adipocytes, osteoblasts, and chondrocytes. Adipogenesis is induced by addition of an adipogenic medium, comprised by Alpha-MEM supplemented with 10% FBS, 1μm dexamethasone, 100μg/mL 3-Isobutyl-1-methylxanthine IBMX, 10μg/mL insulin, and 100μM indomethacin. The adipogenic medium is changed every other day for 3 weeks. Osteoblast differentiation is induced by addition of an osteogenic medium, comprised of Alpha-MEM supplemented with 10% FBS, 1μm dexamethasone, 2μg/mL ascorbic acid, and 10μm beta-glycerophosphate. The osteogenic medium is changed every other day for 3 weeks. Chondrocyte differentiation was induced by addition of chondrogenic medium, that is Alpha-MEM supplemented with 10% FBS, 1μm dexamethasone, 2ug/mL ascorbic acid, 6,25ug/mL insulin, and 10ng/mL TGF-beta. Chondrogenic medium is changed every day three weeks.⁵⁸

IMMUNOHISTOCHEMICAL STAINING METHODS

OIL RED O

Adipogenic differentiation is demonstrated by staining lipid droplets after 3 weeks in culture. The cells are fixed in 4% paraformaldehyde for 30 minutes, washed, dehydrated in 60% isopropanol for 2 to 5 minutes, and stained with 0.5% Oil Red O (O-0625; Sigma) in 100% isopropanol previously diluted in water.⁵⁸

ALIZARIN RED

Osteogenic differentiation is evaluated by Alizarin Red staining after 3 weeks in culture. For Alizarin Red, the cells are fixed in 4% paraformaldehyde for 30 minutes, washed with distilled water, stained with Alizarin Red pH 4.2 for 5 to 10 minutes and thoroughly washed.⁵⁸

TOLUIDINE BLUE

Chondrogenic differentiation is evaluated by toluidine blue staining after 3-week culture. For toluidine blue, the cells are fixed with ethanol 70% for 1 minute, ethanol 90% for 1 minute and absolute ethanol for 1minute, then toluidine blue is added (1g toluidine blue, 1g sodium borate/100mL of water).⁵⁸

DIFFERENTIATION OF hMSC

Human MSCs are also characterized by their ability to differentiate into cells of three different lineages. All five samples obtained either from reusable or disposable filters differentiated into cells of the three lineages.⁵⁸

ADIPOGENIC DIFFERENTIATION

Undifferentiated hMSCs (Figure 22A) are cultured in the presence of adipogenic medium for 21 days, resulting in the formation of cytoplasmic lipid droplets (Figure 21B). For better visualization, cells are stained with Oil Red O, which stains lipid droplets within the cells (Figure 22C).⁵⁸

OSTEOGENIC DIFFERENTIATION

Undifferentiated hMSC are cultured in the presence of osteogenic medium for 21 days, resulting in the formation of a calcium matrix (Figure 22D). For better visualization, cells are stained with Alizarin Red, which stains calcium (Figure 22E).⁵⁸

CHONDROGENIC DIFFERENTIATION

Undifferentiated hMSC are cultured in the presence of chondrogenic medium for 21 days, resulting in the formation of a proteoglycan-rich matrix (Figure 22F). For better visualization, cells are stained with toluidine blue, which stains proteoglycans (Figure 22G).⁵⁸

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Figure-22: (A) Undifferentiated mesenchymal stem cells – CTRL (40x). (B) Lipid droplet formation within cells culture in adipogenic medium (40x). (C) Lipid droplets stained with Oil Red O in cells culture in adipogenic medium (40x). (D) Calcium deposition in cells culture in osteogenic medium (40x). (E) Calcium deposition stained with Alizarin Red in cells culture in osteogenic medium (40x). (F) Morphologic changes in cells culture in chondrogenic medium (40x). (G) Proteoglycan-rich matrix stained with toluidin blue in cells culture in chondrogenic medium (40x).⁵⁸

FEEDER-FREE CULTURES OF HESCs

The hESCs can also be cultured feeder-free on coated plates such as Matrigel TM, laminin⁶⁹ or fibronectin.⁷⁰ In this culture system, mechanical dissociation of hESCs has been replaced with enzymatic dissociation and subsequent passage of cell clumps or single cells. Lack of mEF feeder cells in this culture technique makes it more attractive to stem cell therapy, since the risk of viruses or other macromolecules being transmitted to the human cells is eliminated. However, the system is still not a xeno-free culture system which is a prerequisite for transplantation of hESCs. When culturing hESCs without feeder layers on coated surfaces, it is necessary to use conditioned hESC medium. Such a culture medium is obtained when the hESC medium is incubated on mEF feeder cells, though one can avoid this problem by using hEF cells to make conditioned hESC medium.⁵⁵

XENO-FREE CULTURE OF HESCs

Culture techniques where hESCs can be culture under xeno-free conditions in the absence of an animal feeder layer and animal components are a prerequisite for transplantation of hESCs.⁷¹ To grow hESCs without mEF cells and replace it with human feeder systems,⁷² can be consider as a xeno-free system. However, the presence of the feeder cells in the culture still makes it unsuitable for cell-based therapies and limits large-scale production of hESCs. For this reason it has been critical for researchers to develop novel methods for culturing of undifferentiated hESCs which can keep the promise in tissue engineering and regenerative medicine as a source of tissue-specific cells.⁵⁵

CULTURE OF DP

Freshly extracted DP contains large nerve trunks and blood vessels in the central region of the coronal and radicular pulp (Figure 23A). First outgrowing fibroblast-like cells appeared between three to four days after DP plating (Figure 23B). Long-term culture is performed by mechanical transfer of DP into new culture dish without using enzymatic treatment. After each transfer, DP produces large numbers of outgrowing cells approximately every three or four days, thus allowing constant production of SCs at passage zero (P0) (Figure 23). We obtained successful isolations with all samples (n= 10) of deciduous teeth. We performed multiple DP transfers during, at least, six months (LP of IDPSCs). Both, EP and LP of IDPSCs maintained their morphology (Figure 23C). Transmission electron microscopy revealed two types of IDPSCs morphology: ES-like cells with low cytoplasm-to-nucleus ratio, low cytoplasm density, which are poor of organelles (Figure 23D). IDPSCs of MSC-like cells have a high number of stretched out pseudopodes, which serve to explore substrate and more cytoplasm and organelles when compared with IDPSCs of ES-like cells (Figure 23D, E). Figure 23F documents that IDPSCs showed a relatively uniform population in respect of these two cell types. IDPSCs karyotype is confirmed here to be unchanged, suggesting that during culture, numerical and gross structural chromosomal abnormalities do not occur as shown by routine G-banding technique (Figure 23). Further, in-vitro expansion of IDPSCs is performed using enzymatic treatment and the passages is called P1 (Figure 23).⁶⁶

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Figure-23: The process of DP in-vitro plating (Day 0, P0) followed by DP adherence and cells outgrowth (Day 3–4). This process is followed by enzymatic treatment (P1) of the cells and formation of multiple colonies (CFU-f - Colony Forming Units-fibroblast). After 5 days, enzymatic treatment is performed to harvest multicolony-derived IDPSCs (P2) population. Next, in-vitro expansion of IDPSCs (P3) is performed. Upper numbers represent approximate quantity of harvested IDPSCs in each passage. Vertically, the same process is shown, albeit after multiple DP mechanical transfer.⁶⁶

Stem cells play vital roles in the repair, regeneration and immunomodulation of every organ and tissue through their capacity for self-renewal and differentiation.⁷³

In addition to tissue repair and regeneration, immunomodulatory properties have also recently been identified for MSCs in animals and humans that may be related to therapeutic effects such as angiogenesis, anti-inflammation and anti apoptosis. Furthermore, recent reports suggest that MSCs have low inherent immunogenicity. Therefore, the immunomodulatory properties of MSCs may make them more attractive than other types of stem cells for some applications in cell transplantation. Previous reports demonstrated that human oral tissue derived MSCs, such as DPSCs,⁷⁴ SHED,⁷⁵ PDLSCs,⁷⁶ SCAP⁷⁷ and GMSCs,⁷⁸ have immunomodulatory properties similar to those of BMSCs. In addition, systemically injected GMSCs have been shown to home to the wound site and promote wound repair, and oral mucosal progenitor cells appear to have a more fetal phenotype for immune recognition, with immunomodulation that occurs under a mechanism different from that of BMSCs. Therefore, the gingiva is currently a promising stem cell source that may have wide- ranging potential for future immune-related therapies in addition to regenerative medicine.⁶

SCAFFOLD-BASED TISSUE REGENERATION

The basic concept underlying conventional periodontal regenerative therapy is first to remove the source of infection and then to provide a space into which neighboring cells can grow. To this end, various types of bone grafting materials have been applied to periodontal defects. The most documented material-based regenerative technique for periodontal regeneration therapy is guided tissue regeneration (GTR), in which biocompatible barrier membranes, such as resorbable collagen and poly lactic-co-glycolic acid membranes or non-resorbable expanded poly-tetrafluoro-ethylene and membranes, are surgically implanted to cover and protect the bone defect. In this procedure, connective tissue and bone regeneration then occur within the bone defect, which is protected by the barrier from rapid migration of epithelial tissues into the wound.⁷³

The PLGA and ePTFE polymers and commercially pure titanium membranes are bio inert materials that do not stimulate bone formation and do not directly bond to bone.⁷³

Therefore, alveolar bone augmentation/preservation techniques, such as guided bone regeneration (GBR) and socket preservation, require the use of bioactive materials, such as calcium phosphate (CaP) and collagen-based grafts, to stimulate bone tissue formation and thus provide direct bonding with bone. Representative CaP-based biomaterials include hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate and bovine bone mineral. It should be noted that CaP-based biomaterials are bioactive and osteo conductive, but they are not osteo inductive because they do not induce the formation of de novo bone in non-osseous sites. Clinically, osteo induction by bone grafting substitutes is especially important when applying titanium dental implants to permit accelerated bone formation and enhanced osteo integration of the implants with bone, thereby minimizing implant loosening that could lead to implant failure. Therefore, osteo inductive CaP-based scaffolds have been engineered through the incorporation of osteogenic bioactive factors and have been shown to promote bone formation.⁷³

TISSUE REGENERATION BASED ON GROWTH FACTOR DELIVERY

Growth factor delivery has increased the options for combinatorial approaches with scaffold-based tissue regeneration. It is well known that the sequential bone development cascade is organized by a variety of cells and trophic/growth factors. The tissue regeneration process can be partially considered as a recapitulation of the normal development process; therefore, it is reasonable to use trophic/growth factors to recruit stem cells to tissue defects and stimulate them to achieve regeneration. One representative therapy that uses growth factor delivery to achieve periodontal regeneration is the application of platelet- rich plasma (PRP), which consists of autologous platelets concentrated in a small volume of plasma. PRP contains several different growth factors and matrix elements that may be used to regenerate periodontal defects. Currently, there is great interest concerning the use of PRP in combination with bone grafts or autologous stem cells to obtain predictable periodontal regeneration. A commercially available enamel matrix derivative (EMD) product has also been widely used in periodontal regeneration. EMD is extracted developing porcine tooth buds and has been reported to be composed primarily of amelogenin. Despite its encouraging clinical outcomes, the mechanisms underlying the effects of Emdogain¹ on periodontal regeneration are not yet clear. Several studies suggest that EMD stimulates periodontal fibroblast proliferation/growth and inhibits epithelial cell proliferation/growth, which may thus lead to periodontal tissue regeneration. However, a recent systematic review indicated a lack of additional benefit of a combined therapy of GTR and EMD in infrabony or furcation defects when compared with GTR therapy alone. Because PRP and EMD are composed of various different proteins, recently, several recombinant growth factors have been introduced for periodontal/bone regenerative therapy, including bone morphogenetic protein (BMP)-2, platelet- derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2. BMP was originally characterized by its ability to induce bone formation. Currently, BMPs are also known to play important roles in embryonic patterning and early skeletal formation. Among the members of the BMP family, BMP-2 is famous for its strong ability to induce bone and cartilage formation.⁷³

TOOTH/ROOT REGENERATION

The ultimate goal of tooth regeneration is to develop fully functioning bioengineered teeth that can replace lost teeth. In contrast, the regeneration of the tooth root is a conceivably more realistic and clinical applicable approach, especially for prosthodontists, because the regenerated tooth root can be used as an abutment tooth to permit fixed-prosthetic approaches, such as crown and bridge treatments. Sonoyama et al.⁷⁹ demonstrated that a root/periodontal complex constructed using PDL stem cells (PDLSCs), stem cells from the apical papilla (SCAP) and a HA/TCP scaffold, was capable of supporting an artificial crown to provide normal tooth function in a swine model. In addition, cell sheet technology using DFCs in combination with a dentin matrix-based scaffold has been applied successfully to tooth root reconstruction. New stem-cell-based technology for the regeneration of the tooth root and its associated periodontal tissue may offer clinical opportunities for the treatment of damaged or lost teeth. Regeneration of the entire tooth is expected to be one of the highest achievements in the field of dentistry. Tooth engineering to form dental structures in-vivo has been established using many different types of stem cells from mice, rats. Ikeda et al.⁸⁰ demonstrated a fully functioning tooth replacement in a mouse through the transplantation into the alveolar bone of bioengineered tooth germ reconstituted from epithelial and mesenchymal progenitor/stem cells in a collagen gel. The bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication, and response to noxious stimulation such as mechanical stress and pain in cooperation with other oral and maxillofacial tissues. Using the same cell source used for the bioengineered tooth, the in-vivo reconstruction of a murine ‘‘bioengineered tooth unit’’ was recently demonstrated. Surprisingly, the unit comprised not only a mature tooth and periodontal ligament but also alveolar bone. The unit provided a fully functional tooth with vertical bone regeneration when the unit was transplanted into a vertical alveolar bone defect (Figure-24) in a mouse model. These findings resulted in a new concept in tooth regeneration therapy: the transplantation of a bioengineered tooth has great potential for not only whole-tooth regenerative therapy but also as a treatment in clinical cases where tooth loss is accompanied by a serious alveolar bone defect. One of the major hurdles in the clinical application of tooth regeneration technology is the identification of an appropriate autologous stem cell source in humans. In this regard, iPS cells may be an appropriate cell source because they can be differentiated to dental epithelial and mesenchymal cells and can be prepared from the patients’ own somatic cells.⁷³

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Figure-24: Schematic representation of the current regenerative strategy for mature tooth/organ replacement.⁷³

SALIVARY GLAND REGENERATION

Regeneration of salivary glands by stem cell transplantation is an important study topic for head and neck oncology and surgery because radiotherapy unavoidably impairs salivary gland function and results in xerostomia (dry mouth syndrome) as a side effect. Two main regenerative approaches have been applied to functionally restore damaged salivary glands. One approach is to develop an artificial salivary gland using tissue engineering technologies. Another approach is to apply stem cells to the damaged salivary grand tissue. In a mouse model, adipose-derived MSCs transplanted in irradiated sub mandibular glands restored salivary gland function. Transplantation of BMSCs into the mouse tail vein also repaired the function of irradiated salivary glands. Recently, primitive salivary gland stem cells were isolated from mice, and intra-glandular transplantation of these cells successfully repaired the function of irradiated salivary glands.⁷³

MANDIBULAR CONDYLE REGENERATION

Damage to the temporomandibular joint disc or condyle (condylar osteochondral defect) arising from trauma or arthritis can result in lifelong pain and disturbed masticatory function for patients. Tissue regeneration strategy on these defects can hold promise to affect the quality of life of these patients. In a goat model, the combination of cartilage tissue engineering using cartilage-derived progenitor cells carried in a hydrogel and distraction osteogenesis was successfully used to reconstruct condylar osteochondral defects. Additionally, a human-shaped mandibular condyle was successfully engineered from chondrogenically and osteogenically induced rat BMSCs encapsulated in a biocompatible polymer. BMSCs that were induced to differentiate into chondrogenic and osteogenic cells produced regeneration of rabbit mandibular condyle that was enhanced by low-intensity pulsed ultrasound. These findings may provide an initial proof of concept for the ultimate stem-cell-based tissue engineering of degenerated articular condyles in the context of diseases such as rheumatic arthritis.⁷³

TONGUE REGENERATION

Loss of tongue tissue from surgical resection can profoundly affect the quality of life because the tongue plays a critical role in speech, swallowing and airway protection. Therefore, reconstruction of tongue defects has been a continuing challenge in dentistry. Cell-based reconstruction of the tongue was reported in a rat model where myoblast/progenitor cells carried in a collagen gel were implanted into the hemiglossectomized tongue to provide successful muscle regeneration in the tongue with reduced scar contracture. The tongue is a complex structure that includes skeletal muscle fibers, mucosa with taste buds, and nervous tissue; therefore, functional regeneration is difficult. Egusa et al.⁸¹ demonstrated that applying of cyclic strain to BMSCs greatly accelerated in-vitro skeletal myogenesis to achieve aligned myotube structures, suggesting the importance of cellular alignment for creating physiologically relevant environments to engineer skeletal muscle. Advances in stem cell biology and tissue engineering may enable the reconstruction of the damaged or resected tongue with normal physiological function.⁷³

IN-VIVO BONE REGENERATION

The earliest translational model systems using adipose stem cell (ASCs) focused on the mesodermal potential of the ASC in bone regeneration. In bone repair, (i.e., cranial/parietal, craniofacial palatal, maxillary/mandibular, long bone segmental-tibial, femoral) with ASCs from rodents, rabbits, canines, and humans have been described in the literature for well over the last decade. The first in-vivo paper to describe osseous tissue formation by ASCs was reported in 2004 by Hicok et al., who seeded HA/TCP cubes with osteogenically induced human ASCs and implanted them subcutaneously in athymic mice. Tissue, histologically consistent with osteoid, forms in 80% of the scaffolds. More importantly, their group also confirms the presence of human ASCs within the newly formed osteoid. Cui et al. 2007 report healing in 84% of cranial bone defects in dogs through implantation of dexamethasone-induced ASCs versus 25% healing in acellular defects, with these acellular defects containing fibrous tissues rather than bone tissue as found in the ASC-seeded implants. Osteogenically primed ASCs implanted into palatal defects result in substantial bone formation versus undifferentiated.⁸²

IN-VIVO CARTILAGE REGENERATION

Like bone, most in-vivo studies using ASCs for cartilage regeneration also employ preinduction. One of the earliest studies implanted TGF𝛽1- induced human ASCs into intramuscular pockets in nude mice and noted the histological appearance of hyaline cartilage. Similar results have since been reported using high-density, preinduced ASC monolayers or preinduction through the formation of micromass nodules, followed by seeding onto 3D scaffolds and subcutaneous implantation. Cartilage formation has also been reported upon the subcutaneous implantation of human preinduced ASCs transduced to express TGF𝛽2 and seeded onto alginate or alginate/PLGA scaffolds, suggesting that the ASC can demonstrate chondrogenic potential in-vivo under the influence of the TGF𝛽 family. Using amore translational full thickness cartilage defect, TGF𝛽1-induced rabbit ASCs in fibrin glue scaffolds have been found to form cartilage within the articular surface, with healing continuing down into the subchondral bone. Neocartilage formation with integration with the surrounding host cartilage and bone has also been found in a larger model system using pig ASCs and full thickness articular defects.⁸²

IN-VIVO FAT REGENERATION

The most important application of the ASC would be in its development of soft tissues like adipose tissue. Yet, in comparison to articles on bone and cartilage generation, adipose tissue regeneration is underrepresented. The studies that are found can be divided into two categories: (1) de novo fat formation using ASCs and (2) the use of ASCs for the improvement/maintenance of fat grafts. In category one, ASCs, derived from GFP-transgenic mice and pre induced toward the adipogenic lineage, form tissues histologically confirmed as adipose tissue. Furthermore, the presence of GFP+ve cells can be confirmed in the new tissue, indicating that the ASC is, at a minimum, retained within the forming tissue and has a positive effect on adipose generation. Similar findings have also been reported using ASCs in combination with a variety of scaffolds including silk fibroin, collagen type 1, collagen/gelatin, alginate, polypropylene, and scaffolds modified for the controlled release of growth factors such as bFGF. Human ASCs have also been used to form fat using a “self-assembly” approach in which the ASC is used to produce not only adipocytes but also its own supportive stroma.⁸²

IN-VIVO MUSCLE REGENERATION

The implantation of human ASCs without immunosuppression into murine models of dystrophy can yield good engraftment levels and improvements in muscle function, thus allowing researchers to make more accurate conclusions about the myogenic differentiation of human ASCs in-vivo. Pioneering work from Rodriguez et al. 2005 were among the first to suggest that a xenogeneic transplantation model is possible for muscle repair, with their transplantation of human ASCs into a MDX murine model resulting in substantial expression of human dystrophin in both the injected and adjacent muscle and long-term engraftment without any murine inflammatory infiltration.⁸²

DENTIN-ENAMEL REGENERATION

Tissue engineering using scaffold and cell aggregate methods has been also suggested to produce bioengineered complex dentin-enamel regeneration from dissociated cells. The capability of epithelial cell rests of Malassez (ERM) to regenerate dental tissues by transplanting subcultured ERM seeded onto scaffolds into the omentum of athymic rats. Particularly, in combination with dental pulp cells at the crown formation stage, ERM was coseeded into collagen sponge scaffolds.

Figure-25: Showing cell-based strategy for the development of complex-like mineralized tissue by the coseeding of hDESC and Hdpsc. ⁸³

After 8 weeks transplantation, enamel-dentin complex-like structures were recognized in the implants, as enamel like tissue and the stellate reticulum like structures were observed to some degree, while the tall columnar ameloblast like cells were aligned with the surface of the enamel like tissues. Similar results were observed in our lab with dental epithelial stem populations isolated by fluorescence activation cell sorting (FACS) using previously discovered epithelial stem cell markers and subcultured under serum-free and xenon-free conditions. The collected human dental epithelial stem cells (hDESCs) can generate mineralized tissue in-vivo when coseeded on PLLA scaffolds with human dental pulp stem cells (hDPSCs) and implanted subsequently in the nude mouse. After 10 weeks post implantation mineralization is seen in the implants. Furthermore, complex dental tissues regeneration was investigated with different types of reassociations between epithelial and mesenchymal tissues and/or cells from mouse embryos which were cultured in- vitro before in-vivo implantation. In-vitro the reassociated tissues developed and resulted in jointed dental structures that exhibited normal epithelial histogenesis and allowed the functional differentiation of odontoblasts and ameloblasts. After implantation, the reassociations formed roots and periodontal ligament, the latter connected to developing bone.⁸³

STEM CELLS IN IMMUNO-RECONSTITUTION

The stem cells have been used since many years in immuno-reconstitution following cancer development or following cancer treatments. The high dose chemotherapy has adverse effects on the bone marrow causing myelo suppression. Usually this is followed by the blood cell recovery through the haematopoietic progenitor cells residing in the bone marrow by the complex interactions between the progenitor cells and the marrow microenvironment under the influence of various stimulatory and inhibitory factors. However, time for haematopoietic recovery is proportional to the doses and number of cycles of chemotherapy It has been shown that chemotherapy can induce inhibitory factors such as Tumor Growth Factor (TGF)-β, Interferon(IFN)-γ – IFN-α, Tumor Necrosis Factor(TNF)-α and Interleukin(IL)-4 with cytokines that causes myelosupression. HSCs are the most commonly used and they are the stem cells of choice for the haematopoietic cell transplantation following high dose chemotherapy to restore bone marrow and immune system to pre-chemotherapy levels.⁸⁴

In tissue engineering, the important elements for tissue regeneration are not only stem cells but also biomaterial scaffolds (cell-instructive templates) and growth and differentiation factors (biologically active molecules). In this regard, conventional regenerative dentistry has already developed scaffold and growth factor technologies.⁸⁴

Figure-26: Showing use of stem cell in different disease.⁸⁵

SECRETION OF NEUROTROPHIC FACTORS

MSCs secrete a variety of cytokines and growth factors that promote endogenous neuronal growth, neurogenesis and angiogenesis, encourage synaptic connection and remyelination of damaged axons, decrease apoptosis, and regulate inflammation primarily through paracrine actions Human MSCs are known to secrete neurotrophic factors including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF). After direct transplantation in an animal model of stroke, human MSCs were shown to integrate into host brain, survive, differentiate into neurons and astrocytes, and induce neurobehavioral improvement.⁸⁶

INDUCTION OF NEUROGENESIS AND ASTROGLIAL ACTIVATION

MSCs induce the proliferation of endogenous neural stem/progenitor cells in the subventricular zone (SVZ) and are critical to the survival of newborn cells. They have been shown to be directly involved in neural differentiation after engraftment into damaged tissue and migrate to the CNS to a limited extent. Of particular note, genetically modified MSCs expressing Neurogenin¹, a proneuronal gene that directs neural differentiation, increased the therapeutic effects of MSCs in ischemic brain. In addition, MSCs promote the plasticity of damaged neurons and activate astroglial cells to secrete neurotrophins such as BDNF, GDNF and NGF. In an animal model of stroke, intravenous transplantation of BM stromal cells improved functional outcomes by promoting endogenous repair.⁸⁶

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Figure-27: Potential therapeutic mechanisms of neurorestoration using mesenchymal stem cells. MSCs secrete a variety of neurotrophic factors that promote endogenous neuronal growth, induce angiogenesis, neurogenesis and astroglial activation, encourage synaptic con­nection and axonal remyelination, decrease apoptosis, and regulate microglial activation primarily through paracrine actions. MSCs, mesenchymal stem cells. ⁸⁶

AXONAL SPROUTING AND SYNAPTIC CONNECTION

Extracellular matrix components derived from MSCs can enhance nervous system repair. For example, fibronectin prominently performs essential roles in neuronal survival, axonal sprouting and synaptogenesis following cerebral ischemia. Moreover, extracellular matrix molecules and cell adhesion molecules such as integrin, cadherin, and selectin can promote axonal growth and regeneration.⁸⁶

ANTI-APOPTOTIC EFFECT

Reportedly intravenous transplantation of MSCs reduced apoptotic cells stained with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling in an animal model of middle cerebral artery occlusion. This anti-apoptotic effect, together with the previously described capacity to release neurotrophic molecules, may well explain the remarkable functional recovery obtained with the administration of MSCs in experimental models of stroke as well as spinal cord injury.⁸⁶

IMMUNOMODULATORY EFFECT

MSCs were shown to exert immunomodulatory properties in-vitro.⁸⁷ These features were exploited by researchers in the treatment models of MS and experimental autoimmune encephalomyelitis. In PD, MSCs act as neuroprotectors via an anti-inflammatory response to regulate the activity of microglia and to protect dopaminergic neurons. In ischemic brain, MSCs were also useful as an immunomodulator. MSCs reduced the numbers of Iba-1+ and ED1+ inflammatory cells. In our previous study, intravenously transplanted MSCs did not only decrease the level of pro-inflammatory cytokine IL-1β and the proportion of activated microglia, but also increased the level of anti-inflammatory cytokine IL-10, potentially suggesting that early immunomodulation by MSCs was an underlying mechanism of functional recovery in spinal cord injured rats.⁸⁶

INDUCTION OF ANGIOGENESIS

MSCs secrete a number of growth factors and cytokines, which normally support the proliferation of hematopoietic stem/progenitor cells. In experimental models of cardiovascular diseases such as MI and limb ischemia, the secretion of multiple angiogenic cytokines such as hepatocyte growth factor, basic fibroblast growth factor, insulin-like growth factor 1 and vascular endothelial growth factor was induced from MSCs. Existing evidence suggests that these cells can ameliorate ischemic tissue injury, produce appropriate cytokine milieu to promote angiogenesis, and possibly differentiate into endothelial cells. The evidence seems to point toward the theory that a complex set of trophic factors secreted by MSCs significantly contributes to injury repair in-vivo by stimulating angiogenesis.⁸⁶

MSC IN STROKE AND TRAUMATIC BRAIN INJURY

Neuronal and astroglial damages can occur in cerebro vascular disease resulting from the blockage of blood flow in selected brain areas, leading to motor, sensory and cognitive dysfunctions. It has been shown that stroke-induced endogenous neurogenesis and migration of neural stem or progenitor cells into regions of ischemic damage occurs in humans, but the extent to which neurogenesis is able to replace lost neurons or contributes to functional improvement in stroke patients is largely limited. The limited therapeutic efficacy of endogenous repair processes has encouraged clinicians to incorporate MSCs or BM-derived cells in restorative strategies.⁸⁶

Clinical trials for MSC transplantation to treat stroke and traumatic brain injury are currently ongoing. In patients with middle cerebral artery infarction, the use of autologous MSCs derived from BM has indicated no safety concerns for death, stroke recurrence, or serious adverse events up to 1 year, and trends towards increased functional recovery. This group also reported as a long-term follow-up study for 5 years no serious adverse effects following MSC treatment. Direct administration of MSCs to an injured region following traumatic brain injury has also been performed without adverse events. Briefly, seven patients each received up to 109 expanded MSCs as part of a cranial repair operation. The patients were followed up for six months and demonstrated significant improvements in neurological function.⁸⁶

MSC IN SPINAL CORD INJURY

Depending on the severity and location of injury, patients present with a varying range of functional impairments, arising from both damage to the local circuitry of the spinal cord and disruption of the ascending and descending fiber tracts. All groups who have tested the safety of the transplantation of BM-derived mononuclear cells and stromal cells, or adipose tissue derived MSCs in patients with spinal cord injury indicate that administration of these cells does not cause any serious adverse effects. Geffner, et al.⁸⁸ investigated the improvement in quality of life and bladder function without pain or tumor up to 2 years. Syková, et al.89 reported that five patients who received cells intra-arterially showed improvement up to 1 year.⁸⁶

MSC IN PARKINSON’S DISEASE

PD is a progressive neurodegenerative disease whose dopaminergic neurons selectively degenerate in the substantia nigra. Although a variety of drugs such as L-dopa are available, they only remain effective for a certain period in most patients. The limitation of pharmacologic agents increases the need for cell-based therapy as a restorative strategy. In a study recently reported by Li, et al.⁹⁰ two subjects with PD who underwent transplantation of fetal mesencephalic do­paminergic neurons, which had survived for over 10 years, but later developed α-synuclein-positive Lewy bodies in the engrafted donor neurons, suggesting that the disease can propagate from host to graft cells. On the other hand, when autologous BM-derived MSCs were transplanted into the SVZ by stereotaxic surgery, the results suggested the treatment to be safe, and no serious adverse events occurred after transplantation in PD. Additionally, when patients with multiple system atrophy (MSA) were treated with MSCs, greater improvement was noted on the unified MSA rating scale than in untreated control patients, and no delayed adverse effects related to MSC infusion occurred during the 12-month study period.⁸⁶

MSC IN AMYOTROPHIC LATERAL SCLEROSIS

ALS involves a pathology that causes a selective loss of motor neurons leading to a progressive decline in muscle function and poor prognosis. When Nagano, et al. completed a small double-blind clinical trial to assess the effect of intrathecal administration of IGF-1 on disease progression in nine patients with ALS, the high-dose treatment slowed the decline of motor functions, but not bulbar function or vital capacity. On the other hand, both intravenous and intrathecal administration of autologous MSCs were well tolerated, with some preliminary evidence of efficacy in patients with ALS. However, large controlled clinical studies are needed to assess possibility for this therapeutic strategy.⁸⁶

MSC IN MULTIPLE SCLEROSIS

Mohyeddin, et al.⁹¹ reported iatrogenic meningitis and headache; Yamout, et al.⁹² reported transient encephalopathy and seizure; and Karussis, et al.⁹³ reported fever, headache and aseptic meningitis. Although serious adverse events related with cell transplantation are likely to be extremely uncommon in MS, the therapeutic efficacy in regards to clinical improvement remains controversial.⁸⁶

POSSIBLE TO GROW A TOOTH OR TMJ WITH STEM CELLS

The mandibular condyle consists of two stratified layers of cartilaginous and bone tissues, MSCs were first differentiated into chondrogenic and osteogenic cells. MSC derived chondrogenic and osteogenic cells were encapsulated in a biocompatible hydrogel in two stratified layers molded into the shape and dimensions of an adult human mandibular condyle. Following in-vivo implantation in immunodeficient mice for up to 12 weeks, the retrieved mandibular joint condyles retained the shape and dimensions of the native condyle. The chondrogenic and osteogenic portions remained in their respective layers. The chondrogenic layer was positively stained by chrondrogenic marker, safarnin O, and contained type II collagen. In the interface between cartilaginous and osseous layers, there is a presence of hypertrophic chondrocytes that express type X collagen. In contrast, only the osteogenic markers, such as osteopontin and osteonectin, stained the osseous layer, but not the cartilage layer. Lastly and most importantly, there was mutual infiltration of the cartilaginous and osseous components into each other’s territory, which resembles mandibular condyle. Therefore, the proof of principle has been established to regenerate the human-shaped TMJ condyle.⁹⁴

BONE MARROW STEM CELLS IN ORAL SUBMUCOUS FIBROSIS

Oral submucous fibrosis is a pathological condition which results in poor muscle function and lack of salivation with burning sensation. In a recent study Sankaranarayanan S. et al. aspirated bone marrow from posterior iliac crest of the patient and transported it in Acid Citrate Dextrose and processed for mononuclear cells (MNC) by Fi-coll density gradient centrifugation, following the cGMP protocols. MNC was injected intra orally at various sites in the affected area under local anesthesia. After 4 weeks of stem cell injection. There was gradually relief from burning sensation, increased salivary secretion and a 4mm increase in the mouth opening. H&E stain comparison of pre and post injection biopsy showed signs of angiogenesis with increase in number of capillaries, return of wavy pattern of collagen fibres with less inflammatory cell infiltration indicating the degranulation of the fibrous tissue and loosely organised connective tissue, all indicating a resumption towards a normal histology.⁹⁵

The cellular and molecular requirements for initiation of tumorigenesis are a series of mutations resulting in the acquisition of replication and growth-factor independence, resistance to growth-inhibitory signals, tissue invasion, and metastasis. The mechanisms underlying these mutations have been extensively interrogated; however a unifying “model of tumorigenesis” remains to be completely elucidated.⁹⁶

Tumors have long been recognized to consist of a heterogeneous population of cells differing in proliferative capacity, histologic and immunophenotypic appearance, and tumorigenic potential. Traditionally, this heterogeneity has been hypothesized to be the result of the stochastic accumulation of numerous and varied individual mutations and microenvironmental signals that provide a selective advantage to certain tumor cells. Over the last several years however, a new hypothesis has emerged suggesting that tumor heterogeneity is supported by a stem cell hierarchy. The cancer stem cell hypothesis postulates that tumor heterogeneity with regards to initiation, progression, response to therapy, and metastasis is the result of mutations which either render a normal somatic tissue stem cell cancerous, or cause a cancer cell to become stem cell like.⁹⁷ This mutated CSC is then capable of giving rise both to additional CSCs and to a variety of more differentiated and functionally divergent cancer cells, much like a normal somatic tissue stem cell. Unlike in the traditional stochastic tumorigenesis model, the CSC model proposes that tumorigenicity resides in only a small subpopulation of cancer cells and that these cells, rather than the bulk of the tumor, are responsible for tumor initiation and growth.⁹⁶

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Figure-28: CSC Theory. (a) Origin of CSC. CSC may originate from endogenous stem cells (SC) or reprogramming of the transit amplifying (TA) or differentiated (Diff) cell population. (b) The CSC theory proposes a tumor cell hierarchy with the CSC at the apex. Only CSCs are able to give rise to new tumors and provide support for ongoing tumor growth (Green: CSC, Red: Transit amplifying population, Pink: differentiated cell population). ⁹⁶

As with normal somatic stem cells, CSCs are defined by their ability to self-renew and to give rise to a heterogeneous population of tumor cells. This population of tumor cells consists of rapidly dividing cells (similar to the transient amplifying (TA) cell population in normal tissue) as well as additional CSCs and more differentiated tumor cells. In addition to their replicative capacity, CSCs, like their somatic counterparts, are also more resistant to the effects of cytotoxic chemotherapies and radiation damage.⁹⁸ Defining this stem cell hierarchy and the complex relationship between these cell populations has critical implications, not only for the understanding of the biology of tumor initiation and progression, but also for prognosis and treatment.⁹⁶

CSCs were first experimentally defined in hematopoietic malignancies by John Dick and colleagues in 1994.⁹⁹ Transplantation of a defined subpopulation of human acute myeloid leukemia (AML) cells (CD34hi CD38low) into immunodeficient mice was not only able to recapitulate AML but it was phenotypically and pathologically similar to the patient’s original leukemia. In contrast, the remaining cell populations (CD34low and CD34hi CD38hi) failed to give rise to new leukemia cells.⁹⁶

In the 15 years since the identification of the leukemic stem cell, a number of investigators have identified CSCs in solid malignancies. In 2003, Michael Clarke and colleagues were the first to identify a CSC population in a solid tumor. A subpopulation of CD44 (hi) & CD24 (low) breast cancer cells were able to recapitulate phenotypically heterogeneous breast cancers at very low limiting dilutions in mouse xenograft experiments.¹⁰⁰ Since then a number of other groups have defined CSC populations in other epithelial malignancies including colorectal, prostate, lung, brain, and HNSCC.¹⁰¹ The identification of the cell population responsible for initiating tumorigenesis has significant implications for the prognosis and treatment of cancer. At present, cytotoxic chemotherapies target the rapidly cycling cells of the tumor and result in impressive reduction in tumor size, but leave the largely chemotherapy resistant CSCs untouched.¹⁰²

Additionally, both in-vitro assays and in-vivo monitoring for effectiveness of new experimental cancer therapies are based on reduction in cell number or tumor size. It is therefore theoretically possible that therapies which result in tumor cell death, as currently assayed, will not have any significant effect on the CSC and will therefore not result in long-term disease control or eradication. The ability of the CSC to produce phenotypically diverse tumor cells may also contribute to increased metastatic potential with new mutations selecting for migratory and invasive properties of the tumor.⁹⁶

ENDOGENOUS HEAD AND NECK STEM CELLS AND THE ORIGIN OF CANCER STEM CELLS

The origin of the cancer-initiating cell has long been presumed to be the normal endogenous tissue stem cell. This is based upon their similar behaviors and the notion that only accumulated mutations within a long-lived cell could ultimately result in tumorigenesis. In colorectal cancer there is a strong correlation between induced loss of the Wnt signaling molecule APC in a putative stem cell population and the formation of benign intestinal polyps,¹⁰³ providing evidence that intestinal cancers can arise from a progenitor population. However, it is possible that accumulation of genetic mutations within a differentiated or progenitor cell can allow expression of stem cell behavior, and that this may provide an alternative source of CSCs. For example, oncogene expression driven from myeloid-specific promoters resulted in generation of mouse models of human leukemias.¹⁰⁴ Despite focused examination, the origin of the CSC remains controversial. With the primary focus on identifying CSC markers in HNSCC, little is known about the identity or the location of the normal endogenous stem cell or the stem cell microenvironment. Several studies have examined the putative HNSCC CSC marker CD44 in normal head and neck epithelia with differing conclusions. In one study, isolated CD44hi normal oral keratinocytes were shown to exhibit a G2-block associated with apoptosis resistance, a potential stem cell feature,105 suggesting that CD44 is likely expressed in normal head and neck epithelial stem cells. However, a subsequent study demonstrated that 60%– 95% of the normal epithelia express CD44 (or 60%–80% the splice variant CD44 v6), far too many cells to be considered tissue stem cells. While CD44 populations may indeed harbor a subpopulation encompassing stem cells, by itself it does not appear to be an adequate stem cell marker for normal oral mucosa. The head and neck stem cell identity and niche is clearly underexplored, however, key insights from the skin, airway mucosa and esophagus may guide future investigations into elucidation of this stem cell population.⁹⁶

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Figure-29: A simplified model is suggesting hypothesis about origin of the cancer stem cells. The cancer stem cells may develop when self-renewing normal stem cells acquire mutations and are transformed by altering only proliferative pathways. It is also possible that the cancer stem cells originate by multiple oncogenic mutations in the restricted progenitor cells which acquire the capability of self-renewal.⁸²

1. CANCER STEM CELL IDENTIFICATION

Methods for the identification of CSCs in solid malignancies mirror those strategies employed to differentiate normal stem cells from their differentiated progeny. These include the efflux of vital dyes by multidrug transporters, the enzymatic activity of aldehyde dehydrogenase, colony and sphere-forming assays utilizing specific culture conditions and the most widely used method the expression of specific cell surface antigens known to enrich for stem cells. Once the subpopulation of tumor cells has been identified and isolated, functional characterization through quantitative xenotransplantation assays, the gold-standard for identification of CSCs, are used to assess the tumorigenicity and self renewing potential of the putative CSC population in-vivo.⁹⁶

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Figure-30: CSC Identification. (a) Tumor sphere formation by CSCs. Differentiated tumor cells (pink) are unable to give rise to new clonally derived tumor spheres, whereas CSCs (green) give rise to new tumor spheres, (b) Tumor subpopulations are identified through differing mechanisms, including cell surface markers, aldehyde dehydrogenase activity, side population and are isolated with fluorescence-activated cell sorting (FACS). Xenotransplantation assays in immunocompromised mice demonstrate tumorigenic, self-renewing and differentiation properties of putative CSC population. ⁹⁶

1.1. SURFACE ANTIGENS

The most common method of identifying CSCs has relied on the expression of specific cell surface antigens that enrich for cells with CSC properties. Many of these antigens were initially targeted because of their known expression on endogenous stem cells. While a multitude of studies have identified CSC markers across a variety of solid malignancies, relatively few of these markers have been studied in HNSCC.⁹⁶

1.1.a. CD133.

A pentaspan transmembrane glycoprotein localized on cell membrane protrusions, is a putative CSC marker for a number of epithelial malignancies including brain, prostate, colorectal, and lung.106 In HNSCC cell lines, CD133 hi cells display increased clonogenicity, tumor sphere formation and tumorigenicity in xenograft models when compared to their CD133 low counterparts. While CD133 expression has been noted in primary human HNSCC tumors, quantitative xenotransplantation assays utilizing CD133+hi cells from fresh tumors has yet to be performed. Given the artificial environment of cell culture, these findings will need to be substantiated using primary tumor samples before any definitive conclusions can be made about the usefulness of CD133 as a CSC marker in HNSCC.⁹⁶

1.1.b. CD44.

One of the well-recognized CSC markers is a large cell surface glycoprotein that is involved in cell adhesion and migration. It is a known receptor for hyaluronic acid and interacts with other ligands such as matrix metalloproteases.107 Initially identified as a solid malignancy CSC marker in breast cancer, Prince et al. demonstrated that CD44 expression could also be used to isolate a tumor subpopulation with increased tumorigenicity in HNSCC. In their study they were able to show that as few as 5,000 CD44hi HNSCC cells could form a tumor when transplanted into the flank of immunocompromised mice, whereas higher concentrations of CD44low cells failed to form tumors. Additionally, these tumors recapitulated the original tumor’s cellular heterogeneity and could be serially passaged, characteristics that define CSCs. Although CD44 expression enriches for cells with CSC properties, the relatively high number of cells required for tumor formation as compared with known CSC populations from other epithelial malignancies raises questions about whether CD44 expression alone is sufficient for isolation of a pure CSC population. For instance, in breast cancer, as few as 100 CSCs injected into the mammary fat pads of immunocompromised mice generated tumors. It is important to note that in the Prince study, two thirds of HNSCC samples were initially passage through immunocompromised mice to generate a sufficient number of tumor cells for cell sorting, which has the potential for altering native CSC expression patterns. Using primary human tumor samples as well as utilizing a more natural host microenvironment through an orthotopic xenograft model might reduce the number of cells needed to generate tumors. However, it is likely that expression of multiple cell surface markers or the combination of marker expression with functional assays will be needed to further enrich the CSC population.⁹⁶

1.2. ALDEHYDE DEHYDROGENASE ACTIVITY

Aldehyde dehydrogenase (ALDH) is an intracellular enzyme normally present in the liver. Its known functions include the conversion of retinol to retinoic acids and the oxidation of toxic aldehyde metabolites, like those formed during alcohol metabolism and with certain chemotherapeutics such as cyclophosphamide and cisplatin.108 ALDH activity is known to enrich hematopoetic stem/progenitor cells and more recently has been shown to enrich cells with increased stem like properties in solid malignancies. Chen et al. showed that ALDH activity correlated with disease staging in HNSCC and that higher enzymatic activity correlated with expression of epithelial to mesenchymal transition (EMT) genes as well as enriching cells with CSC properties. In addition, ALDH activity appears to enrich for CSCs in HNSCC to a higher degree than that currently provided by cell sorting based on surface antigen expression. Clay et al. demonstrated that as few as 500 ALDHhi cancer cells could give rise to new HNSCC tumors when transplanted into immunocompromised mice, tenfold fewer cells than isolation by CD44 positivity. Most of the ALDHhi cells were also CD44high, suggesting that ALDH activity defines a subset of HNSCC CD44high cells with increased tumorigenicity.⁹⁶

1.3. SIDE POPULATION

Hoechst-33342 is a fluorescent DNA binding dye that preferentially binds to A-T rich regions. It is actively pumped out of cells by members of the ATP-binding cassette (ABC) transporter super family. Once stained with Hoechst dye, cells can be sorted by fluorescent-activated cell sorting (FACS) based upon the activity level of these multidrug transporters. Originally noted to enrich bone marrow for long-term hematopoetic stem cells, this method has also been used to identify cells within solid tumors with increased tumorigenicity. Side population (SP) cells from oral squamous cell carcinoma have been shown to have increased clonogenicity and tumorigenicity in xenotransplantation assays. Furthermore, HNSCC SP cells displayed higher expression of known stem cell related genes-Oct4, CK19, BMI-1 and CD44 and lower expression of involucrin and CK13, genes associated with a differentiated status.⁹⁶

1.4. TUMOR SPHERE FORMATION

Under serum-free culture conditions CSCs can be maintained in an undifferentiated state, and when driven toward proliferation by the addition of growth factors, form clonally derived aggregates of cells termed tumor spheres. The ability of CSCs but not the remaining tumor bulk to form tumor spheres has been used extensively in neural tumors to identify populations enriched for CSCs. In HNSCC, these spheres have been shown to be enriched for stem markers, including CD44hi,109 Oct-4, Nanog, Nestin, and CD133hi,110 as well as exhibiting increased tumorigenicity in orthotopic xenografts.⁹⁶

2. CANCER STEM CELLS AND DISEASE PROGRESSION

While there exists significant data defining the presence of CSCs within a variety of tumor types and many aspects of the cell and molecular biology of CSC have been elucidated, the manner in which this unique cell population influences clinical disease progression remains unclear. Given that metastases can be formed from implantation of a single tumor cell, it seems likely that CSCs, as the progenitor of all tumor cell types, would be responsible for metastatic spread. Central to the CSC hypothesis is the presence of a unique stem cell “niche” or environment necessary to support the growth of stem cells. It has been shown that a pre metastatic niche is established by the attraction of bone marrow derived cells to the future site of metastases by the secretion of factors from cancer cells and that blocking the creation of this pre metastatic niche prevents metastases. What these secreted factors are and whether they are secreted by CSCs or one of their progeny remains an open question; however, creation of this niche, possibly for the arrival of CSCs to form a metastasis, appears to be a crucial step in metastatic spread. The strongest evidence that CSCs are responsible for metastases comes not in HNSCC but in colorectal cancer. In this tumor, a unique CSC population that is CD26hi appears to be tightly linked with metastases. Not only are CD26hi cells found in both primary and metastatic tumors, but the presence of CD26hi cells in the primary tumor predicted future development of metastases. In a mouse xeno-graft study, CD26hi CSCs implanted into the cecal wall of a nude mouse formed a tumor in the colon as well as liver metastases, while CD26low CSCs formed a tumor at the site of implantation without developing liver metastases. Similarly, injection of CD26hi CSCs into the portal vein led to liver metastases, while similar injection of CD26low CSCs did not. Thus, these CD26hi CSCs appear to be the cells responsible for metastatic spread in this tumor population. Another stem cell marker, CD44, has also been implicated in metastatic spread and disease progression in HNSCC, although the CD44 story is more complex. Recently, three different isoforms, CD44 v3, v6, and v10, have been shown to be associated with progression and metastasis of HNSCC. Increased CD44 v3 expression in primary tumors was associated with lymph node metastasis, while CD44 v10 expression was associated with distant metastasis and CD44 v6 expression was associated with perineural spread. In cell culture, blockade of these CD44 isoforms with isoform specific antibodies inhibited cellular proliferation, with the greatest inhibition seen with blockade of CD44 v6. Finally, increased expression of CD44 v6 and v10 was associated with shortened disease free survival. These studies suggest that alteration in CSC phenotype through variation in CD44 isoform expression may alter the interaction of CSCs with the surrounding microenvironment. This may allow CSCs to more readily invade surrounding tissues or metastasize, thereby promoting disease progression.⁹⁶

Table-3: MARKERS USED TO IDENTIFY CSCS IN VARIOUS TISSUES111

Abbildung in dieser Leseprobe nicht enthalten

CANCER STEM CELLS AND CHEMOTHERAPY RESPONSE

Tumor recurrence after initial response to chemotherapy is a major clinical issue. Recurrent tumors usually show heterogeneity in both the population of CSCs and non- CSCs, and also in histologic and cytogenetic appearances. This may be due to the survival of CSCs within the original tumor, which despite chemotherapy and removal of the bulk of the tumor, have repopulated the recurrent tumor.¹¹¹

Abbildung in dieser Leseprobe nicht enthalten

Figure-31: Cancer Stem Cells (CSCs) survive chemotherapy or radiotherapy, relapse follows.¹¹¹

Although cancer stem cells constitute about 1% of tumor cells, they can generate tumors similar to the original one when xenotransplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. It has been reported that this property is not observed with the remaining non-CSC bulk tumor cells.¹¹¹

Most cytotoxic therapies induce DNA damage or disrupt mitosis leading to cell death in dividing cancer cells. CSCs are protected against anti-neoplastic drugs through multiple defense mechanisms. These mechanisms can be divided into two groups: CSC-intrinsic and CSC-extrinsic. CSC-intrinsic mechanism can be due to more efficient DNA repair mechanisms, expression of drug pumps, and altered cell cycle. CSC-extrinsic mechanisms refer to the effects of tumor microenvironment on CSCs (Maugeri-Sacca et al., 2011).¹¹¹ Furthermore, it seems that CSC population is enriched following chemotherapy, as in a study it was shown that breast cancer stem cell markers including temozolomide, etoposide, carboplatin and paclitaxel, whereas non-CSCs were responsive (Liu et al., 2006).¹¹¹

Embryonic and adult stem cells have more robust DNA repair systems compared to progenitor and differentiated cells (Bracker et al., 2006; Maynard et al., 2008).¹¹¹ However, aged stem cells have reduced capability to repair DNA lesions and as a result accumulate genetic and epigenetic mutations. This may lead to increased incidence of various cancers with aging (Rossi et al., 2007).¹¹¹ Activation of DNA-damage checkpoint and DNA-damage repair pathways have been proposed as a mechanism of chemotherapy resistance in various cancers (Gallmeier et al., 2011).¹¹¹ When lung CSCs are exposed to genotoxic agents, they activate CHK1 and CHK2, but more differentiated lung cancer cells are responsive to these drugs. Use of CHK1 inhibitors along with chemotherapy, can induce cell death in CSC compartment (Maugeri-Sacca et al., 2011).¹¹¹

Epithelial tumor cells secrete Interleukin-4 which contributes in an autocrine manner to apoptosis resistance (Todaro et al., 2008).¹¹¹ Colon CSCs showing resistance to fluorouracil and oxaliplatin, were made responsive to these chemotherapy drugs with the use of antibodies against IL-4 (Todaro et al., 2007).¹¹¹ It would appear that autocrine IL-4 may function as a survival factor in cancer stem cells and therefore clearly could play a role in chemotherapy resistance.¹¹¹

Expression of ATP-binding cassette (ABC) transporters is elevated in both normal stem cells and cancer stem cells. These include multidrug resistance transporter 1 (MDR1) and breast cancer resistance protein (BCRP) (Moitra et al., 2011).¹¹¹ Acute myeloid leukemia (AML) CSCs can extrude daunorubicin and mitoxantrone more efficiently than non-CSCs (Wulf et al., 2001).¹¹¹ Likewise, neuroblastoma stem cells can extrude mitoxantrone with high efficiency (Hirschmann-Jax et al., 2004).¹¹¹ Vinblastine and paclitaxel can be expelled by MDR1 and imatinib mesylate and methotrexate can be removed by BCRP (Eyler and Rich, 2008).¹¹¹

Metabolic alterations also may contribute to drug resistance. Aldehyde dehydrogenase 1 (ALDH1) is overexpressed in leukemic stem cells (Pearce et al., 2005)¹¹¹ and it was shown that ALDH1 gene transfer can lead to cyclophosphamide resistance in normal stem cells (Magni et al., 1996);¹¹¹ so ALDH1 may also play a role in chemotherapy resistance.¹¹¹

Normal stem cells are usually in a state of quiescence and do not exhaust their proliferative ability, unless the tissue encounters injury. During this period of quiescence, CSC population can repair damaged DNA. Likewise, CSCs are mostly quiescent and therefore can escape chemotherapy induced cytotoxicity which acts on dividing cells (Maugeri-Sacca et al., 2011).¹¹¹ It was shown that ovarian CSCs proliferate more slowly and display more resistance to cisplatin relative to non-CSC population (Gao et al., 2010).¹¹¹

EMT has a role in development of metastasis and chemotherapy resistance. EMT can be induced by the activation of a transcriptional network involved in stem cell self-renewal, including Notch, Wnt, and Hedgehog (CD44+/CD24-) were expressed more abundantly after neoadjuvant chemotherapy of primary breast cancer patients (Li et al., 2008).¹¹¹ In another study, glioma CSCs showed resistance to multiple chemotherapeutic drugs (Maugeri-Sacca et al., 2011).¹¹¹ Cells undergoing EMT usually reside at the tumor-stroma interface and acquire stem cell markers and clonogenic properties (Mani et al., 2008).¹¹¹ Hypoxia within the tumor can induce angiogenesis through HIF-1 pathway, but abnormal organization of the newly formed vessels leads to low concentration of the chemotherapeutic drugs in the tumor and can be a possible mechanism of therapeutic resistance (Maugeri-Sacca et al., 2011).¹¹¹

CANCER STEM CELLS AND RADIOTHERAPY RESPONSE

Radiotherapy yields a curative potential in many solid tumors. Radiotherapy alone or in combination with chemotherapy can cure locally advanced, unresectable head and neck carcinoma and non-small cell lung cancer in about 10-50 percent of cases. In early stages of tumor progression, radiotherapy alone or in combination with chemotherapy can control local recurrence of tumor similar to surgery (Krause et al., 2011).¹¹¹ CSC population enriched after radiotherapy and it shows that CSC population survived more compared to non-CSC compartment. Radiotherapy induced the same amount of DNA damage to both CSCs and non-CSCs, but CSCs were able to repair damage more robustly. Furthermore, non-CSC population went through apoptosis more after radiation. Genotoxic stress activates ATM, CHK1 and CHK2 checkpoint proteins which in turn activate DNA repair pathway. CSCs display a basal level of checkpoint activation, which means that they are ready to respond to genotoxic insults. Use of CHK1 and CHK2 inhibitors resulted in radiosensitivity of CSC population (Bao et al., 2006).¹¹¹

It seems that Wnt/β-catenin has a role in radiotherapy resistance. Radiation led to enrichment of stem cells in a murine mammary epithelial cell line, which had high levels of activated β-catenin and survivin (an antiapoptotic protein). These cells displayed elevated self-renewal in mammosphere formation assay (Chen et al., ²⁰⁰⁷ ; Woodward et al., ²⁰⁰⁷ ).¹¹¹

Globally, stem cell science continues to progress at an astounding rate. Countries differ in their regulatory approaches to the science, yet it remains an international endeavor. Research teams are composed of scientists from around the globe, and there is a growing need to have access to cell lines that have been created in other parts of the world.¹¹²

As countries place restrictions on which cell lines may be used for research and under what conditions they may be used, it is increasingly important for scientists to have access to a registry of detailed information about existing cell lines. Several countries, states, and institutions are beginning to house and catalogue stem cell lines to address these questions. These initiatives sometimes differ in both nature and purpose. Facilities that physically house samples are commonly known as banks. Perhaps the best known stem cell bank is the UK Stem Cell Bank (UKSCB), which houses research- and clinical-grade stem cell lines. In the United States the National Stem Cell Bank is hosted by WiCell, a non-profit foundation in Madison Wisconsin, which holds the National Institutes of Health (NIH) stem cell lines. Recently WiCell founded the International Stem Cell Bank (ISCB) to hold a broader spectrum of stem cell lines including the induced pluripotent stem cell lines (iPS) derived at the University of Wisconsin in 2007.¹¹²

Stem cell registries catalogue stem cell lines, they may differ in two major respects. First, they may differ in purpose. Certain registries are created for regulatory purposes and contain information relevant to regulatory standards, such as whether or not proper research ethics board (REB) or stem cell research oversight (SCRO) committee approval was obtained. Regulatory registries may create distinctions between lines according to their designated use (clinical vs. research) or their ability to be supported by government funds according to specific government criteria. Other registries may be created for research purposes that are specific to an organization, such as the International Stem Cell Forum (ISCF). Such research registries may or may not make the information they hold publicly available.¹¹²

At present, there are five registries that catalogue stem cell lines available for research and commercial purposes. These include the UKSCB’s registry, ISCF’s International Stem Cell Characterization Initiative (ISCI) registry. The United States’ NIH Human Pluripotent Stem Cell Registry. The European Union Human Embryonic Stem Cell Registry (hESC reg), and the International Stem Cell Registry launched by the government of Massachusetts.¹¹²

REGULATORY STEM CELL REGISTRIES

THE UNITED KINGDOM STEM CELL BANK AND REGISTRY

Under the Human Fertilisation and Embryology Act, 1990, all hES cell lines created in the United Kingdom using donated oocytes must be created pursuant to a license granted by the HFEA. It is a condition of HFEA licenses that a sample of all hES lines derived in the United Kingdom be deposited in the UKSCB. Perhaps the best known stem cell bank in the world, the UKSCB was created in 2003 to act as curator for ethically sourced, quality-controlled human stem cell lines from all sources, including adult and fetal stem cell lines. Stem cell lines are divided according to their potential use into research-grade and clinical grade lines. The bank stores and maintains detailed information about the stem cell lines. To register a cell line with the UKSCB, a long list of information must be submitted, ranging from details on the license holder and provider of the stem cell line to morphological characteristics in the culture of the cell line.¹¹²

The UKSCB has a Code of Practice that lays out detailed criteria on issues related to ethics and consent. Although primarily a United Kingdom repository, the UKSCB will register hES lines from international sources.¹¹²

PLURIPOTENT STEM CELL REGISTRY

To date, the NIH has kept a registry of stem cell lines that are eligible for US federal funding, providing investigators with a unique NIH code for each stem cell line and contact information to facilitate their acquisition of stem cell lines. From August 9, 2001 to March 9, 2009, only hES lines derived before August 9, 2001 were eligible for federal funding in the United States. Although President G. W. Bush stated there were approximately 60 lines available at the time of the registry’s creation, there are, in fact, only 21 lines available for federal funding, of which only a smaller subset are suitable for research leading to potential treatments in humans. The NIH maintains relatively detailed derivation, investigator contact, and characteristic information on all the cell lines in the Registry.¹¹²

INTERNATIONAL STEM CELL FORUM (ISCF)

The ISCF was founded in 2003 by an international group of 21 funders of stem cell research to encourage international scientific and funding collaboration. Its stated aims include the promotion of global good practice and the acceleration of scientific advance in the field. To that end the ISCF has established a set of global criteria for the derivation, characterization, and maintenance of hES lines, called the International Stem Cell Initiative (ISCI).¹¹²

In June 2007 the ISCF launched an electronic registry to record quality hES cell lines. The registry contains detailed information on subculture protocols, media, and feeder cells, with more limited information on provision of the embryos.8 Thus far, only laboratories that have participated in the characterization study may register their cell lines. The ISCF has approved funding for a second phase of the ISCI that will focus on comparing the how hES culture in different media and on any genetic changes that accumulate in hES as they live in cell lines. ISCI also includes provision for collecting data on new hES lines and incorporating these data into the ISCI registry. The Canadian Institutes of Health Research has established an ethics working party to provide the ISCF with legal and ethical advice.¹¹²

STEM CELL REGISTRY (HESCREG)

In March 2007 the European Commission proposed the creation of a registry for hES lines available in Europe and funded by the European Union’s (EU) Research Framework Programme (a European-wide funding initiative). The European hES cell registry is jointly operated by the Centre of Regenerative Medicine in Barcelona and the Berlin-Brandenburg Centre for Regenerative Therapies in Berlin. One of the central organizations in the hESCreg is the Banco Nacional de Lineas Celulares (the Spanish National Stem Cell Bank). One of the first national stem cell banks, formed in 2003, the bank holds both adult and embryonic stem cell lines. The 10 EU countries that currently allow hES research are eligible to register cell lines and must provide a limited data set on each line, including news on clinical trials, information on hES projects from EU-funded projects, cell line origin and derivation methodology, and different parameters used for characterization.¹¹²

AMERICAN UNIVERSITY INITIATIVES

The US National Academies of Science (NAS) published ethical guidelines for hES research in 2005. These guidelines recommend that all hES research be subject to oversight by an embryonic stem cell research oversight (ESCRO) committee. The NAS recommended that ESCRO committees “maintain registries of hES research conducted at the institution and hES cell lines derived or imported by institutional investigators” and “include on their registry a list of cell lines that have been imported from other institutions or jurisdictions and information on the specific guidelines, regulations, or statutes under which the derivation of the imported cell lines was conducted.” As a result of these recommendations, a number of institutions have proposed establishing internal and external registries, including the University of California, San Francisco and The Harvard Stem Cell Institute. Few registries, however, are currently operational.¹¹²

THE TRI-INSTITUTIONAL STEM CELL INITIATIVE

In May 2005, the Starr Foundation announced that it would donate $50 million over three years to Rockefeller University, Weill Medical College of Cornell University, and Memorial Sloan-Kettering Cancer Center to be used in part to create a registry of stem cell lines being used on the three campuses. That registry is currently available as an intranet site for faculty and staff.¹¹²

THE INTERNATIONAL STEM CELL REGISTRY

In September 2008, the Government of Massachusetts launched a searchable database of hES lines and other pluripotent stem cell lines. Called the International Stem Cell Registry, the registry currently includes profiles of over 190 hES and induced pluripotent stem (iPS) cell lines. The profiles contain data on the derivation, availability and characteristics of each line.¹¹²

PROPOSED STEM CELL REGISTRIES

At present, proposed stem cell registries include a database by the International Society for Stem Cell Research (ISSCR) for published stem cell lines and a national registry for Canadian hES lines from the Canadian Institutes of Health Research (CIHR).¹¹²

THE CANADIAN INSTITUTE OF HEALTH RESEARCH

(CIHR) EMBRYONIC STEM CELL REGISTRY

In 2006, the CIHR proposed the creation of a national electronic registry for all Canadian hES lines created using CIHR federal funding. As with the UK Stem Cell Bank, participation in the Canadian Stem Cell Registry is to be a prerequisite for CIHR funding of hES research protocols. All hES lines created using CIHR funds are to be made available by the researcher to other researchers, subject to “reasonable cost-recovery charges” as a means of minimizing the need to generate large numbers of cell lines and, therefore, the need for donation of large numbers of embryos. Registration criteria are outlined in the Guidelines for Human Pluripotent Stem Cell Research and include information related to derivation and consent. In 2008 the Stem Cell Oversight Committee of Canada recommended that the registry be expanded to house any hES lines derived by institutions receiving funds from the Canadian research agencies, not just those receiving CIHR funding.¹¹²

STEM CELL BANKING IN INDIA

Stem cells banks are present, and even some of these banks do not only freeze eord stem cells but also dental stem cells of baby teeth. This can be done easily when a child's anterior milk tooth is shedding, the tooth is extracted by the dentist and preserved in a special kit provided from the stem cell bank company who then in their turn transfer the tooth to their special labs to harvest the dental stem cells and store them in their bank for each child confidentially until they are needed later for the child himself or a member of his family.¹⁶

The first-ever dental stem cell bank in India called "Store your Cells" has now been launched. This unique bank is the venture started by dentists at Dhruv Polyclinic, Mumbai. The venture was formed under the guidance of Dr. Kedar Gadgil, who is a successful implant dentist practicing at London (UK), Kent (UK), and Mumbai. After having established presence in Mumbai and New Delhi, Stemade Biotech, the country's first private dental stem cell bank, is expanding its reach to at least 10 more cities such as Pune, Hyderabad, Surat, Chennai in the next 8-10 months buoyed by initial response at the Bangalore launch of operations. The collection of stem cells from bone-marrow presents multiple collection frequency options but the procedure is invasive and quite painful. On the other hand, dental stem cells can be collected between 6-12 years for children and from adults extracting their third molars. According to researchers the right time to recover baby teeth with stem cells is before the teeth become very loose. It is necessary for these stem cell banks to be licensed and patented. The patent gives the technology for extraction, multiplication and banking of the dental stem cells. Deciduous teeth especially the canines are the best sources and 12 deciduous teeth are sufficient and for the adults a minimum of 2 molars during the extraction of the third molars. Once collected, the dental stem cells should arrive at the storage facility within 16 hours, much ahead of the 72- hour deadline. The teeth are preserved in the Special Cryogenic storage facilities. Once stored, the dental stem cells can be pulled from inventory and shipped within 24 hours much alike like the cord blood stem cells. Therefore there is no extra effort involved for the concept of stem cell banking is gaining interest in view of its aftordability, easy extraction, awareness among parents to safe guard their child's health and emphasis in modern medical research towards therapies from regenerative tissues.¹⁶

THE STATUS OF THE HUMAN EMBRYO

One of the most heated ethical issues surrounding embryonic stem (ES) cell research is independent of the research goals or outcome possibilities. This ethical issue involves the status of the human embryo. The discussion about the status of the embryo has been in the forefront for many years in the United States. The debate becomes more impassioned during legal proceedings regarding abortion and political campaigns. Since the ethical issue regarding the status of the embryo has never been resolved, it continues to be contentious and rises to the surface whenever people discuss stem cell research. In general, people adopt one of four stances towards using human embryos for research, and ES cell research in particular.¹¹³

POSITION #1:

Embryos are human individuals and should not be used or destroyed for research purposes.

One position contends that embryos are human individuals and therefore deserve the same respect and protection as all human beings. From this perspective, a human embryo ought to enjoy all the rights and protections as any other human being. This position considers the destruction of a human embryo to be immoral and often equates it with other types of murder. People who adhere to this belief oppose ES cell research, because the process of extracting stem cells destroys the human embryo. One argument offered by supporters of this position is that researchers should exhaust less ethically controversial sources of stem cells like stem cells found in human adults or animals before considering the use of human embryos for scientific and medical advances.¹¹³

A subset of individuals subscribing to this belief do not believe destroying an embryo is equal to murder, but still consider its destruction reprehensible and immoral. Many state that while they understand the value that stem cell research could one day yield, the ends (potential benefits) do not justify the means (destroying a human embryo), particularly considering that the ends in this instance are hypothetical benefits that could one day yield helpful medical therapies.¹¹³

One possible way to continue to conduct stem cell research without destroying embryos is to use stem cell lines for research derived from embryos that have already been destroyed. Defenders argue that it is acceptable to use ES cells that have already been derived, as long as no new human embryos are destroyed. The argument presented in favor of this position is that while the actual act of destroying a human embryo is wrong, it cannot be reversed, and therefore the stem cells from embryos that have already been destroyed are permissible for use in research. This is current federal policy for federal funding, announced by President Bush on August 9, 2001. Some criticize this position saying that using the by-products of a destroyed embryo means the user is “complicit” in the destruction of that embryo, or at least taking advantage of someone else’s immoral act. Therefore, using ES cells already derived from embryos is in direct conflict with the belief that destroying embryos in order to extract their stem cells is wrong. The United States’ policy on federal funding for research seems too many to be inconsistent in its argument because of this implied complicity.¹¹³

POSITION #2:

Embryos do not have the same status as a fetus or a baby and can be used for research.

A second position regarding human embryo status holds that embryos are nothing, but they don’t have the same status as a fetus or a baby and can therefore be used to derive stem cells for research. From this position, embryos do not deserve the same protections as a fetus or a baby, and therefore the rights and potential benefits for people that are currently alive outweigh the rights of the embryo.¹¹³

Supporters of this position believe that embryos are unique and have special properties because of their potential to become human beings, but they are not as valuable as the lives of living human beings suffering from disease or illness who might benefit from ES cell research outcomes. Many persons who support using embryos for stem cell research contend that stem cell research is so valuable that medical advances will be held back without their use, even if research with alternatives, such as adult stem cells, continues.¹¹³

POSITION #3:

Embryos should not be created for research, but can be used if they are left over from in-vitro fertilization (IVF) procedures.

A subset of people who believe that embryos can be used and destroyed for stem cell research also believe that embryos should not be created or cloned for use in research but can be used and destroyed for research if they are left over from in-vitro fertilization procedures and are going to be unused anyway. This position is referred to as the “nothing is lost” principle. The “nothing is lost” principle means if an embryo is not going to be used for its original purpose of reproduction and would be discarded in the future, then science should be allowed to make use of the embryo prior to its destruction for research that might benefit people who are alive and suffering from a disability or illness. Many believe using embryos destined to be destroyed in the future for research is justified because it is simply varying the method and timing of the embryo’s destruction and not the fact of whether it will be destroyed or not. ¹¹³

There are currently more than 200,000 “excess embryos” frozen in fertility centers around the United States. Excess embryos are created because harvested eggs cannot be frozen for later use, while embryos can. Therefore, unless the eggs are fertilized and made into embryos, a woman would need fresh eggs harvested at each fertility treatment attempt. When the people seeking infertility treatment complete the IVF process and do not plan to pursue reproductive treatments further, they decide what to do with their remaining embryos. One possible option is to donate their embryos to research.¹¹³

POSITION #4:

Embryos are clusters of cells no different from other cells and can be created specifically for use in research.

The final viewpoint regarding the status of the human embryo is that embryos are a mere cluster of cells no different from any other cluster of cells in the body and they can be derived, created, and used in any way people see fit to use them. Supporters argue that even if the embryo deserved special deference because it has the information inside of it to create a human life, that it is this very property which makes the embryo so valuable for research.¹¹³

Embryos created for research are either produced by in-vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) procedures. Supporters of this viewpoint frequently present the argument that if it is ethical to use human embryos in research, then it should be considered ethical to create embryos for that purpose, the intent of the original creation of the embryo is effectively irrelevant. Many advocates of ES cell research (and even some who traditionally adopt a pro-life position) support the creation of human embryos by somatic cell nuclear transfer. One potential of ES cell research with SCNT created embryos is related to the possible medical therapies capable of generating tissues that could be incorporated into a failing organ or tissue.¹¹³

A variety of organizations and individuals object to creating human embryos for research purposes for several reasons. One primary argument is that the potential for exploitation and abuse is too great to open the door to creating embryos for research, particularly if there is potential for monetary benefit for the person donating the egg, sperm, or embryo. Another ethical concern regarding the creation of embryos for ES cell research is donation of surplus embryos for research should be, and is in fact, different from creating embryos explicitly for research. Persons opposed to creating embryos for research argue that creating a human embryo simply to destroy it is an immoral and disrespectful action. Others who object to creating embryos for research have said it is simply “unnatural” to create anything for the sole purpose of destroying it.¹¹³

Research conducted with cloned embryos created via SCNT procedures raises unique issues from research with embryos created via IVF procedures. The concerns stem from a fundamental moral concern about opening the door to cloning humans for reproductive purposes. President Bush, in his August 2001 remarks to the nation regarding stem cell research stated, “I strongly oppose human cloning, as do most Americans. We recoil at the idea of growing human beings for spare body parts, or creating life for our convenience.” According to popular opinion polls, Americans make distinctions between cloning for reproductive purposes and research, or therapeutic, cloning. In a 2002 Gallup Poll of over 1,000 adult Americans, 90% were opposed to reproductive cloning. However, 61% objected to cloning human embryos for use in medical research, while the same poll shows 51% favoring the cloning of human cells taken from adults for use in medical (including SCNT) research. Donating embryos while the use of surplus embryos remaining from infertility treatments is less controversial than the creation of embryos for research, there are other ethical issues to consider. One group of ethical issues involves the donation of human embryos for embryonic stem (ES) cell research. These issues are often similar to those involved in organ donation. U.S. federal law makes payment for organ and tissue donation illegal under the National Organ Transplantation Act (NOTA) of 1984. People are encouraged to give their organs and tissues, or those from a deceased loved one, out of a feeling of obligation and sympathy for people suffering from disease. Concerns center on the possible exploitation of potential donors and inequitable, or uneven, distribution of organs to those with greater ability to pay. Financial incentives for renewable tissues do exist. For example, women can currently be compensated for donating their eggs for fertility treatments to women who cannot use their own eggs to have children. In addition, people are compensated for donating blood or plasma, with no real ethical objections being raised in regard to this practice. Even though eggs and oocytes are considered renewable tissues and can be donated for compensation, many people have qualms about financial incentives for egg donation. People fear ES cell research will force the burden of donation to fall disproportionately on poor women, while the benefits of research outcomes would be enjoyed by all. This is due to the fact that eggs are necessary for both SCNT and IVF procedures.¹¹³

As is true with all research situations, researchers working with ES cells have a variety of incentives. Possibilities for financial gain and prestige motivate researchers. If these incentives are overwhelming, researchers might be encouraged to practice less than ideal (flawed or unethical) research. Incentives for research come from the belief in the potential therapeutic uses of stem cells. If researchers believe they are only a few steps away from curing Parkinson’s disease or leukemia, there is a fear that they would overlook some of the ethical safeguards in order to more quickly attain their goals. Curing one of these diseases would bring prestige within the field and monetary rewards and these incentives can be difficult to put aside or overlook in the quest for knowledge and ethically sound research. The incentives of prestige and financial gain might become even more controversial if there are allowances for patents of ES cells and their products. Many people object to giving patents for stem cell lines and the products of stem cells because it might be equated with patenting the function of a pancreas or a lung. For this reason, several countries have banned the issuance of patents for stem cells and products of stem cell derivatives and the European Union has recommended that patents be issued only for stem cell lines that have been “modified by inventive processes for industrial use.”¹¹³

FEDERAL FUNDING FOR HUMAN EMBRYONIC STEM (ES) RESEARCH

On August 9, 2001, President George W. Bush announced that federal funding for embryonic stem (ES) cell research in the United States would be limited to research with approved stem cell lines derived on or before that date. Research with existing stem cell lines derived from human embryos can be federally funded if the stem cells are included on an official list of authorized lines. Authorized stem cell lines have to meet the following criteria, in addition to being derived prior to August 9, 2001, as stated by the national institute of health.¹¹³

The stem cells must have been derived from an embryo that was created for reproductive purposes, the embryo was no longer needed for these purposes, informed consent must have been obtained for the donation of the embryo, and no financial inducements were provided for donation of the embryo. The announcement of this federal policy had two direct impacts on stem cell policy and research. First, it prompted public discussion and proposed legislation around human stem cell research. Each chamber of the United States Congress was quick to introduce legislation to address the following issues: use of embryonic stem (ES) cells in research, the donation of ES cells for research, the creation of embryos for ES cell research, cloning of embryos for ES cell research, and federal funding of ES cell research. None of these legislative efforts have been successful so far, although this continues to be an active policy issue.¹¹³

Second, the impact of President Bush’s policy is being felt by researchers. Testimony from researchers demonstrates difficulty in obtaining stem cell lines from approved sources. There are only 78 available and eligible stem cell lines which are approved for use in federally funded research. Only 27 of these are in the United States, making physical procurement of the ES cell lines cumbersome, while a mere 9 of these 27 stem cell lines are available to researchers. In addition, all of the eligible stem cell lines were derived using private money and these private holders are under no obligation to share or sell their stem cell lines. As a result, the cost of both the administrative support needed to work with organizations to buy stem cells lines and the cost of the stem cells themselves rose sharply, according to testimony before the United States Senate. Other difficulties with the Bush policy include doubts researchers have about the quality of the eligible stem cells and their genetic diversity.¹¹³

Federal funding for ES cell research receives a great deal of attention because a large proportion of biomedical research funding conducted in the United States comes from the federal government. About 45% of medical and health research is conducted with federal dollars. The remaining 55% comes from private business, foundations, and charitable giving. Within the global community, the United States government spends more than any other country on biomedical research. Since the federal government is the largest single source of funding for biomedical research in the world, many see the issue of U.S. federal funding for ES cell research as critical and crucial to the hopes of medical therapies from these research efforts.¹¹³

The United States funded some research with human embryos resulting from IVF procedures until 1975, shortly after the Roe v. Wade abortion case re-sparked the debate on the status of the human embryo. At that time, a moratorium was placed on federal funding for IVF and embryo research. Since that time, there have been several boards and commissions appointed at the national level to make recommendations regarding policies for the use of human embryos in research. The results have been published, discussed, and partially implemented periodically since the mid 1970s. It is an ongoing, politically-charged process resulting in fragmented state laws and federal regulations about what is and is not acceptable practice.¹¹³

In September of 1999, the National Bioethics Advisory Commission (NBAC) published recommendations concerning the use of human embryos for use in stem cell research. The NBAC’s first recommendation was the continuation of federal funding for embryonic germ (EG) cell research, which uses tissues from aborted embryos/fetuses. This recommendation was consistent with the Fetal Tissue Transplantation Act of 1993 which allows federal funding for research on discarded fetal tissue. The second recommendation from the NBAC was to federally fund embryonic stem (ES) cell research with embryos left over from in-vitro fertilization treatments, but to not federally fund research with embryos created specifically for research. Funding for ES cell research using embryos remaining after in-vitro fertilization treatment hinged on whether the donors of the embryo were made fully aware that: a) research will destroy the embryo, b) the embryo will not be implanted in another woman, c) the funding source for the research, d) the intention and context of the research, and e) that decisions to donate will not impact the donor’s current or future medical care. Shortly after the release of the NBAC report, in December of 1999, the National.¹¹³

Institutes of Health (NIH) drafted guidelines for federally funding research on ES cell lines derived in the private sector. President Clinton backed the guidelines and they were finalized in August of 2000. At the time the guidelines were finalized, then-candidate for President, George W. Bush, denounced the guidelines and made statements opposing the destruction of human embryos for research purposes. After his inauguration and a review of the NIH guidelines, President George W Bush announced the current federal policy. He also formed a new bioethics advisory group to address bioethical issues including cloning and stem cell research, called the President’s Council on Bioethics. The Council’s mission is to advise the President regarding policies involving ethical issues, including human stem cell research.¹¹³

December 2002 there had been no formal recommendations concerning embryonic stem cells research, but the Council did publish their recommendations (based on a 10-8 majority) on stem cell research using cloned or SCNT embryos: “We propose a congressionally enacted four-year national moratorium (a temporary ban) on human cloning-for-biomedical-research.” This ban would apply to all organizations and individuals in the United States whether or not they are receiving funding from the federal government.¹¹³

Regarding privately funded ES cell research, opinions are varied. Many suggest that if federal funding of ES cell research doesn’t apply to private research, there will be no rules to ensure an ethical standard. They believe policies would be more effective if they applied to all forms of research and not limited to only federally funded projects. On the other side of the debate are those who state that the “freedom to conduct research” is a right, and private research should be encouraged to be conducted both ethically and publicly. Traditionally, federal regulations on research involving human subjects have strongly influenced the ethical conduct of research in the private sector and the same result could occur for policies regarding ES cell research with human embryos.¹¹³

In September 2002, California passed a law allowing state funding of stem cell research projects and the derivation of ES cells from human embryos, including embryos created through SCNT procedures. The legislation includes a stipulation for the establishment of ethical review committees to review research projects in order to ensure ethically responsible research. The unique aspect of this legislation is that the law also applies to research conducted by private organizations, which will be subject to ethical review as well.¹¹³

OPINIONS

The opinions expressed below do not necessarily represent the opinions of all individuals or organizations within a specific group, but rather the majority or most popular opinion.¹¹³

PRESIDENT GEORGE W. BUSH

President Bush has allowed federal funding to go towards research on stem cell lines derived prior to August 9, 2001. His policy states, “This allows us to explore the promise and potential of stem cell research without crossing a fundamental moral line, by providing taxpayer funding that would sanction or encourage further destruction of human embryos that have at least the potential for life.”¹¹³

EUROPEAN UNION

The European Union’s European Group on Ethics (EGE) released an opinion on stem cell research in November of 2000. Their findings concluded that: 1) the use of adult stem cells should require the same ethical consideration as tissue donation, 2) retrieving stem cells from umbilical cord blood after delivery should require the consent of the fully informed donor, 3) using foetal tissues to derive stem cells requires both informed consent and the rule that no abortion be induced for the purpose of obtaining tissues for stem cell research, and 4) the use of embryos for stem cell research should be up to each country within the European Union to decide.¹¹³

UNIVERSITY OF MINNESOTA

The University of Minnesota is actively pursuing stem cell research through its stem cell institute. Many research innovations have come out of the University including the first bone marrow stem cell transplant in the 1960s. The University follows all state and federal laws and policies relating to stem cell research, and hosts the Stem Cell Ethics Advisory Board to address ethical issues around stem cell research.¹¹³

PRESIDENT’S COUNCIL ON BIOETHICS

As of October 2002, the President’s Council on Bioethics is currently still hearing from experts in the field and debating the ethical points for and against using embryonic stem cells for research, but they did recommend a 4-year moratorium on research with cloned (SCNT) human embryos.¹¹³

CATHOLIC CHURCH

Richard Doerflinger, in his 1999 opinion for the Catholic Church states, “Stem cell research that requires the destruction of human embryos is incompatible with Catholic moral principles, and with any ethic that gives serious weight to the moral status of the human embryo.”¹¹³

JUDAISM

In testimony to the National Bioethics Advisory Commission in 1999, Rabbi Elliot Dorff explained that one condition of Judaism is the body has to be preserved and health pursued. Rabbi Dorff interprets this to mean that God would encourage research of any kind that would help preserve one’s body. Of course, many Jewish persons may not agree with his statement but he does recommend the advancement of stem cell research because of its great potential to do good.¹¹³

PROTESTANT

There are many different views held within by Protestants. The more conservative Protestant denominations would consider it unethical to destroy human embryos for use in research. Some of the more liberal denominations might be more permissive in their opinions regarding the use of an embryo, but would most likely still place limits on research.¹¹³

ISLAM

The Islamic tradition in general considers research with human pluripotent stem cells (from embryonic germ cells) obtained from an aborted fetus acceptable, with several conditions placed on using embryonic or fetal cells from different stages of development as stated in the Koran.¹¹³

GENERAL PUBLIC

In a public opinion poll taken by the Pew Research Center (n = 2,002 adults over 18) in April of 2002, 50% of people think the federal government should fund stem cell research, while 35% think that stem cell research should not be funded by the federal government. When asked what they thought was more important, research towards medical cures or not destroying human embryos, 47% thought research was more important versus 39% who favored not destroying embryos.¹¹³

OTHER ETHICAL ISSUES

The ethical issues around embryonic stem cell research, embryonic germ cell research, cloned (SCNT) embryo research, and adult stem cell research are varied and complex. The debate becomes more intense when people consider special situations ranging from current medical therapies developed with stem cells to issues that may occur in the future. Below are a few of the current and future ethical issues that require attention. When working with SCNT embryos, there is currently no genetic manipulation, but that might not always be the case. Some worry that stem cell research is laying the foundation that will give scientists the knowledge they require to genetically alter human cells and thus change, fundamentally, the building blocks of humanity so significantly that the new cells created are no longer human.¹¹³

XENOTRANSPLANTED STEM CELLS

Xenotransplanted stem cells are stem cells from animals that are used to create tissues and cells. These cells would then be used in medical therapies for humans. Ethical issues involving the abuse and exploitation of animals and animal rights concern many people. Medical concerns about using Xenotransplanted stem cells involve the possibility of animal-human pathogens and illnesses that might not be anticipated by researchers.¹¹³

CREATING A LIFE TO SAVE ANOTHER

In 2000, the University of Minnesota used preimplantation diagnosis to create a child in order to harvest stem cells from the umbilical cord at birth for use in treatment for the child’s sibling. Preimplantation diagnosis occurs when embryos are fertilized in-vitro and then screened for a particular characteristic. In this case, the sibling had a rare genetic disorder and needed a healthy donor from which to collect genetically-matched umbilical cord stem cells. Ethical concerns involved in cases like this one might include: debate over the pre-implantation selection, the intentions and motivations of the parents, and the creation of a child as a means to gather the stem cells from the umbilical cord.¹¹³

PARTHENOGENESIS

A new and innovative process of developing blastocysts in order to extract their stem cells for research is parthenogenesis. In this process, oocytes are activated through the use of chemical stimulation without requiring sperm or an SCNT procedure. Researchers recently derived stem cell lines from monkeys using this procedure, but have not been able to stimulate human oocytes into progressing this far. Scientists are doubtful that parthenogenesis could ever develop an oocyte into an advanced embryo or a fetus, and therefore, this potential source of stem cell lines could be used without creating a human embryo that could develop into a human being, which could serve to eliminate a great deal of ethical objections arising from the use of human embryos for research. The future of stem cell research is in question. While federal funding is limited to a few available stem cell lines, private research continues at a rapid pace and federally funded research with adult stem cells and embryonic germ cells also progresses. The future of stem cell research will offer new and additional scientific and medical advances, along with many ethical challenges. Society’s challenge is how to balance them all.¹¹³

Every tissue in the body contains some type of stem cell. From here arose the idea of medical and specifically dental cell based stratergies for tissue repair. The focus of stem cell research as it applies to dentistry is on facial reconstruction. Recent findings and scientific research supports the use of these very powerful mesenchymal stem cells found within teeth and other accessible tissue harvested from the oral cavity for use in regenerative medicine. While we can see the promise of human stem cell therapies for the future, dentists should know how important it is to harvest and store these mesenchymal stem cells, making these opportunities available to their child, adolescent, and adult patients for future regenerative therapies.

Stem-cell therapy has been accepted as the effective treatments for many blood diseases, certain types of Cancers and several other diseases in the world over the last 10 years. It is believed that Stem-cell therapy may eventually offer remedies for Brain diseases such as Stroke, Parkinson's and Alzheimer's diseases, Spinal cord injury, autoimmune diseases, Mitral stenosis, Osteoarthritis, Degenerative diseases, and certain forms of Cancer and Heart disease. One of the most important potential applications using this material is for the treatment of paralysis due to spinal cord injury which has already been done using Mesenchymal stem cells from other sources. Storing these cells for our self and our child is an excellent way to ensure our and our child's future biological needs in case of disease or injury.

Stem-cells derived from all sources hold immense medical promises. Stem-cell therapies have virtually unlimited medical and dental applications. We have moved on from the surgical model of care to the medical model and are likely to move onto the biological model of care. Stem-cell therapy is no longer science fiction. Recent developments in the technique of Stem-cell isolation and expansion together with advances in growth factor biology and biodegradable polymer constructs have set a stage for successful tissue engineering of tooth/tooth-related tissues. Stem-cell therapy has brought in a lot of optimistic hope amongst researchers, doctors, and not to forget the patients who are the chief beneficiary of this innovation. Stem-cells regenerate hope and not all that is happening in research is hype.

Adult stem cells ( Somatic stem cells):- A relatively rare undifferentiated cell found in many organs and differentiated tissues with a limited capacity for both self renewal (in the laboratory) and differentiation. Such cells vary in their differentiation capacity, but it is usually limited to cell types in the organ of origin. This is an active area of investigation.

Bone marrow stem cells (skeletal stem cells):- A multipotent subset of bone marrow stromal cells able to form bone, cartilage, stromal cells that support blood formation, fat, and fibrous tissue.

Cell-based therapies:- Treatment in which stem cells are induced to differentiateinto the specific cell type required to repair damaged or destroyed cells or tissues.

Cell culture:- Growth of cellsin-vitroin an artificial medium for research or medical treatment.

Differentiation:- Process whereby an unspecialized embryonic cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

Directed differentiation:- The manipulation of stem cell culture conditions to induce differentiation into a particular cell type.

DNA:- Deoxyribonucleic acid, a chemical found primarily in the nucleus of cells. DNA carries the instructions or blueprint for making all the structures and materials the body needs to function. DNA consists of bothgenesand non-gene DNA in between the genes.

Ectoderm:- The outermostgerm layer of cells derived from theinner cell mass of theblastocyst gives rise to the nervous system, sensory organs, skin, and related structures.

Embryo:- In humans, the developing organism from the time offertilization until the end of the eighth week of gestation, when it is called afetus.

Embryonic stem cells:- Primitive (undifferentiated) cells that are derived frompreimplantation-stageembryos, are capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.

Embryonic stem cell line:- Embryonic stem cells, which have been cultured underin-vitroconditions that allow proliferationwithoutdifferentiationfor months to years.

Gene:- A functional unit of heredity that is a segment of DNA found on chromosomes in the nucleus of a cell. Genes direct the formation of an enzyme or other protein.

Human embryonic stem cell (hESC):- A type ofpluripotentstem cells derived from early stage human embryos, up to and including the blastocyststage, thatare capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primarygerm layers.

Induced pluripotent stem cell (iPSC):- Type of pluripotent stem cells, similar to an embryonic stem cell, formed by the introduction of certain embryonic genes into somatic cells.

In-vitro:- Latin for "in glass"; in a laboratory dish or test tube; an artificial environment.

In-vitro fertilization:- A technique that unites the egg and sperm in a laboratory instead of inside the female body.

Long-term self-renewal:- The ability of stem cells to replicate themselves by dividing into the same non-specialized cell type over long periods (many months to years) depending on the specific type of stem cell.

Mesenchymal stem cells:- A term that is currently used to define non-blood adult stem cells from a variety of tissues, although it is not clear that mesenchymal stem cells from different tissues are the same.

Microenvironment:- The molecules and compounds such as nutrients and growth factors in the fluid surrounding a cell in an organism or in the laboratory, which play an important role in determining the characteristics of the cell.

Multipotent:- Having the ability to develop into more than one cell type of the body.

Oligopotent:- Stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.

Parthenogenesis:- The artificial activation of an egg in the absence of a sperm; the egg begins to divide as if it has been fertilized.

Pluripotent:- The state of a single cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development.

Regenerative medicine:- A field of medicine devoted to treatments in which stem cells are induced todifferentiateinto the specific cell type required to repair damaged or destroyed cell populations or tissues.

Stromal cells:- Connective tissue cells found in virtually every organ. In bone marrow, stromal cells support blood formation.

Surface markers:- Proteins on the outside surface of a cell that are unique to certain cell types and that can be visualized using antibodies or other detection methods.

Totipotent:- Having the ability to give rise to all the cell types of the body plus all of the cell types that make up the extra embryonic tissues such as the placenta. (See also PluripotentandMultipotent).

Transdifferentiation:- The process by which stem cells from one tissuedifferentiateinto cells of another tissue.

Umbilical cord blood stem cells:-Stem cells collected from the umbilical cord at birth that can produce all of the blood cells in the body (hematopoietic). Cord blood is currently used to treat patients who have undergone chemotherapy to destroy their bone marrow due to cancer or other blood-related disorders.

Undifferentiated:- A cell that has not yet developed into a specialized cell type.

Unipotent:- cells can produce only one cell type, their own, but have the property of self-renewal, which distinguishes them from non-stem cells (e.g., muscle stem cells).

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