Characterisation of the effects of hypoxia on proliferation, immune phenotype and differentiation potential of adipose tissue-derived, bone-marrow derived mesenchymal stromal cells and HUVECs

This project practical consists of two pilot projects:

- Characterisation of the effects of hypoxia on proliferation, immune phenotype and differentiation potential of adipose tissue-derived mesenchymal stromal cells
- Towards Cell-based therapies for diabetes: Characterisation of the effects of hyperglycemia on proliferation, immune phenotype and differentiation potential of adipose tissuederived mesenchymal stromal cells

Introduction

Human mesenchymal stem cells (MSC), which are first isolated from bone marrow (BM)¹ and subsequently isolated from other tissues such as adipose tissue, cutaneous tissue, fetal hepatic and pulmonary tissue[1, 2, 3] are pluripotent progenitors for a variety of tissues includ- ing bone, cartilage, tendon, fat, and muscle⁵. The adipocytic pheno- type is characterized by intracellular accumulation of lipid droplets as well as transcription of adipocyte-specific genes. Previous studies have shown that MSCs play an important role during bone formation and bone remodeling and also have been indicated as potential resources for many clinical applications. Therefore, mesenchymal stromal cells (MSCs) hold great potential in skeletal tissue engineering and regener- ative medicine. 1

Date: December, 2014.

2 KLIMENT SANDJAKOSKI, MD

Recently it was shown that MSC recruit from the perivascular niche which represents a tight network throughout the vasculature of the whole body. These perivascular cells lack endothelial/hematopoietic markers (CD31, CD34) but express CD146, PDGF-R beta, and alkaline Phosphatase⁸.

Project Rationale

(1) In the first part of this project practical we aimed to evaluate the effect that changing glucose concentration, medium change and partial preassure(pp) of O2 have on HUVECs and/or ECFCs in vitro.
(2) In the second part an effort was made to make analogous eval- uation in respect to MSCs derived from adipose tissue(LA) and bone marrow(BM).

Methods

Isolation and expansion of MSCs MSCs used in this study were isolated from BM aspirates. Human BM was obtained from the iliac crest of four healthy donors (median age, 24; range, 869 years), and MSC cultures were established. BM cells for the experiment were supplied by thawing BM103FCSp1/1 cells.

Adipose tissues were taken from the various region during cosmetic surgery and always from one patient (single donors). Adipose tissues were washed extensively with equal volumes of Dulbecco’s phosphate- buffered saline (DPBS; HyClone, Logan, UT), and the extracellular matrix was digested with collagenase A(250 μ l in 50ml PBS) at 37C for 35-45 min. Enzyme activity was neutralized with (PBS).(AB medium. containing 10%

Red cell lysis was attempted by using NH4Cl solution. Samples were then centrifuged for 10 min. The cell pellet was washed with DPBS and filtered through a 100-m nylon mesh (mounted on a Falcon tube). Cells were seeded on uncoated T175 culture flasks. The cells were incubated in a humidified atmosphere at 37C with 5% CO2, and the medium was changed every 3 to 4 days. After 7-10 days, when reached confluency, cells were phenotyped.

Isolation and expansion of HUVECs Endothelial cells (ECs) are isolated from umbilical vein vascular wall by a collagenase treatment, then seeded on fibronectin-coated plates and cultured in a medium with Earles’ salts and fetal calf serum (FCS), but without growth fac- tor supplementation(endothelial baseic medium, EBM), for 7 days in

a 37 C5% CO2 incubator. Cell confluency can be monitored by phasecontrast microscopy.

Human umbilical chords are sup- plied by the local clinic, and kept at 4 ◦ C for the next day when the harvesting procedure is performed.

Adipogenic differentiation of MSCs Adipogenic medium was made by supplementing complete

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Figure 1. Experimental setup

DMEM with 10% v/v FCS, 10% v/v horse serum, 0.5 mmol/L isobutylmethylxanthine (IBMX), 0.06 mmol/L indomethacin, 50 pmol/L hydrocortisone, 2 mmol/L L-glutamine and antibiotics, as previously de- scribed. Adipogenic differentiation assays were performed in 96-well plates, with the use of constant cell seeding density of 4 104 cells per cm2. Adipogenic medium was changed twice weekly (one-half media changes) during a 21-day time course, and samples were taken for analysis at different time points as specified below.

Oil red staining for adipogenesis assay Before staining, cells were washed twice with PBS and fixed in 10% (v/v) formalin. After fixation and two further washes in PBS, oil red solution [0.5% oil red (w/v) (Sigma) in isopropanol] was added for 10 min. After incubation at room temperature (RT), oil red was removed, and cells were washed twice in PBS and stained with Harris hematoxylin for 3-5 min. Subsequently, cells were washed in tap water and immediately viewed on microsope Ph-2.

Osteogenic differentiation of MSCs One of the most noteworthy characteristics of mesenchymal stem cells (MSCs) is their ability to differentiate into osteoblasts in vitro and in vivo. In vitro, this is eas- ily achieved by culturing in the appropriate induction medium. It is because of the reliability and ease of this process that osteogenic dif- ferentiation has become a popular assay for the demonstration of MSC plasticity. The cells were cultured in MSC growth medium (MSCGM; the cell system components consisted of the MSC basal medium and the SingleQuots growth supplements MCGS, which contained fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin in a hu- midified incubator in the presence of 95% air and 5% CO2 at 37C. Af- ter 24 h, the media was changed to MSC osteogenic induction medium (MSCOIM; the cell system components consisted of MSC basal medium that contained FBS, L-glutamine, penicillin/streptomycin, dexametha- sone, ascorbate, and -glycerophosphate. The media was changed twice a week.

ODA staining for osteogenesis assay Before staining, cells were fixed in 10% (v/v) formalin. After fixation and two further washes in distilled water, AgNO3 was added for 20 min. After incubation at room temperature (RT), AgNO3 was removed, and cells were washed twice in Aqua dest. and stained with 1% pyrogalol for 2-5 min. Sub- sequently, cells were washed in destilled water, sodium thiosulphate 5 % was added, washed twice with Aqua dest. and immediately viewed on microsope Ph-2.

Immunofluorescence in HUVE cells Endothelial cells were grown to confluence on coated coverslips in 6-well plates. The wells were washed with serum-free medium and cells were fixed in 4% paraformalde- hyde in serum-free medium with or without 0.1% Triton X-100 to per- meabilize the cell membrane and allow intracellular staining. The cov- erslips were blocked with 2% BSA in PBS, and stained with the indi- cated primary and secondary antibodies. The cells were imaged using using an inverted-stage microscope (Zeiss), equipped with a high-speed digital cameraa nd later, analyzed using AxioVS40 V 4.8.0.0. (Zeiss, Jena, Germany) software.

Matrigel assay for HUVECs For this assay, Matrigel was pro- vided(frozen at -80 ◦ C). All the reagents and materials used in this pro- tocol were kept in fridge(4 ◦) and the procedures were performed on ice.

On the start of the exper- iment, the matrigel was

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Figure 2. Aproximate cell-density on thawed(4 ◦ C) overnight.

matrigel(1.7x10⁴ ) After applying it on the slide(Fig 2.), it is put in incubator(37 ◦ C, 5 % CO2) to become hard. The following procedures were performed accord- ing to the protocol and HUVECs, after 2 days, formed tube-like formations in the gel.

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Experimental design

As mentioned before, three culture conditions were applied for exam- ining cells’ response to Hyperglycemia state. However, hyperglycemia in DM patients(> 200mg/L) is not the one that was used in our ex- periments, which was 10-15 times higher(4g/L). One reason for that is to get clear response to Glucose overload in the media. In diabetic patients Hyperglycemic state can last for relatively long period(many years) even in some treated patients with low compliance to treatment protocols suggested by physicians. In this experiment this period was reduced to 2-3 weeks, so to compensate for this discrepancy, Glucose concentration was markedly increased. Same concentration of Manni- tol was applied in the second culture condition, as a positive control. Finally, as a negative control, normal medium with physiological con- centration of glucose was applied. In hypoxia conditions, cells were incubated at 1% oxigen, 37 ◦ C, while in normoxia conditions the per- centage of oxigen content was 21%. For the tube formation assay on matrigel, cells were incubated in Normoxic or Hypoxic conditions for 2- 3 weeks, and after their transfer on matrigel, they were also incubated under the same conditions.

Results

Observation and data analysis for the Immunofluorescence in ADA and ODA MSCs

ADA&ODA stainings Observing stained samples of differentiated cells in these assays, a problem of particular interest was the ability of ODA- stain to penetrate and stain cells(nuclei) in the cell culture, thereby making it impossible to compare the cellularity vs. matrix deposition in these samples. In adipogenesis assay, however, an obvious gradient in differentiation degree was observed going from normal(highest) through High Glucose(middle), to Mannitol(low level) (Figure 10.).

TECAN measurement and analysis Differentiation may depend on pro- liferation rate in a cell culture and vice versa. To normalize for the possible interaction between the two variables, first it was necessary to titrate the concentration of staining solution in both assays and then analyse data, generated by TECAN-software. For this purpose, serial dilutions of different statining solutions was made according to a scheme(Table 3). The normalization analysis to the DNA content was performed, in Excel(the output is presented on p.19-20, Appendix, Table).

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Table 3. Serial dilution scheme for Hoechst Staining solutions in Adipored and Osteoiamge

Control IF microscopy Control pictures on immunofluorescence microscope were made for Adipored and Osteoimage assays due to their unequal distribution through the well’s bottom(Fig. 14).

vWF in HUVECs Weibel-Pallades bodies in endothelial cells were stained with green immunofluorescent dye and DAPI was used to stain the nuclei(blue). When observing the intracellular distribution in dif- ferent culture conditions, a more periferal localization(distant from cell-nuclei) of those bodies was appreciated in Normoxic High Glucose culture condition. No other significant differences in these conditions could be observed(Appendix p.16).

FACS on HUVECs Statistical analysis of FACS results. Medians for background was subtracted from medians for different antigens. The distribution of those antigens is presented graphically for both conditions: Normoxia and Hypoxia. Differential expression of various antigens can be observed in cells exposed to different partial pressure of O2(refer to p.17, Appendix, ”FACS analysis”).

Aknowledgements

The collective knowledge generated from applied research, summa- rized in various references has been critical in the creation of this report. My most sincere thanks go to Elvers-Hornung Susanne and Stefanie Uh- lig for their orientation remarks in the realization this practical task.

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¹ Williams JT, Southerland SS, Souza J, et al. Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. Am Surg 1999; 65:226.

² Gronthos S, Zannettino AC, Graves SE, et al. Differential cell surface expres- sion of the STROP-1 and alkaline phosphatase antigens on discrete develop- ment stages in primary cultures of human bone cells. J Bone Miner Res 1999; 14:4756.

³ Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-Like cells from umbilical cord. Stem cells 2003; 21:10510.

⁴ Aldridge A1, Kouroupis D, Churchman S, English A, Ingham E, Jones E. Assay validation for the assessment of adipogenesis of multipotential stromal cells- a direct comparison of four different methods. Cytotherapy. 2013 Jan;15(1); 89:101