A Comparative Analysis of Multipotent Mesenchymal Stromal Cells derived from Different Sources, with a Focus on Neuroregenerative Potential

Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton’s jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H2O2 treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.


Materials and methods
To promote osteogenic and adipogenic differentiation, minimally three different BM-MSCs, AT-MSCs and WJ-MSCs samples at passage three (P3) were initially seeded in six-well plates (60,000 cells/well) in a complete culture medium (CCM) at 37°C and 5% CO2 in a humidified atmosphere containing 95% air. After reaching 90% confluence, the culture medium was removed and the appropriate differentiation media were added to the cultures. To induce osteogenic differentiation, the cells were cultured for minimally 3 weeks in an induction medium composed of α-MEM, supplemented with with 10% fetal bovine serum (Biosera), 50 U/ml penicillin, 50 µg/ml streptomycin, 10 mM glycerol-2-phosphate, 0.1 μM dexamethasone and 100 μM L-ascorbic acid. The medium was changed twice weekly.
Osteogenesis was assessed by detection of calcified extracellular matrix deposits using Alizarin Red S staining.
Adipogenesis was assessed using conventional Oil Red O staining to visualize lipid droplets in the cytoplasm.

Results
The induction of adipogenic, osteogenic or chondrogenic differentiation resulted in corresponding changes in cell morphology and the appearance of specific signs of differentiation: the accumulation of lipid droplets in adipogenic cultures, matrix mineralization in osteogenic and accumulation of extracellular matrix in chondrogenic conditions (Fig. S1).

Materials and methods
To perform a migration assay, xCELLigence RTCA DT Instrument (Acea Biosciences Inc. San Diego, CA, USA) was used. Prior to the migration assay, cells were left for 24 hours in a media without PL. On the day of the experiment, a medium with SDF-1α (20 ng/ml) was placed into a lower chamber of CIM-Plate 16, and MSCs were then pipetted into the upper chamber (10 5 cells in 100 µl of media without supplements). The recorded impedance signal at 6 hours was normalized to migration towards supplement-free media and was then used to assess the rate of cell migration. The assay was repeated five times.

Results
Migratory capacity is one of the essential MSC properties that enables their trafficking and thus allows them to act at the site of injury. In a transwell culture, we confirmed the capacity of all MSCs to migrate towards SDF-1α. The migration rate did not differ between individual cell types (Fig. S2).

Materials and methods
The conditioned medium for secretome analysis was collected after 24 hrs cultivation of WJ-

MSCs, BM-MSCs and AT-MSCs in a PL-free medium to detect factors released specifically by
MSCs rather than present in PL supplement. However, to avoid the serum starvation associated changes in gene expression and secretory activity, shown previously, the culture medium was additionally supplemented with 1% Insulin-transferrin-selenium supplement

Results
The remarkable increase in the secretion of IL6, IDO, sICAM-1, sVCAM-1 and BDNF was detected after cytokine treatment, independently on the source of cell isolation (Fig. S4).
These results confirm the previously published data 1,3 . In turn, among the growth factors tested SDF-1α and VEGF-A were secreted constitutively regardless of culture conditions.