Paper Title
The Effect of Pooled Human Platelet Lysate and Growth Factors on Urinary Mesenchymal Stem Cells Proliferation and Differentiation

Mesenchymal stem cells (MSCs) are promising candidates for novel cell therapeutics application. MSCs are immunosuppressive and hypo-immunogenic. Remarkably, stem cells were also found in the urine, as a novel cell source, urine stem cells provide advantages for regenerative medicine and tissue engineering applications. Large scale of MSCs expansion in vitro involves supplementing the growth media with fetal bovine serum (FBS). Human platelet lysate (pHPL) is a successful approach in replacing FBS in stem cells culture. It does not only reduce the immunogenicity, but it is also enriched with many growth factors that enhance cells’ growth. This study aims to investigate the effect of pooled HPL on the proliferation of urine MSC in vitro. In addition, the effect of three growth factors (EGF, TGF-β and VEGF) on the proliferation and differentiation of USCs into urothelial cells was tested. Materials and Methods: UMSCs were isolated from relatively healthy donors, and characterized according to the NIH criteria for MSCs classification. The effect of different concentration of pHPL on the proliferation of UMSCs compared with FBS and the effect of EGF, TGF-β, VEGF and EFG inhibitors on UMSCs proliferation were measured by MTT assay. Gene expression analysis for cell cycle markers CDK2, CDK4, and CCND2, cell senescence marker (RB transcriptional corepressor like 2, RPL2) and mitotic double-strand break repair protein, MRE-11, were detected by Real Time RT-qPCR. UMSCs were induced into urothelial cells using both CM and keratinocyte serum free medium (KSFM). mRNA expression of urothelium markers (K-18, K-19 and UP-2) was detected by quantitative RT-PCR. Protein expression of the urothelium markers (cytokeratin-18 (K-18), uroplakin-1a (UP-1a) and uroplakin-2 (UP-2)) was detected by immunofluorescence. Results: Isolated cells were conformed to be MSCs according to a set of criteria by National Institution of Health. Expansion of UMSCs in pHPL resulted in a preserved flattened and spindle-shape. UMSCs cultured in different concentration of pHPL at passage 2, demonstrated a significantly lower proliferation rate than the standard FBS (p < 0.05). However, 10% pHPL considered to be the most suitable concentration. All tested growth factors had a significant negative effect on the proliferation rate compared with the control . UMSCs induced with TGF-β and VEGF inhibitors exhibited a lower differentiation potential towards urothelial lineage in terms of K-18 and K-19 expression with more than 2-fold difference compared to negative control. Index Terms- Growth Factors, Mesenchymal Stem Cells, Platelet Lysate