Identification of small molecules that enhance murine bone marrow derived mesenchymal stem cell expansion

Mesenchymal stem cells became a great candidate for transplantation-based therapies with their immunomodulatory traits (ability to regulate immune response), homing capability (migration to injured sites), differentiation ability to all three embryonic lineages; only after perfecting isolation protocols and expension tecniques. They are present in the adult body, they can self-renew themselves and exhibit multipotency. They can be obtained from a variety of tissue types including; bone marrow, adipose tissue, umbilical cord, dental tissue. On the other hand, there are major challenges on mobilization, expansion and understanding the differentiation mechanism. If these challenges can be overcome, MSCs show great potential for experimental and clinical applications. In our study we focused on expansion of mBM-MSCs with small molecule treatment. With that, we hope to achive increased mobilization results as well. We selected four effective molecules primarily by WST-1 cell viability assay, the assay was supported by hoechst staining for cell counting; SKF96265, SB203580 (P38-MAPK Inhibitor), GSK-3 Inhibitor and StemReginin 1 (AhR Antagonist). To see the effects on cell mechanism; cell cycle analysis with DAPI and Hoechst staining, apoptosis analysis with Annexin V-FITC and PI staining were conducted. The results did not suggest any misconduct on our MSC culture by the expanding effects of selected small molecules except SFK96265, a CCE inhibitor, which caused G1-phase arrest and the cell population treated by had increased cell death rates. Next, we performed RT-PCR to check if there were any negative changes on HDR, CDKI, S-Phase-related gene expressions. SFK96265 also showed highly significant upregulation of RAD51 and PCNA HDR-related genes expressions. Our first molecule is a GSK-3 inhibitor that stabilizes free cytosolic β- catenin and inhibits differentiation. Next candidate is a p38-MAPK inhibitor, which is the most effective small molecule. In conclusion, we layed a foundation for a safe and reliable way of in vitro expansion of murine BM-MSCs. Our next step is treatment of the mixture of compatible small molecules, in vivo trials by small molecule treated stem cell transplanation for GvHD and immunomodulation therapy. With all this, our goal is to carry this knowledge to therapeutic field.