The mechanism of the differentiation of stem cells (SCs) into smooth muscle cells (SMCs) is not yet fully understood, although recent research in this area has made significant strides. In previous studies, human embryonic mesenchymal stem cells (hEMSCs) were shown to differentiate by induction with transforming growth factor beta (TGF-Β) through Smad and serum response factor (SRF), p38 mitogen-activated protein kinase (p38 MAPK), and PI3k Phosphoinositide 3-kinase (PI3k).3 Human amniotic stem cell derived smooth muscle cells do not show continuous differentiation and are functionally different as compared to embryonically derived cells.2 This may be influenced by different differentiation mechanisms. This experiment will attempt to identify several mechanisms by which amniotic stem cells are differentiated into smooth muscle cells that have been shown in embryonic stem cells to drive differentiation to smooth muscle cells. This study will synthesize and characterize smooth muscle cells from human amniotic stem cells (h-AMSCs) and then characterize the TGF-B induced MAPK pathway by inhibiting possible pathway constituents.
Amniotic stem cells will be differentiated into smooth muscle cells using platlet derived growth factor (PDGF-BB) and TGF-B1. The differentiation of h-AFSCs will be analyzed at varying concentrations of TGF-B in order to determine the most effective concentration specific to h-AFSCs. SMC differentiation will be measured by measuring the absence of AFSC specific antibodies and gene expression. These SMCs will then be analyzed using reverse-transcription polymerase chain reaction, western blot analysis, contractility assays, and immunoflouresecent staining using the antibodies for smooth muscle a- actin, smooth muscle myosin heavy chain, Smad 2, Smad 3, and calponin. Intracellular Ca2+ will be measured using calcium reagent solution as well as luciferase assay. The activation or absence of these genes may show differences in the differentiation of SMC from ASC.
The TGF-B-MAPK pathway previously shown to be involved in h-EMSC differentiation into SMCs will be examined.2 By measuring or knocking out the constituents of the MAPK pathway differences in the pathway of h-AFSCs a compared to h-EMSCs can be inferred. This information will provide for a better understanding of the differentiation of stem cells in general and may make it possible to differentiate stable SMC from amniotic stem cells. By determination of the effect of the constituents of the MAPK signaling pathway, it may be possible to determine an approach that allows the production of stable and more fully functional SMCs from h-AFSCs. It may be that the MAPK pathway in h-AFSC differentiation functions differently than h-EMSCs differentiation to SMC in-vivo, however it is also possible that h-AFSC differentiation functions through similar pathways in-vivo.
The determination of the...