AbstractThe generation of induced pluripotent stem (iPS) cells is a useful tool for regenerative medicine. However, the risk of tumor development of the aforementioned cells should be addressed before they can be used for clinical applications. During the reprogramming process a number of signal pathways are activated, which may lead to direct differentiation of specific cell lineages prior to the cells reaching the pluripotent state. In order to test this hypothesis we designed a combined protocol of reprogramming and differentiation in an attempt to achieve direct differentiation of fibroblasts to specific cell lineages. Human fibroblasts were shortly reprogrammed by overexpression of four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) and maintained in reprogramming media on a gelatin substrate for four days. These cells were defined as partially induced pluripotent stem (PiPS) cells. PiPS cells did not form tumours in vivo and differentiated into smooth muscle cells (SMCs) when seeded on a Collagen IV substrate and maintained in differentiation media (DM). The PiPS-SMCs expressed a panel of SMC markers such as SMA, SM22 and Calponin at mRNA and protein levels. Immunofluorescent staining of PiPS-SMCs showed positive staining for the above markers, demonstrating a typical SMC morphology. These cells displayed a greater potential to differentiate into SMCs than iPS cells. In order to elucidate the mechanism of PiPS cell differentiation into SMCs, data from a series of experiments indicated that the gene DKK3 was involved in SMC differentiation of PiPS cells. DKK3 was expressed in parallel with SMC markers, while its overexpression or stimulation induced SMC marker expression. Furthermore, DKK3 silencing resulted in downregulation of SMC markers on both the mRNA and protein levels.
Finally, additional experiments revealed that the upregulation of SMC markers by DKK3 is mediated by activation of Wnt signalling through interaction of DKK3 with the transmembrane receptor Kremen 1. Therefore, we developed a protocol to generate SMCs from PiPS cells through a DKK3 signal pathway. These findings provide a new insight into the mechanisms of SMC differentiation with therapeutic potential to vascular disease.
|Date of Award
|Qingbo Xu (Supervisor) & Rene Botnar (Supervisor)