Directed differentiation protocol of embryonic stem cell tries to recapitulate in-vivo environment of organogenesis in an in-vitro setting. The key factors affecting developmental process are soluble growth factors, extracellular matrices and intercellular signaling. Likewise, soluble factors and ECM have been shown to have significant effect in the in-vitro differentiation of ESCs to various lineages. The effects of intercellular signals in the context of pancreatic differentiation of ESCs however remain less explored. In pancreatic embryogenesis, it has been reported that developing pancreatic cells secrete VEGF to attract endothelial cells, which in turn secrete ECM components that promote islet maturation via integrin mediated pathways . Endothelial cells have also been demonstrated to induce Ptf1a expression and sustain Pdx1 expression in the dorsal pancreatic bud .
The current study investigates the effect of endothelial cell in the differentiation of hESC to pancreatic islet-like cells. A directed differentiation approach has been developed, involving definitive endoderm induction, followed by pancreatic progenitor specification and final maturation into insulin producing cells. For the last step, the hESC-derived pancreatic progenitor cells have been co-cultured with rat micro-vascular endothelial cells and human umbilical vein endothelial cells, which results in dramatic up-regulation of insulin. Comparison with parallel control groups with fibroblast co-culture or without co-culture results in significantly lower expression of insulin. Furthermore, the insulin expression elicits an increasing trend with increased endothelial cell co-culture ratio. Different co-culture configurations have been tested in an attempt to understand the mechanism of differentiation. Both transwell co-culture and addition of endothelial cell conditioned media shows high up-regulation of insulin compared to the control, although contact co-culture has by far the best performance.
These preliminary studies establish novel means of maturing ESC derived pancreatic progenitor cells to functional insulin producing cells. Current research is underway in identifying mechanistic details of the process.
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