Impact of co-culture on pancreatic differentiation of embryonic stem cells.
Promise of cellular therapy for type 1 diabetes has inspired the search for transplantable cell sources, and embryonic stem cells (ESCs) have emerged as strong candidates. We have developed a directed differentiation protocol to obtain insulin-producing cells from ESCs. The ESCs are first induced towards a homogeneous monolayer of definitive endoderm-like cells by co-culture with primary hepatocytes. Pancreatic commitment is induced by plating the ESC-derived endoderms on Matrigel, along with Sonic hedgehog inhibition and retinoid induction. More than 70% of differentiated cells positively upregulated Pdx-1, along with pro-endocrine transcription factors Ngn3, β2/neroD1, Nkx2.2 and Nkx6.1. Final maturation to islet-specific cells is achieved by co-culturing the ESC-derived pancreatic endocrine cells with endothelial cells, which resulted in Insulin 1 upregulation in 60% of the cell population, along with high levels of IAPP and Glut2. The differentiated cell population also secreted high levels of insulin. Our findings illustrate the significant effect of co-culture in different stages of differentiation and maturation of ESCs in vitro. Such a high yield of pancreatic islet cells has not yet been reported. Our findings establish a robust protocol for islet differentiation.
Banerjee
Ipsita
I
Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA. ipb1@pitt.edu
Sharma
Nripen
N
Yarmush
Martin
M
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D019070:Cell Lineage / Q000187:drug effects; D018920:Coculture Techniques / Q000379:methods*; D003094:Collagen / Q000494:pharmacology; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004707:Endoderm / Q000166:cytology / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007797:Laminin / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects; D051379:Mice; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects; D011509:Proteoglycans / Q000494:pharmacology; D051381:Rats; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D014704:Veratrum Alkaloids / Q000494:pharmacology
D004338:Drug Combinations; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; D007797:Laminin; C000715627:Nkx2-2 protein, mouse; C000715628:Nkx2-2 protein, rat; D011509:Proteoglycans; D015534:Trans-Activators; D014704:Veratrum Alkaloids; C084010:pancreatic and duodenal homeobox 1 protein; C059485:matrigel; D014212:Tretinoin; D003094:Collagen; C000541:cyclopamine