Inhibition of gamma-secretase activity promotes differentiation of embryonic pancreatic precursor cells into functional islet-like clusters in poly(ethylene glycol) hydrogel culture.
We assessed the ability of a gamma-secretase inhibitor to promote the in vitro differentiation of induced embryonic pancreatic precursor cell aggregates into functional islet-like clusters when encapsulated within a three-dimensional hydrogel. Undifferentiated pancreatic precursor cells were isolated from E.15 rat embryos, dissociated into single cells, and aggregated in suspension-rotation culture. Aggregates were photoencapsulated into poly(ethylene glycol) hydrogels with entrapped collagen type 1 and cultured for 14 days with or without a gamma-secretase inhibitor. Gene expression, proinsulin content, and C-peptide release were measured to determine differentiation and maturation of encapsulated precursor cell aggregates. In the control medium, scattered breakthrough beta cell differentiation was observed; however, cells remained largely insulin negative. Upon addition of a gamma-secretase inhibitor the majority of cells in clusters became insulin positive, and insulin per DNA and glucose-stimulated insulin release measurements for these cultures were comparable with those for adult rat islets. Cluster counts after culture day 14 were 88% of those initially encapsulated, demonstrating excellent cluster survival in hydrogel culture. These results indicate that concerted differentiation of pancreatic precursor cell aggregates into functionally mature islet-like clusters can be achieved in poly(ethylene glycol)-based hydrogel cultures by blocking cell contact-mediated Notch signaling with a gamma-secretase inhibitor.
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, USA.
Mariah N
MN
Mason
Melissa J
MJ
Mahoney
D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049108:Cell Enlargement; D002470:Cell Survival; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000201:enzymology*; D005843:Geography; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology*; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering / Q000379:methods*
D011092:Polyethylene Glycols; D053829:Amyloid Precursor Protein Secretases