Abstract
Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 104 cells/cm2 to 5.3 x 104 cells/cm2 followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1, PTF1a, NGN3, ARX, and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression, followed by pancreatic endocrine specification marker expression (BRN4, PAX4, ARX, NEUROD1, NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols.
Highlights
Human islet transplantation is a potential cure for type 1 diabetes, limited cadaveric islet availability precludes widespread clinical application [1,2]
Definitive Endoderm Induction To examine the effect of initial cell seeding density on subsequent differentiation to definitive endoderm and later pancreatic endocrine hormone-producing cells, we applied an established culture protocol (Figure 1A) known to yield polyhormonal pancreatic endocrine cells [18] from the CA1S human embryonic stem cells (hESCs) line
In this study we examined the effect of modifying initial cell seeding density at the start of pancreatic endocrine differentiation of hESCs
Summary
Human islet transplantation is a potential cure for type 1 diabetes, limited cadaveric islet availability precludes widespread clinical application [1,2]. Culture conditions have been designed to mimic developmental signalling pathways reported to induce progenitor cell development in various model organisms. Using this approach, the approximately three stage framework for forming pancreatic endocrine competent progenitor cells from hESCs has become TGF-beta signalling (Activin A) dependent induction of definitive endoderm [19,20], FGF7 or FGF10 enhanced patterning to endodermal gut tube [5,6], and retinoic acid dependent induction of PDX1 [5,21,22] with concurrent BMP and sonic hedgehog inhibition [5,14,15,21]. A considerable range of signalling molecules has been applied to coax endocrine cell development from endocrine-competent progenitors; these include exendin-4, IGF1, HGF, noggin, bFGF, BMP4, VEGF, WNT and various inhibitors of sonic hedgehog, TGF-beta, and NOTCH signalling pathways [5,14,23]
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