Initial Cell Seeding Density Influences Pancreatic Endocrine Development During in vitro Differentiation of Human Embryonic Stem Cells

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.

Overexpression of PAX4 reduces glucagon expression in differentiating hESCs

“…4 Based on this timeline, and one of the presumed PAX4 targets being ARX, we performed infections of developing hESC cultures at day 11 with either control virus expressing enhanced green fluorescent protein (Ad eGFP) or PAX4 virus (Ad PAX4) at a multiplicity of infection (MOI) of 6 or 60 based on a day 11 cell density of 4.3 x 10 5 cells/cm. 2,4 By day 12, eGFP-positive cells were observed in the control group, and eGFP expression was maintained until day 21 (Fig. 1B).…”

“…2 The majority of in vitro hESC-derived pancreatic endocrine cells express multiple hormones within the same cell. [3][4][5][6][7][8] While these polyhormonal cells are a natural component of human development, [9][10][11] the eventual bias of these cells, which most notably co-express glucagon and insulin, is to an α-cell fate. 3,[12][13][14] Therefore, new methods that shift the polyhormonal nature of cells away from a glucagon-positive lineage could significantly improve the generation of functional β-cells in vitro.…”

Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation

“…Various protocols have been developed to generate DE in vitro by recapitulating in vivo embryogenesis, but their efficiency varies widely [6][7][8][9][10][11]. It has been reported that the initiating DE density is influential for the final differentiation yield [12]. In view of the variability in DE production outcomes, validation and improvement of these protocols are needed.…”

Advances and challenges in the differentiation of pluripotent stem cells into pancreatic β cells

“…It has been previously shown that the capacity of PSCs to differentiate into pancreatic endocrine cells is highly affected by the seeding density of initial cultures. For example, Gage et al [47] demonstrated that high density hESCs led to remarkable increase in that rate of cell differentiation into PDX1-and NGN3-positive cells as compared to low density cultured cells. In contrast to PDX1, NGN3 expression is stimulated by a reduction in NOTCH signaling.…”