Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells

Abstract

Embryonic pancreatic bud cells, the earliest pancreas-committed cells, generated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into mature pancreatic β-cells in vivo, indicating the feasibility of hESC/iPSC-based cell therapy for diabetes. However, the key factors required for the differentiation of these cells into pancreatic bud cells are incompletely understood. The purpose of this study was to establish culture conditions that efficiently induce PDX1(+)NKX6.1(+) pancreatic bud cells from hESCs/iPSCs. We differentiated a hESC line, KhES-3, into pancreatic lineages with a stepwise protocol recapitulating developmental process. The induction rate of PDX1(+)NKX6.1(+) cells was correlated with cell density in adherent cultures, and markedly improved with cell aggregation cultures. The positive effects of cell aggregation cultures on the differentiation of pancreatic bud cells were reproduced in multiple hESC/iPSC lines. The human PDX1(+)NKX6.1(+) cells developed into pancreatic epithelia after implantation into immunocompromised mice. Moreover, human C-peptide secretion into mouse bloodstream was stimulated by glucose challenges after in vivo maturation. Taken together, these results suggest that cultures with high cell density are crucial for the differentiation of pancreas-committed progenitor cells from hESCs/iPSCs. Our findings may be applicable for the development of hESC/iPSC-based cell therapy for diabetes.

Development of an Alginate Array Platform to Decouple the Effect of Multiparametric Perturbations on Human Pluripotent Stem Cells During Pancreatic Differentiation

“…This suggests that formation of tight cell‐cell contacts from pre‐aggregation prior to encapsulation may be promoting differentiation. This is supported by the report by Toyoda et al showing that aggregation of human embryonic stem cells strongly enhances pancreatic bud differentiation …”

“…Lee et al showed controlling hPSC colony size could control specification to mesoderm (1200 µm in diameter) or endoderm in the presence of soluble differentiation factors, linking cellular organization to hPSC differentiation . More specific to pancreatic differentiation, Toyoda et al demonstrated enhancement of pancreatic induction of hPSCs with increasing cell density in adherent (2D) cultures, which was further improved in aggregate culture . Thus clinical translation of encapsulated hPSCs will require a thorough evaluation of optimum parameters supporting hPSC growth and differentiation …”

iPSC technology‐based regenerative therapy for diabetes

“…Scale bars, 100 μm. Adapted from Toyoda et al . with permission (licensed under Creative Commons Attribution).…”

Lacrimal gland development: From signaling interactions to regenerative medicine

“…By providing appropriate growth factors and substrates that closely mimic the relevant environment during embryonic development, IPS cells have been successfully coaxed to differentiate into numerous cell types, including those constituting glandular organs such as the liver and pancreas (Pagliuca et al, 2014; Ishikawa et al, 2015; Pellegrini et al, 2015; Toyoda et al, 2015). A similar strategy to that used for human embryonic stem cells (hESCs) (Lee et al, 2007; Dincer et al, 2013; Leung et al, 2013) can be applied to IPS cells to derive lacrimal gland tissues.…”