Potential for Pancreatic Maturation of Differentiating Human Embryonic Stem Cells Is Sensitive to the Specific Pathway of Definitive Endoderm Commitment

Abstract

This study provides a detailed experimental and mathematical analysis of the impact of the initial pathway of definitive endoderm (DE) induction on later stages of pancreatic maturation. Human embryonic stem cells (hESCs) were induced to insulin-producing cells following a directed-differentiation approach. DE was induced following four alternative pathway modulations. DE derivatives obtained from these alternate pathways were subjected to pancreatic progenitor (PP) induction and maturation and analyzed at each stage. Results indicate that late stage maturation is influenced by the initial pathway of DE commitment. Detailed quantitative analysis revealed WNT3A and FGF2 induced DE cells showed highest expression of insulin, are closely aligned in gene expression patterning and have a closer resemblance to pancreatic organogenesis. Conversely, BMP4 at DE induction gave most divergent differentiation dynamics with lowest insulin upregulation, but highest glucagon upregulation. Additionally, we have concluded that early analysis of PP markers is indicative of its potential for pancreatic maturation.

Capsule stiffness regulates the efficiency of pancreatic differentiation of human embryonic stem cells

“…The stage-wise induction protocol for the pancreatic differentiation of hESCs was identical to our previous study, ending at the pancreatic progenitor stage instead of the maturation stage [4].…”

“…Alternately, β cells can be derived from human pluripotent stem cells (hPSCs), which have the capacity to self-renew and differentiate, making them attractive for regenerative medicine and cell therapy applications. A significant amount of work has focused on deriving β -like cells from pluripotent stem cells, including work done in our own lab [3][4][5][6]. Recently, the Melton group and the Kieffer group have reported a significant breakthrough in generating glucose responsive insulin producing β cells from hPSCs in vitro [3,7].…”

Network Analysis Identifies Crosstalk Interactions Governing TGF-β Signaling Dynamics during Endoderm Differentiation of Human Embryonic Stem Cells

“…The differentiation media were made using DMEM/F12 (Life Technologies, Grand Island, NE, USA), supplemented with 0.2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 1xB27 (Life Technologies, Grand Island, NE, USA). The induction protocol for endoderm was adopted from our previous study [2,9].…”

“…In differentiating hESCs to lineages of pancreas, liver, etc., an important first step is the induction of Definitive Endoderm (called endoderm henceforth) [1]. The quality of later stage maturation is dictated by the initial pathway of endoderm differentiation [2]. Extensive research over a decade have given rise to alternate protocols for endoderm induction of hESCs that employ unique combinations of growth factors and small molecules [3][4][5][6][7].…”

“…Among these commonly used supplementary constituents, inhibition of PI3K pathway has been consistently reported to enhance endoderm differentiation of hESCs in conjunction with Activin induction [9]. The resulting endoderm cells show good potential to transform into pancreatic β-like cells with continued differentiation [2,10].…”