Differentiation of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors

Abstract

BackgroundThe expression of a specific combination of transcription factors (TFs) in the multipotent progenitor cells (MPCs) is critical for determining pancreatic cell fate. NKX6.1 expression in PDX1+ MPCs is required for functional β cell generation. We have recently demonstrated the generation of a novel population of human pluripotent stem cell (hPSC)-derived MPCs that exclusively express NKX6.1, independently of PDX1 (PDX1−/NKX6.1+). Therefore, the aim of this study was to characterize this novel population to elucidate its role in pancreatic development.MethodsThe hPSCs were exposed to two differentiation protocols to generate MPCs that were analyzed using different techniques.ResultsBased on the expression of PDX1 and NKX6.1, we generated three different populations of MPCs, two of them were NKX6.1+. One of these NKX6.1 populations coexpressed PDX1 (PDX1+/NKX6.1+) which is known to mature into functional β cells, and an additional novel population did not express PDX1 (PDX1−/NKX6.1+) with an undefined role in pancreatic cell fate. This novel population was enriched using our recently established protocol, allowing their reorganization in three-dimensional (3D) structures. Since NKX6.1 induction in MPCs can direct them to endocrine and/or ductal cells in humans, we examined the coexpression of endocrine and ductal markers. We found that the expression of the pancreatic endocrine progenitor markers chromogranin A (CHGA) and neurogenin 3 (NGN3) was not detected in the NKX6.1+ 3D structures, while few structures were positive for NKX2.2, another endocrine progenitor marker, thereby shedding light on the origin of this novel population and its role in pancreatic endocrine development. Furthermore, SOX9 was highly expressed in the 3D structures, but cytokeratin 19, a main ductal marker, was not detected in these structures.ConclusionsThese data support the existence of two independent NKX6.1+ MPC populations during human pancreatic development and the novel PDX1−/NKX6.1+ population may be involved in a unique trajectory to generate β cells in humans.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0834-0) contains supplementary material, which is available to authorized users.

CHIR-99021 regulates mitochondrial remodelling via β-catenin signalling and miRNA expression during endodermal differentiation

“…Definitive endoderm is the common progenitor to epithelial cells in diverse internal organs, including the liver, pancreas and intestines (Tremblay and Zaret, 2005). To examine their multilineage differentiation potential, we cultured hEPCs in pancreatic (Aigha et al, 2018) or intestinal (Spence et al, 2011) induction medium for four days and then evaluated the cells for expression of pancreatic lineage-specific transcription factors pancreatic and duodenal homeobox1 (PDX1) (Offield et al, 1996) and caudal type homeobox 2 (CDX2), a member of the caudal-related homeobox transcription factor gene family. CDX2 is a major regulator of intestine-specific genes involved in cell growth and differentiation (Gao et al, 2009).…”

Keratinocytes Derived from Patient-Specific Induced Pluripotent Stem Cells Recapitulate the Genetic Signature of Psoriasis Disease

“…Immunostaining was performed as previously reported [22,23]. The antibody details are described and listed in Supplementary Table S1.…”

Stem Cell Therapy for Diabetes: Beta Cells versus Pancreatic Progenitors

“…Therefore, in vitro differentiation strategies employ a combination of small molecules or recombinant proteins to turn on or off these pathways at specific stages of the protocol [8,22]. Multiple groups have differentiated hPSCs to PDX1 and NKX6.1 co-expressing pancreatic progenitors in monolayer cultures [16,23], yielding even up to 90% of PDX1+/NKX6.1+ co-positive pancreatic progenitors [24,25] (Figure 3). Prior to the most recent improvements in protocols employing monolayer method, studies showed that cellular aggregation during differentiation process could improve the efficiency of pancreatic progenitors as compared to monolayer cultures [19,26].…”

“…Recently, Aigha et al has generated a novel population of pancreatic progenitor population expressing NKX6.1 in the absence of PDX1 (PDX1-/NKX6.1+) from hPSCs. The initial characterization of this population indicates the potential of PDX1-/NKX6.1+ cells to generate islet endocrine cells [25]. In vitro expanded islets undergoing de-differentiation have been shown to generate INS+ beta cells on extended culture [38,39], as well as progenitor cells with high aldehyde dehydrogenase activity extracted from the fetal and adult pancreas [40,41].…”