PDX1⁻/NKX6.1⁺ progenitors derived from human pluripotent stem cells as a novel source of insulin‐secreting cells

Abstract

Aim Beta cell replacement strategies are a promising alternative for diabetes treatment. Human pluripotent stem cells (hPSCs) serve as a scalable source for producing insulin‐secreting cells for transplantation therapy. We recently generated novel hPSC‐derived pancreatic progenitors, expressing high levels of the transcription factor NKX6.1, in the absence of PDX1 (PDX1−/NKX6.1+). Herein, our aim was to characterize this novel population and assess its ability to differentiate into insulin‐secreting beta cells in vitro. Materials and Methods Three different hPSC lines were differentiated into PDX1−/NKX6.1+ progenitors, which were further differentiated into insulin‐secreting cells using two different protocols. The progenitors and beta cells were extensively characterized. Transcriptome analysis was performed at different stages and compared with the profiles of various pancreatic counterparts. Results PDX1−/NKX6.1+ progenitors expressed high levels of nestin, a key marker of pancreatic islet‐derived progenitors, in the absence of E‐cadherin, similar to pancreatic mesenchymal stem cells. At progenitor stage, comparison of the two populations showed downregulation of pancreatic epithelial genes and upregulation of neuronal development genes in PDX1−/NKX6.1+ cells in comparison to the PDX1+/NKX6.1+ cells. Interestingly, on further differentiation, PDX1−/NKX6.1+ cells generated mono‐hormonal insulin+ cells and activated pancreatic key genes, such as PDX1. The transcriptome profile of PDX1−/NKX6.1+‐derived beta (3D‐beta) was closely similar to those of human pancreatic islets and purified hPSC‐derived beta cells. Also, the 3D‐beta cells secreted C‐peptide in response to increased glucose concentrations indicating their functionality. Conclusion These findings provide a novel source of insulin‐secreting cells that can be used for beta cell therapy for diabetes.

Stepwise differentiation of functional pancreatic β cells from human pluripotent stem cells

“…With combinations of signaling pathway regulators for later step, eventually resulting in the generation of β cells with high efficiency (Liu et al, 2021 ). Intriguingly, a novel population of pancreatic progenitors expressing NKX6-1 but not PDX1 has been identified during in vitro pancreatic differentiation from human PSCs (Memon et al, 2018 ; Aigha et al, 2018 ), which can further differentiate into glucose-responsive INS-positive β cells in vitro (Memon et al, 2021 ). These PDX1-negative/NKX6-1-positive cells are non-epithelial with high expression of NESTIN but lacking the pancreatic epithelial marker CDH1, and similar to progenitor cells resident in the pancreatic mesenchyme, indicating that there is an alternative β cell determination route distinct from that of the PDX1 and NKX6-1 co-expressing progenitors during development (Memon et al, 2021 ).…”

A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors

“…centage of PDX1 + and PDX1 + /NKX 6.1 + target cells in a confluent monolayer and minimized commitment to alternate lineages (Figure 4). [46] This strategy provides important insights into the transcriptional programs that tightly regulate this process. This further suggests that HA induces synchronous EMT-MET, thereby homogenizing the cell response to differentiation factors supplemented in the differentiation medium and consequently increasing the reproducibility of the culture.…”

Loss of FOXA2 induces ER stress and hepatic steatosis and alters developmental gene expression in human iPSC-derived hepatocytes

“…Immunocytochemistry was performed as we previously reported [ 44 , 46 ]. Cells were washed with DPBS (ThermoFisher Scientific) and fixed with 4% PFA (Sigma–Aldrich) at room temperature for 20 min, followed by washes with tris-buffered saline containing 0.5% Tween-20 (TBST) (Sigma–Aldrich) twice and permeabilized using phosphate buffered saline with 0.5% Triton (PBST) (Sigma–Aldrich) at room temperature for 20 mins.…”