Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2 + Human Pancreatic Progenitors

Abstract

Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2) as a specific cell surface marker for isolating pancreatic endoderm cells (PECs) from differentiated hESCs and human fetal pancreas. Isolated GP2 PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK) inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2 PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

Nicotinamide promotes differentiation of pancreatic endocrine progenitors from human pluripotent stem cells through poly (ADP-ribose) polymerase inhibition

“…Fig. 2), which has been shown to enrich for endocrine progenitors [24], [25]. Levels of acetylated lysine were not significantly reduced in GP2+ cells compared to GP2-cells, indicating similar levels of sirtuin activity in both populations (Fig.…”

Pluripotent Stem Cell-Derived Pancreatic Progenitors and β-Like Cells for Type 1 Diabetes Treatment

“…In an effort to identify human PP populations by cell surface markers, flow cytometry was used to monitor expression kinetics of E-cadherin (ECAD), pancreatic secretory granule membrane major glycoprotein 2 (GP2), and sushi domaincontaining 2 (SUSD2) during late embryonic/early fetal period (7-12 wpc) (56). Confirming recent data from hPSC-directed differentiation, the multipotent pancreatic epithelium (defined by ECAD expression) at 7 wpc is uniformly positive for GP2 (56), a recently discovered human PPs marker (2,17). Snapshot expression profiles of tissues collected between 8 and 13 wpc indicate that GP2 expression correlates with acinar commitment, whereas loss of GP2 is associated with NGN3 expression and the emergence of the endocrine program as early as 8.4 wpc and can be tracked by upregulation of SUSD2 and more robust NGN3 expression at 8.6 wpc (56).…”

“…This suggests that lineage commitment to the acinar and endocrine program occurs over several weeks in humans and that putative hMPCs might persist postnatally. Additional studies are needed to further explore the dynamics and biological significance of these novel human PP populations described in vivo and in vitro (2,17,56).…”

“…However, the major limitation of these approaches is represented by the lack of specificity of both CD24 and CD142 to PP cells, posing potential risks associated with the isolation of non-target cell types. Recently, GP2 was described as a specific hESCderived PPs cell surface marker by transcriptomics and proteomics analysis (2,17). Importantly, hESC-derived GP2ϩ PP generate higher percentages of ␤-like cells in vitro compared with unsorted populations, suggesting that GP2 might be used to purify ␤-cell progenitors (2,17).…”

New imaging probes to track cell fate: reporter genes in stem cell research

“…[104–106]. Differentiation protocols have been developed to generate beta cells from stem or progenitor cells [107,108]. The resulting cells may only be a first approximation, however, since recently emerging evidence suggests that beta cells are not homogenous but consist of heterogeneous subpopulations exhibiting morphological, functional and gene expression differences [109].…”