Single-Cell RNA-Seq Reveals that CD9 Is a Negative Marker of Glucose-Responsive Pancreatic β-like Cells Derived from Human Pluripotent Stem Cells

Abstract

To date, it remains unclear if there are specific cell-surface markers for purifying glucose-responsive pancreatic b-like cells derived from human pluripotent stem cells (hPSCs). In searching for this, we generated an efficient protocol for differentiating b-like cells from human embryonic stem cells. We performed single-cell RNA sequencing and found that CD9 is a negative cell-surface marker of b-like cells, as most INS + cells are CD9 À. We purified b-like cells for spontaneous formation of islet-like organoids against CD9, and found significantly more NKX6.1 + MAFA + C-PEPTIDE + b-like cells in the CD9 À than in the CD9 + population. CD9 À cells also demonstrate better glucose responsiveness than CD9 + cells. In humans, we observe more CD9 + C-PEPTIDE + b cells in the fetal than in the adult cadaveric islets and more Ki67 + proliferating cells among CD9 + fetal b cells. Taken together, our experiments show that CD9 is a cell-surface marker for negative enrichment of glucose-responsive b-like cells differentiated from hPSCs.

Harnessing Proliferation for the Expansion of Stem Cell-Derived Pancreatic Cells: Advantages and Limitations

“…Attempts made at expanding the pancreatic progenitor population have found that PDX1 cells generated do not necessarily express NKX6-1 and that pancreatic marker expression varied with passage (44,46). The inverse relationship between proliferation and functional maturation of b-like cells was further supported in a recent study examining the cell surface CD9 marker (52). Depleting the stem cell-derived population of CD9 + b-like cells removed the population of immature b-like cells, leaving cells that demonstrated higher expression of genes associated with b-cell maturity and insulin secretion.…”

“…The end goal of stem cell-derived b-like cell research is to develop a transplantable system of cells that fully replicate b-cell function. Ideal tactics to achieve this would be to optimize current differentiation protocols, such as through manual purification of mature populations or treatments that increase final b-like cells (10,12,41,52,60,61), and to improve in vivo site support for transplanted b-like cells (62)(63)(64). Despite the limitations listed above, expanding b-like cells or their progenitors in vitro still presents a promising strategy to further support these approaches.…”

“…A general issue that arises with the use of stem cell-derived cell sources is ensuring that proliferative capacity within end-stage β-like cells is tightly regulated to avoid uncontrolled cell growth. While cellular outgrowths have been occasionally detected following transplantation of cultures containing pancreatic progenitors ( 3 , 7 ), endocrine progenitors ( 10 ), or end-stage cultures that failed to commit to the endocrine lineages ( 52 ), no outgrowths have been reported from transplantation of end-stage populations sorted for insulin-expressing cells ( 10 ). These findings from insulin-purified cell sorts are not surprising as human embryonic stem cell (hESC)-derived endocrine cells are typically post-mitotic, and there has been evidence that expanded pancreatic progenitors do not induce outgrowth when transplanted to mice ( 44 ).…”

Towards a Functional Cure for Diabetes Using Stem Cell-Derived Beta Cells: Are We There Yet?

“…Further studies on TGF-β signaling in beta cells are needed, but, with respect to the differentiation of highly functional beta cells from stem cells, TGF-β fulfills a dual role, as its inhibition is required at early stages while its signaling is beneficial at late stages. Recently, Yoshihara et al demonstrated that non-canonical WNT4 signaling drives the metabolic maturation of beta-like cells—essential for robust insulin secretion—in large part through the induction of an ERRgamma gene network [ 63 ], while Li et al developed a beta cell differentiation protocol that is based on three previously published protocols [ 52 , 53 , 64 ] to obtain beta cells with high glucose-responsiveness and insulin production [ 65 ].…”

“…Sorting strategies while using a GFP reporter under the control of the insulin gene promoter or an antibody to the beta cell surface marker CD49a enabled obtaining 80–90% pure beta cells [ 57 , 66 ]. Alternatively, the cell-surface marker CD9 can be exploited for negative selection to enrich for glucose-responsive human beta-like cells [ 65 ]. These strategies may also prove to be useful for sorting other hormone-producing cells, such as alpha cells, which contribute to disease etiology by elevating blood glucose levels [ 67 , 68 , 69 ].…”