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Controlling human stem cell-derived islet composition using magnetic sorting

ABSTRACT: Stem cell-derived islets (SC-islets) hold significant promise for treating diabetes, but optimizing their functional maturity remains a challenge. CD49a, an integrin subunit involved in cell adhesion and signaling, has been identified as a potential marker associated with islet cell differentiation. In this study, we employed magnetic-activated cell sorting (MACS) to isolate CD49a-enriched and CD49a-depleted populations from SC-islets. These two distinct populations were evaluated for functional and molecular differences using single-cell RNA sequencing (scRNA-seq), in vitro glucose-stimulated insulin secretion (GSIS), and in vivo transplantation. Our scRNA-seq analysis revealed distinct transcriptional profiles between the CD49a-enriched and CD49a-depleted populations, with the CD49a-enriched population exhibiting higher expression of key β cell lineage markers, including insulin. Functional assays demonstrated that CD49a-enriched SC-islets had significantly improved GSIS, indicative of enhanced β cell functionality. Additionally, when transplanted into mice, the CD49a-enriched SC-islets exhibited superior graft performance, as evidenced by human C-peptide secretion. These findings support that CD49a enrichment through MACS represents a promising approach to enhance the functional maturity of SC-islets. Further investigations into the role of CD49a in islet cell differentiation and function may facilitate the development of more effective stem cell-based therapies for diabetes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE278800 | GEO | 2025/01/28

REPOSITORIES: GEO

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Project description:Objective: Transcriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Our prior work highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult β-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in β-cells and primary islets (mouse and human) to impact β-cell target genes MafA and Glp1R. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 would have critical roles in pancreatic islet cell development and function in vivo, similar to the Isl1::Ldb1 complex. Methods: We first generated a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse to constitutive Cre lines (Pdx1- and Pax6-driven) to recombine of SSBP3 in the developing pancreas and islet (SSBP3DeltaPanc and SSBP3DeltaIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of β-cell identity and function. Using an inducible Cre system we also deleted SSBP3 in the adult β-cell, a model termed SSBP3Deltaβ-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Deltaβ-cell we conducted RNA-Seq and compared our results to published datasets for similar β-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. Results: SSBP3DeltaPanc and SSBP3DeltaIslet neonates present with hyperglycemia. SSBP3DeltaIslet mice are glucose intolerant by P21 and exhibit a reduction of β-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of β-cell SSBP3 in SSBP3Deltaβ-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week SSBP3Deltaβ-cell islets revealed a decrease in β-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult β-cells. Conclusions: SSBP3 drives proper islet development and identity, where its loss causes altered islet-cell abundance and glucose regulatory function. β-cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.

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Controlling human stem cell-derived islet composition using magnetic sorting

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