Introduction: The endocrine β-cells, are the only cells in the body that can produce insulin, a hormone responsible for controlling glucose metabolism and loss or dysfunction of β-cells leads to diabetes. Currently available therapies require external insulin delivery or glucose-lowering drugs together with lifelong glucose monitoring. Therefore, huge efforts are put into the creation of a robust cell source for β-cells replacement therapy. Data from animal studies and the preliminary findings from human clinical trials indicate that physiological therapy for diabetes could be achieved by transplanting β-cells derived from human pluripotent stem cells (hPSCs). Despite recent progress, hPSCs cannot yet be reliably coaxed into functional β-cells in sufficient purity, number, and costs. The ideal type of cells represents an expandable population of human differentiated β-cells that maintains its functional capacity over long-term.

Methods: hPSCs cells were differentiated towards β-cells using a modified 3D protocol. Cells were treated with recombinant SPOCK2, MMP2, a JNK inhibitor, an MMP2 inhibitor, and mitogens (CHIR99021, Harmine, GW788388, WS6). Proliferation and function were assessed via immunofluorescence, flow cytometry, western blot, immunoprecipitation, zymography, GSIS assays. Transplantation of β-cells was performed under the kidney capsule of SCID-Beige mice. Transcriptomic profiling included single-cell RNA-seq with downstream bioinformatic analysis.

Results: We identified SPOCK2, an extracellular matrix (ECM) protein, as a key inhibitor of immature β-cell proliferation. The proliferating SPOCK2 KO SC-β-cells expressed higher INS, NKX6-1 and PCSK1 levels, suggesting further maturation. Importantly, we showed that SPOCK2 deficiency increases glucose-stimulated insulin secretion (GSIS) from β-cells. SPOCK2 KO SC-β-cells exhibited elevated expression and activity of MMP2, which triggered the β-integrin–FAK–c-JUN signaling pathway. Administration of recombinant MMP2 protein led to substantial short- and long-term expansion of SC-β-cells and markedly improved their glucose-stimulated insulin secretion both in vitro and in vivo with physiological competence comparable to that of human cadaveric islets.

Conclusion: These findings highlight the role of ECM as essential regulator of pancreatic β-cell proliferation, differentiation, and function. Our findings identify SPOCK2, an ECM protein, as a key modulator of fetal β-cell maturation and expansion. In summary, we uncover a molecular mechanism that governs SC-β-cell proliferation and function, pointing to  a unique signaling environment that can be leveraged to enhance the generation of fully functional β-cells for therapeutic transplantation.