Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions
Luka Jarc,
Manuj Bandral,
Elisa Zanfrini,
Mathias Lesche,
Vida Kufrin,
Raquel Sendra,
Daniela Pezzolla,
Ioannis Giannios,
Shahryar Khattak,
Katrin Neumann,
Barbara Ludwig,
Anthony Gavalas
Affiliations
Luka Jarc
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; German Centre for Diabetes Research (DZD), Munich, Germany
Manuj Bandral
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; German Centre for Diabetes Research (DZD), Munich, Germany
Elisa Zanfrini
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; German Centre for Diabetes Research (DZD), Munich, Germany
Mathias Lesche
Dresden Concept Genome Centre (DcGC), TU Dresden, Dresden, Germany; Center for Molecular and Cellular Bioengineering (CMCB) Technology Platform, TU Dresden, Dresden, Germany
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany
Daniela Pezzolla
German Centre for Diabetes Research (DZD), Munich, Germany; Center for Regenerative Therapies Dresden (CRTD), Faculty of Medicine, TU Dresden, Dresden, Germany
Ioannis Giannios
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; German Centre for Diabetes Research (DZD), Munich, Germany
Shahryar Khattak
Stem Cell Engineering Facility, (SCEF), CRTD, Faculty of Medicine, TU Dresden, Dresden, Germany
Katrin Neumann
Stem Cell Engineering Facility, (SCEF), CRTD, Faculty of Medicine, TU Dresden, Dresden, Germany
Barbara Ludwig
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; German Centre for Diabetes Research (DZD), Munich, Germany; Center for Regenerative Therapies Dresden (CRTD), Faculty of Medicine, TU Dresden, Dresden, Germany; Department of Medicine III, University Hospital Carl Gustav Carus and Faculty of Medicine, TU Dresden, Dresden, Germany
Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; German Centre for Diabetes Research (DZD), Munich, Germany
The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell-derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and identified conditions for the robust and unlimited expansion of hPS cell-derived PP cells under GMP-compliant conditions through a hypothesis-driven iterative approach. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling, and inhibition of selected branches of the TGFβ and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over 10 passages and 40–45 d, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cell lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant β-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell-derived PP cells. They will enable the establishment of large banks of GMP-produced PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine, and cell therapies.
