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-1 Human induced-pluripotent stem ells (hiPSC) enveloped in elastin-like recombinamer microcapsules for the cell therapy of type 1 diabetes mellitus (T1D) preliminary data

Riccardo Calafiore MD, Italy

Diabetes Research Foundation
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Human induced-pluripotent stem ells (hiPSC) enveloped in elastin-like recombinamer microcapsules for the cell therapy of type 1 diabetes mellitus (T1D) preliminary data

Giuseppe P Basta MD1, Pia Montanucci PhD1, Teresa Pescara PhD1, Alessia Greco PhD1, Riccardo Calafiore MD2.

1Medicine and Surgery, University of Perugia, Perugia, Italy; 2Research, Diabetes Research Foundation, Perugia, Italy

Introduction: Autoimmune destruction of 70-100% of pancreatic islet beta-cells in T1D may warrant attempts for their replacement for the disease's cure. Human induced pluripotent stem cells (hIPSC), derived by reprogramming somatic cells into a pluripotent state, by over-expression of a key set of transcriptional factors, could be considered for beta-cell surrogates for replacement purposes. In order to prevent hiPSC graft-directed immunity, cell coating with  new polymer technologies consisting of human elastin-based recombinamers (ELRs) might be used (1).

Materials and Methods: Thawed hiPSC were cultured on dishes coated with Vitronectin XF™, and NutriStem XFTM. We then started hiPSC differentiation into beta cell-like spheroids according to Millman protocol. For coating purpose, spheroids were sequentially dipped in ELRs, starting with RGD-N3 and VKV-cyclo, to a total of 6 bilayers. The product was assessed by analysis of preparation viability, immunocytochemistry, transmission electron microscopy, insulin output/content, qPCR and cytofluorimetry. The spheroids  were grafted intraperitoneally in NOD/SCID as well as CD-1 mice.

Results: Immunofluorescence for pluripotency markers confirmed the presence of Oct4, Sox2, Nanog, c-Myc and Ki67 on all cells of each cluster and cytofluorimetry confirmed that over 90% of cells were triple positive for Sox2, Oct4 and Nanog. qPCR for the various differentiation factors (PDX-1, NKX6.1, SOX9, PTF1a, FOXA2, SOX17, CXCR4, c-KIT, Glut and Glucokinase), and those expressed by beta-cells (MafA, MAfB, NKX2.2 and NKX6.1) showed modulation of most of them at day 27 of maturation. Static incubation with glucose confirmed that the majority of batches, of correctly differentiated hiPSCs, were able to respond physiologically to glucose concentration shifts. The coated (6 layers RDG-N3/VKV-Cyclo), differentiated cell spheroids showed no differences in terms of morphology, viability (always greater than 95%), or hormones content or messengers, or functional performance, as compared to controls. The spheroids were transplanted intraperitoneally into immune-incompetent NOD/SCID mice to determine their ability to survive in vivo: we observed slight differences in terms of material survival, although the uncoated, unlike coated spheroids, showed areas of dead cells and lower morphologic integrity. We also grafted ELR-microencapsulated cell spheroids into immunocompetent xenogeneic-discordant CD1 mice: Histological examination, at 7,14 and 42 d of TX, showed preservation of cell morphology and viability while the amount of produced insulin resulted either increased or unchanged. On the contrary, uncoated spheroids provoked lymphocytic and macrophagic reaction to the transplanted tissue.

Conclusion: We have provided initial evidence that hiPSC might serve for beta-cell surrogates. ELRs might constitute a new generation of immunoprotective conformal microcapsules, the long-term performance in pre-clinical animal models pending.

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