Select your timezone:

Best Abstracts Session

Monday April 24, 2023 - 14:45 to 16:25

Room: Riverfront


.7 Complete suspension culture biomanufacturing of human pluripotent stem cell-derived islets for diabetes cell therapy

Nidheesh Dadheech, Canada

Senior Research Lead
University of Alberta


Complete suspension culture biomanufacturing of human pluripotent stem cell-derived islets for diabetes cell therapy

Nidheesh Dadheech 1, Nerea Cuesta Gomez1, Mario Bermúdez de León2, Ila Tewari Jasra1, Rena Pawlick1, Braulio Marfil Garza1, Kevin Verhoeff1, Sandhya Sapkota1, Haide Razavy1, Jasmine Maghira3, Patrick MacDonald3, AM James Shapiro1.

1Department of Surgery, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada; 2Departamento de Biología Molecular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, , Mexico; 3Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada

Introduction: Advanced bioengineering in the generation of stem cell-derived islets (SC-islets) enabled clinical testing in patients. Due to planar (2D) or combined suspension (3D) culturing, optimized methods still have limitations in terms of preserving cell loss, minimizing heterogeneity (quality), and scalability (quantity). We developed a fully scalable suspension SC-islets biomanufacturing protocol using patients’ induced pluripotent stem cells (iPSC).

Methods: Combining optimal protocols, we performed modifications to generate iPSC- islets in complete suspension differentiation using a vertical-wheel® bioreactor system. Generated SC-islets were assessed for molecular (flow, histology, gene expression), electrophysiological, and functional (insulin secretion) characterization. In-vivo functional assessment for glucose control was evaluated post-transplantation in SCID-beige mice.

Results: We created a protocol for directed differentiation of patients’ iPSCs that resulted in functional, metabolically active, and transcriptionally mature SC-islets. In a single batch of small 0.1L and large 0.5L bioreactors, ~35,000 to 110,000 IEQ SC-islets are produced. Without modifying the cytoarchitecture of SC-islets, entire suspension method reliably differentiated cells and controlled dramatic cell loss (<50%) at all stages. Cell composition in SC-islets indicated a mixture of functional endocrine cells, including α cells (10% glucagon), β cells (42% insulin), γ cells (8% somatostatin). Single-cell visualization of SC-islet mass flowcytometry with viSNE and FlowSOM dimensional reductionality revealed >90% ChgA+NKx6.1+ cells, ~63% Cpep+ISL+NKX6.1+ β-like cells with fewer SLC18A1+ EC-like cells. Gene-array profiling reflected transcriptional maturity for islet-specific genes (INS, UCN3, NKX6.1, GP2, CHGB, MAFB, PAX4) and upregulated functional secretory and exocytotic genes (PCSK1/2, G6PC2, CHGB, CPE, GCK, ZnT8, TSPAN1). Dynamic insulin perifusion traces from SC-islets show a 2-fold increase in insulin secretion in response to glucose, a stimulator (exendin-4), and membrane depolarization. Fully mature S6-like cells exhibited Na+/Ca2+ current and exocytosis electrophysiological properties similar to donor islets. Finally, in diabetic mice (n=20), suspension-generated SC-islets provided physiological glucose control and functional regulation. Eight weeks post-transplantation, mice have improved glucose tolerance, graft maturity, and stimulated human C-peptide secretion. Maturation-associated genes like INS, GCG, MAFB, ZnT8, ISL1, PCSK1, ABCC8, ITGA1, and NEUROD1 were significantly upregulated in graft-harvested cells (120 days) compared to SC-islets pre-transplantation.

Conclusion: The development of a complete suspension differentiation protocol and further optimization to scale up SC-islet biomanufacturing has resulted in the large-scale generation of functional and mature SC-islets. Vertical-wheel® bioreactors provide a reliable source for clinical grade SC-islet cell production with high precision at large scale.

Juvenile Diabetes Research Foundation, Canada. Canadian Institutes of Health Research. Canadian Donation and Transplantation Research Program. Alberta Diabetes Foundation. University Health Foundation. Diabetes Research Institute Foundation Canada (DRIFCan).

Organized by

A section of

© 2024 BetaCells 2023