Production Of Induced Pluripotent Stem Cells (iPSCs) For The Generation Of Insulin-Producing β Cells

The goal of this interventional study is to investigate how induced pluripotent stem cells (iPSCs) can be efficiently generated from somatic cells and subsequently differentiated into insulin-producing β cells, with the broader aim of improving the understanding of pancreatic β cell biology and supporting the development of future cell-based therapies for diabetes. The study involves the collection of biological samples from both healthy donors and patients affected by metabolic or genetic disorders that impact pancreatic β cell function, such as type 1 diabetes, type 2 diabetes, Maturity Onset Diabetes of the Young (MODY), Wolfram syndrome, and pancreatogenic diabetes.

The primary objective is to establish a reliable and reproducible protocol for the production of iPSCs from somatic cells obtained from different donor groups and to evaluate their potential to differentiate into functional β cells. By comparing iPSC-derived β cells from patients with those derived from healthy controls, the study aims to identify disease-specific cellular and molecular characteristics that may contribute to β cell dysfunction and diabetes pathogenesis. These findings will serve as a foundation for improving strategies for β cell replacement and regenerative medicine.

Participants enrolled in the study will provide a single biological sample, collected at Ospedale San Raffaele in Milan. Depending on the laboratory's technical requirements and the participant's clinical condition, the sample may consist of a 3 mm skin punch biopsy, a small volume of peripheral blood (up to 20 mL), or a urine sample (up to 300 mL). These procedures are all standard clinical techniques performed under sterile conditions by trained healthcare personnel. The risks associated with the procedures are minimal and limited to mild pain, bruising, or, in the case of skin biopsy, a small scar. Participants will receive post-procedure care instructions and will not be required to attend follow-up visits after the sample collection.

Once collected, the somatic cells (such as fibroblasts, peripheral blood mononuclear cells, or urine-derived epithelial cells) will be isolated, expanded, and cryopreserved. These cells will then be reprogrammed into iPSCs using a non-integrating RNA-based reprogramming system (StemRNA™ 3rd Gen Reprogramming Kit, Reprocell). The success of the reprogramming process will be assessed by measuring cell viability and the expression of specific pluripotency markers, including SSEA4, OCT4, and NANOG, through flow cytometry. Only iPSC lines that meet predefined quality criteria-viability above 60% and pluripotency marker expression above 80%-will be considered successful and preserved for further use.

Subsequently, the established iPSC lines will be differentiated into insulin-producing β cells through a stepwise process that mimics pancreatic development. The resulting β-like cells will be analyzed for their ability to produce and secrete insulin in response to glucose stimulation, as well as for other molecular and functional properties. The comparison between β cells derived from healthy donors and those from patients with different forms of diabetes will help elucidate the mechanisms underlying β cell dysfunction and loss in these diseases.