(622o) Xeno-Free Differentiation of Human Pluripotent Stem Cells Toward Insulin-Expressing Pancreatic Progeny

Diabetes is a chronic disease caused by the loss of insulin-producing beta-cells due to autoimmune destruction (Type 1) or by insulin resistance and associated functional defects in beta-cells (Type 2). Human pluripotent stem cells (hPSCs) including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can serve as a renewable sources for pancreatic beta-cells. Yet, stem cell-based diabetes therapies will rely on the efficient differentiation under xeno-free conditions of hPSCs to genetically normal and functional progeny resembling pancreatic endocrine cells. In this work, we employed both hESC and hiPSC lines aiming to develop a protocol for guiding stem cells along pancreatic cell lineages under defined conditions free of components of animal origin.

 A stage-wise differentiation strategy was established to sequentially differentiate H9 hESCs and IMR90 hiPSCs into definitive endoderm, primitive gut tube, posterior foregut, and insulin-expressing pancreatic cells. Differentiation was induced in xeno-free media with growth factors involved in embryonic pancreas development. Different basal media and combinations of factors were screened and selected by standard design-of-experiments methodology based on the expression of stage-specific makers. The differentiating hPSC populations were analyzed by quantitative PCR, immunostaining and flow cytometry.

Human PSC monolayers were coaxed to definitive endoderm-like cells. Based on the choice of basal medium, a higher percentage of viable cells was obtained after 4-day differentiation with more than 60% cells co-expressing FOXA2 and SOX17. Subsequent induction led to the emergence of HNF1B⁺/HNF4A⁺ (foregut) cells after 4 days while over 30% of PDX1⁺cells appeared on day 11 of the differentiation. These cells were further induced into cells displaying insulin and c-peptide when examined on day 19.

A protocol was established in this work for the efficient generation of insulin-expressing pancreatic progeny from hPSCs under complete xeno-free conditions and without the formation of embryoid bodies. These findings are an important step toward the establishment of strategies for the generation of clinically relevant pancreatic beta-cells for diabetes therapies.