429641 Xeno-Free Differentiation of Human Pluripotent Stem Cells Toward Insulin-Expressing Pancreatic Progeny

Wednesday, November 11, 2015
Exhibit Hall 1 (Salt Palace Convention Center)
Yongjia Fan1, Fan Zhang1 and Emmanuel S. Tzanakakis1,2, (1)Chemical and Biological Engineering, Tufts University, Medford, MA, (2)Tufts Clinical and Translational Science Institute, Tufts Medical Center, Boston, MA

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.

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