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Differentiation of Human Liver-Derived, Insulin-Producing Cells Toward the ß-Cell Phenotype

From the Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel

ß-Cell transplantation is viewed as a cure for type 1 diabetes; however, it is limited by the number of pancreas donors. Human stem cells offer the promise of an abundant source of insulin-producing cells, given the existence of methods for manipulating their differentiation. We have previously demonstrated that the expression of the ß-cell transcription factor pancreatic duodenal homeobox 1 (PDX-1) in human fetal liver cells activates multiple aspects of the ß-cell phenotype. These cells, termed FH-B-TPN cells, produce insulin, release insulin in response to physiological glucose levels, and replace ß-cell function in diabetic immunodeficient mice. However, they deviate from the normal ß-cell phenotype by the lack of expression of a number of ß-cell genes, the expression of non–ß-cell genes, and a lower insulin content. Here we aimed to promote differentiation of FH-B-TPN cells toward the ß-cell phenotype using soluble factors. Cells cultured with activin A in serum-free medium upregulated expression of NeuroD and Nkx2.2 and downregulated paired box homeotic gene 6 (PAX-6). Glucokinase and prohormone convertase 1/3 were also upregulated, whereas pancreatic polypeptide and glucagon as well as liver markers were downregulated. Insulin content was increased by up to 33-fold, to 60% of the insulin content of normal ß-cells. The cells were shown to contain human C-peptide and release insulin in response to physiological glucose levels. Cell transplantation into immunodeficient diabetic mice resulted in the restoration of stable euglycemia. The cells continued to express insulin in vivo, and no cell replication was detected. Thus, the manipulation of culture conditions induced a significant and stable differentiation of FH-B-TPN cells toward the ß-cell phenotype, making them excellent candidates for ß-cell replacement in type 1 diabetes.

Abbreviations: 1-AT, 1-antitrypsin; Act-A, activin A; BrdU, 5-bromo-2-deoxyuridine; ELISA, enzyme-linked immunosorbent assay; HGF, hepatocyte growth factor; hTERT, catalytic subunit of human telomerase; KRB, Krebs-Ringer buffer; PAX-6, paired box homeotic gene 6; PC, prohormone convertase; PDX-1, pancreatic duodenal homeobox-1; PP, pancreatic polypeptide; RIA, radioimmunoassay; SFM, serum-free medium; STZ, streptozotocin

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