In Vitro Insulin Production and Analysis of Pancreatic Transcription Factors in Induced Human Hepatic Progenitor Cells Aleem A. Khan, Ph.D., 1 Alaganuru Rajendraprasad, M.Sc., 1 N. Parveen, Ph.D., 1 Mahaboob V. Shaik, M.Sc., 1 Santosh K. Tiwari, Ph.D., 1 G. Srinivas, M.Sc., 2 T. Avinash Raj, M.Sc., 2 M.A. Habeeb, M.D., D.M., 1 G. Pande, Ph.D., 2 and C.M. Habibullah, M.D., D.M. 1 Abstract Background: b-Cell destruction and=or insufficient insulin production are the hallmarks of diabetes mellitus (type 1 diabetes). A hepatic progenitor from developing liver is sought to be one of the surrogate sources of insulin production as the pancreas and the liver share a common precursor and signals from the cardiac mesoderm. Production of insulin is possible by transfecting pancreatic transcription factors that play important roles in development of the pancreatic b-cell. But, there is always the fear of using genetically manipulated cells for therapeutics. Hence, the present study was designed to analyze the feasibility of using primary human fetal hepatic progenitors as a potential source for insulin production. Methods: Human fetal hepatic progenitors were enriched using CD-326 magnetic cell sorting. The sorted cells were cultured with different concentrations of glucose (5–30 mM) in Dulbecco’s modified Eagle’s medium. The amount of insulin production was estimated in the cultured cells by the chemiluminescence method. Total RNA isolated from sorted epithelial cell adhesion molecule (EpCAM)-positive cells was reverse-transcribed, and the expression of different b-cell-producing transcriptions factors was analyzed by polymerase chain reaction (PCR). Immunocytochemical analysis was performed in cultured cells using specific insulin antibodies. Results: The viability of the total liver cells isolated was found to be 95%. The average number of EpCAM- positive cells in the total liver was found to be *15%. An insulin kinetics study using glucose induction with different concentrations showed increased insulin secretion in response to glucose concentrations up to 20 mM. Furthermore, results of immunocytochemical analysis demonstrated intense insulin expression in EpCAM- positive cultured cells. Expression studies of the cultured EpCAM-positive cells using reverse transcription-PCR showed positive expression of the pancreatic transcription factors essential for insulin production. Conclusions: The present study demonstrates that in vitro differentiation of induced human hepatic progenitors into insulin-producing cells without genetic manipulations may promote strategies for the treatment of type 1 diabetes. Introduction b-Cell destruction or insufficient insulin production are the hallmarks of diabetes mellitus (type 1 diabetes). The up- surge in the prevalence of diabetes mellitus has led to the consideration of diabetes as an epidemic of the 21 st century affecting over 246 million people worldwide and is expected to affect 380 million by 2025. 1 Therapeutic strategies available for treating type 1 diabetes include but are not limited to daily insulin injections combined with meticulous blood glucose monitoring and diet adaptations. The continued failures in the gene therapy initiative to treat type 1 diabetes paved the way for cell-based therapy using isolated islets of Lang- erhans. 2 However, owing to significant limitations such as requirement for lifelong immunosuppression, shortage of donated organs, operative risk, post-transplant rejection, cost-intensive procedure, and limited survival time of trans- planted cells, it is still not considered a surrogate for insulin replacement therapy. 3 These results therefore warrant the need for the development of new alterative therapeutic strategies that can overcome limitations of b-cell transplan- tation. 1 Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences; and 2 Center for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India. DIABETES TECHNOLOGY & THERAPEUTICS Volume 12, Number 5, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=dia.2009.0083 373