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In vitro quantitative and relative gene expression analysis of pancreatic transcription factors Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1 in trans-differentiated human hepatic progenitors.

ABSTRACT: AIMS/INTRODUCTION:Diabetes is a major health concern throughout the world because of its increasing prevalence in epidemic proportions. ?-Cell deterioration in the pancreas is a crucial factor for the progression of diabetes mellitus. Therefore, the restoration of ?-cell mass and its function is of vital importance for the development of effective therapeutic strategies and most accessible cell sources for the treatment of diabetes mellitus. MATERIALS AND METHODS:Human fetuses (12-20 weeks gestation age) were used to isolate human hepatic progenitor cells (hHPCs) from fetal liver using a two-step collagenase digestion method. Epithelial cell adhesion molecule-positive (EpCAM+ve)-enriched hHPCs were cultured in vitro and induced with 5-30 mmol/L concentration of glucose for 0-32 h. Pdx-1 expression and insulin secretion was analyzed using immunophenotypic and chemifluorescence assays, respectively. Relative gene expression was quantified in induced hHPCs, and compared with uninduced and pancreatic cells to identify the activated transcription factors (Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1) involved in ?-cell production. RESULTS:EpCAM+ve cells derived from human fetal liver showed high in vitro trans-differentiation potential towards the ?-cell phenotype with 23 mmol/L glucose induction after 24 h. The transcription factors showed eminent expression in induced cells. The expression level of transcription factors was found significantly high in 23 mmol/L-induced hHPCs as compared with the uninduced cells. CONCLUSIONS:The present study has shown an exciting new insight into ?-cell development from hHPCs trans-differentiation. Relative quantification of gene expression in trans-differentiated cells offers vast possibility for the production of a maximum number of functionally active pancreatic ?-cells for a future cure of diabetes.

SUBMITTER: Vishwakarma SK

PROVIDER: S-EPMC4188105 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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In vitro quantitative and relative gene expression analysis of pancreatic transcription factors Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1 in trans-differentiated human hepatic progenitors.

Vishwakarma Sandeep Kumar SK Rahamathulla Syed S Bardia Avinash A Tiwari Santosh K SK Srinivas Gunda G Raj Avinash A Tripura Chaturvedula C Sandhya Annamaneni A Habeeb Mohammed Aejaz MA Khan Aleem A AA Pande Gopal G Reddy K Pratap KP Reddy P Yugandhar PY

Journal of diabetes investigation 20140129 5

<h4>Aims/introduction</h4>Diabetes is a major health concern throughout the world because of its increasing prevalence in epidemic proportions. β-Cell deterioration in the pancreas is a crucial factor for the progression of diabetes mellitus. Therefore, the restoration of β-cell mass and its function is of vital importance for the development of effective therapeutic strategies and most accessible cell sources for the treatment of diabetes mellitus.<h4>Materials and methods</h4>Human fetuses (12 ...[more]

PMID: 25411615

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Project description:Although they are expandable in vitro, hepatic progenitors are immature cells and share many immunomarkers with hepatocellular carcinoma, raising potential concerns regarding maltransformation after transplantation. This study investigated the effects of hepatic nuclear factor (HNF) 4α on the proliferation, migration, and maltransformation of hepatic progenitors and determined the feasibility of using these manipulated cells for transplantation.The effects of HNF4α on rat hepatic progenitors (i.e. hepatic oval cells) were analyzed by HNF4α overexpression and HNF4α shRNA. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice injured by carbon chloride (CCl4) were then transplanted with control, HNF4α-overexpressing or HNF4α-suppressing hepatic oval cells. Finally, the engraftment of these cells in the recipient liver was analyzed.Rat hepatic progenitors (i.e. hepatic oval cells) expressed HNF4α, although less than that in hepatocytes. When HNF4α was overexpressed in these cells, the proliferation and migration of hepatic oval cells were reduced; but when HNF4α was suppressed by shRNA, the proliferation and migration, and even anchorage-independent growth, of these cells were accelerated. RNA microarray and gene functional analysis revealed that suppressing HNF4α not only impaired many biosynthesis and metabolism pathways of hepatocytes but also increased pathways for cancer. When transplanted into CCl4-injured NOD/SCID mice, few HNF4α-suppressing hepatic oval cells localized into the liver, while control cells and HNF4α-overexpressing cells engrafted into the liver and differentiated into albumin-positive hepatocytes. Interestingly, the hepatocytes derived from HNF4α-overexpressing cells were less migrative and expressed less c-Myc than the cells derived from control cells.HNF4α constrains proliferation, migration, and maltransformation of hepatic progenitors, and HNF4α-overexpressing hepatic progenitors serve as an optimal candidate for cell transplantation.

Dataset Information

In vitro quantitative and relative gene expression analysis of pancreatic transcription factors Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1 in trans-differentiated human hepatic progenitors.

Publications

In vitro quantitative and relative gene expression analysis of pancreatic transcription factors Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1 in trans-differentiated human hepatic progenitors.

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