In vitro insulin production and analysis of pancreatic transcription factors in induced human hepatic progenitor cells.
BACKGROUND
beta-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 beta-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 beta-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 approximately 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.
Khan
Aleem A
AA
Centre for Liver Research and Diagnostics , Deccan College of Medical Sciences, India.
Rajendraprasad
Alaganuru
A
Parveen
N
N
Shaik
Mahaboob V
MV
Tiwari
Santosh K
SK
Srinivas
G
G
Raj
T Avinash
TA
Habeeb
M A
MA
Pande
G
G
Habibullah
C M
CM
D002478:Cells, Cultured; D015870:Gene Expression; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D010179:Pancreas / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D014157:Transcription Factors