The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin-producing cells derived from adipose-tissue stem cells
The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin-producing cells derived from adipose-tissue stem cells
Dian Dayer,1,*Mohammad Reza Tabandeh,2Mahmud Hashemi Tabar,3Eskandar Moghimipoor,4
1. Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 3. Department of Anatomy, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 4. Department of Pharmaceutics, Faculty of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Introduction: Pancreatic and duodenal homeobox 1 (Pdx1) and Sonic hedgehog (Shh) are
the key regulators of beta-cell function. In vitro experiments have shown that
there is significant cooperation between Pdx1 and Shh with regard to the production
and maintenance of insulin-producing cells (IPCs). In this study, the
combined effect of Pdx1 overexpression and Shh manipulation on the function
of adipose tissue-derived IPCs was determined.
Methods: A eukaryotic expression
vector (Pdx1-pCDNA3.1(+)) was constructed and transfected into a Chinese
hamster ovary (CHO) cell line. Adipose tissue-derived mesenchymal stem
cells (ADMSCs) obtained from rats were assigned to two groups [control (C)
and manipulated (M)] and differentiated into IPCs. Manipulated cells were
treated with a mixture of FGF-b and cyclopamine and recombinant Shh protein
at days 3 and 11, respectively, and transfected with Pdx1-pCDNA3.1(+)
at day 10. The expression of multiple genes related to function of beta cells
was analyzed using real-time PCR. The functionality of IPCs in vitro was
analyzed through dithizone (DTZ) staining and ELISA. IPCs were injected
into the tail vein of diabetic rats, and blood glucose and insulin concentrations
were measured.
Results: CHO cells transfected with Pdx1-pCDNA3.1(+) showed
a significantly higher expression of Pdx1 compared with nontransfected cells.
Manipulated IPCs exhibited a significantly higher expression of MafA,
Nkx2.2, Nkx6.1, Ngn3, insulin, and Isl1 and a higher insulin secretion in
response to glucose challenge in relation to control cells. Rats that received
manipulated IPCs exhibited a higher ability to normalize blood glucose and
insulin secretion when compared to controls.
Conclusion: Our protocol might be used for
more efficient cell therapy of patients with diabetes in the future.