The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells

Abstract

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 ( IPC s). In this study, the combined effect of Pdx1 overexpression and Shh manipulation on the function of adipose tissue‐derived IPC s was determined. A eukaryotic expression vector ( Pdx1‐ pCDNA 3.1(+)) was constructed and transfected into a Chinese hamster ovary ( CHO ) cell line. Adipose tissue‐derived mesenchymal stem cells ( ADMSC s) obtained from rats were assigned to two groups [control (C) and manipulated (M)] and differentiated into IPC s. Manipulated cells were treated with a mixture of FGF ‐β and cyclopamine and recombinant Shh protein at days 3 and 11, respectively, and transfected with Pdx1‐ pCDNA 3.1(+) at day 10. The expression of multiple genes related to function of beta cells was analyzed using real‐time PCR . The functionality of IPC s in vitro was analyzed through dithizone ( DTZ ) staining and ELISA . IPC s were injected into the tail vein of diabetic rats, and blood glucose and insulin concentrations were measured. CHO cells transfected with Pdx1‐ pCDNA 3.1(+) showed a significantly higher expression of Pdx1 compared with nontransfected cells. Manipulated IPC s 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 IPC s exhibited a higher ability to normalize blood glucose and insulin secretion when compared to controls. Our protocol might be used for more efficient cell therapy of patients with diabetes in the future.

The Roles of Different Multigene Combinations of Pdx1, Ngn3, Sox9, Pax4, and Nkx2.2 in the Reprogramming of Canine ADSCs Into IPCs

“…Many studies have reported that transferring key genes that regulate the development of islet β-cells into ADSCs can induce them to differentiate into IPCs 18,29,30 . However, only part of the cascade-regulated gene expression can be found in the induced IPCs, and the ability to secrete insulin from IPCs is limited.…”

“…ADSCs contain biologically active substances that regulate blood sugar balance, such as leptin, adipocytokines, and visfatin, and are better sources of seed cells for diabetes cell transplantation therapy compared with other MSCs 27,28 . Many studies have reported that transferring key genes that regulate the development of islet β-cells into ADSCs can induce them to differentiate into IPCs 18,29,30 . However, only part of the cascade-regulated gene expression can be found in the induced IPCs, and the ability to secrete insulin from IPCs is limited.…”

“…For example, Pdx1 can activate the regulation of early embryonic pancreatic development 14 , Ngn3 can initiate the differentiation of pancreatic precursor cells into pancreatic endocrine cells 15 , Pax4 plays an important regulatory role in the production of islet progenitor cells and the differentiation of islet β-cells 16 , and Nkx2.2 is necessary for the phenotypic determination of mature β-cells 17 . A large number of studies have confirmed that transfection of Pdx1, Ngn3, Pax4, or Nkx2.2 into stem cells can induce their differentiation into IPCs [18][19][20] . A Pdx1-Ngn3 combined transfection can mediate the reprogramming of mesenchymal stromal cell differentiation into pancreatic endocrine cells in vitro 12 .…”

MafA-positive insulin-producing cells derived from rat adipose stem cells are efficient in glucose hemostasis in vitro

“…On day 14 of differentiations, the differentiated and control groups were treated with KRB buffer and high glucose concentration glucose as described previously (32). The supernatant was used for the Insulin assay.…”

Redefining the signaling pathways from pluripotency to pancreas development: In vitro β‐cell differentiation

“…The previous studies regarding transplantation of the adipose‐derived PSCs using the IPC protocols and pdx1 gene transgene to STZ‐induced diabetic rats have shown that the blood glucose concentration (92 ± 1.2 mg/ml) can be normalized within 2 weeks. Then, a period of instability in blood glucose (raised to 315 ± 1.9 mg/dl) had occurred, during the third to fifth week and at the sixth week posttransplantation, and the average glucose concentration became stable at 147 ± 1.1 mg/ml (Hashemi Tabar et al, ). This was in comparison with the diabetic rats that had received adipose‐derived PSCs in an IPC‐differentiation protocol, and in combination with transgenic mafA transfection.…”