Combined transfection of the three transcriptional factors, PDX-1, NeuroD1, and MafA, causes differentiation of bone marrow mesenchymal stem cells into insulin-producing cells.

AIMS: The goal of cell transcription for treatment of diabetes is to generate surrogate ß-cells from an appropriate cell line. However, the induced replacement cells have showed less physiological function in producing insulin compared with normal ß-cells. METHODS: Here, we report a procedure for induction of insulin-producing cells (IPCs) from bone marrow murine mesenchymal stem cells (BM-mMSCs). These BM-mMSCs have the potential to differentiate into insulin-producing cells when a combination of PDX-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1), and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) genes are transfected into them and expressed in these cells. RESULTS: Insulin biosynthesis and secretion were induced in mMSCs into which these three genes have been transfected and expressed. The amount of induced insulin in the mMSCs which have been transfected with the three genes together is significantly higher than in those mMSCs that were only transfected with one or two of these three genes. Transplantation of the transfected cells into mice with streptozotocin-induced diabetes results in insulin expression and the reversal of the glucose challenge. CONCLUSIONS: These findings suggest major implications for cell replacement strategies in generation of surrogate ß-cells for the treatment of diabetes.

[Altered expressions of embryonic stem-related genes in pancreatic cancer stem cell].

OBJECTIVE: To explore the altered expressions of embryonic stem-related genes Oct4 and Nanog in pancreatic cancer stem cells (CSCs). METHODS: The uni-cell suspension of human pancreatic cancer cell line PANC-1 was prepared and incubated with CD24 and CD44 antibodies. Flow cytometer was used to separate CD24(+)CD44(+) pancreatic cancer stem cells. Tumor cell spheres were observed under light microscope. Then CSCs were induced to differentiate with 10% fetal bovine serum and the expressions of CD24 and CD44 re-evaluated by flow cytometer. Finally the cells were divided into 2 groups, group 1: CD24(+)CD44(+) and group 2: non-separated group. RT-PCR (reverse transcription-polymerase chain reaction) and Q-PCR (quantitative-polymerase chain reaction) were used to examine the transcriptions of Oct4 and Nanog in CSCs. The immunofluorescence was employed to examine the expressions of Oct4 and Nanog. Chemo-sensitivity to gemcitabine was determined by CCK8 assay in each group. RESULTS: About 1%-3% CD24(+)CD44(+) CSCs were separated from cell line PANC-1. The sorted cells were cultured in a stem cell culture medium to observe the spheroid-forming capacity. And they showed a higher colony-forming efficiency than the unsorted cells [(122 ± 6)‰, P < 0.05]. When cultured in medium with serum, these cells gradually returned to the status of parental cells with a low expression of CD24 and CD44. Both Oct4 and Nanog were highly expressed in CD24(+)CD44(+) stem cells. And the CD24(+)CD44(+) subgroup demonstrated a higher resistance to gemcitabine. CONCLUSION: Subpopulation cells CD44(+)CD24(+) have the properties of tumor stem cells. The up-regulated levels of Oct4 and Nanog may be highly correlated with the multi-potency and a higher drug-resistance of pancreatic CSCs.