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Abstract
Background
Despite a recent breakthough in human islet transplantation for treating type 1 diabetes mellitus, the limited availability of donor pancreases remains a major obstacle. Endocrine cells within the gut epithelium (enteroendocrine cells) and pancreatic β cells share similar pathways of differentiation during embryonic development. In particular, K-cells that secrete glucose-dependent insulinotropic polypeptide (GIP) have been shown to express many of the key proteins found in β cells. Therefore, we hypothesize that K-cells can be transdifferentiated into β cells because both cells have remarkable similarities in their embryonic development and cellular phenotypes.
Methods
K-cells were purified from heterogeneous STC-1 cells originating from an endocrine tumor of a mouse intestine. In addition, a K-cell subclone expressing stable Nkx6.1, called "Kn4-cells," was successfully obtained. In vitro differentiation of K-cells or Kn4-cells into β cells was completed after exendin-4 treatment and serum deprivation. The expressions of insulin mRNA and protein were examined by RT-PCR and immunocytochemistry. The interacellular insulin content was also measured.
Results
K-cells were found to express glucokinase and GIP as assessed by RT-PCR and Western blot analysis. RT-PCR showed that K-cells also expressed Pdx-1, NeuroD1/Beta2, and MafA, but not Nkx6.1. After exendin-4 treatment and serum deprivation, insulin mRNA and insulin or C-peptide were clearly detected in Kn4-cells. The intracellular insulin content was also increased significantly in these cells.
Figures and Tables
Fig. 1
Design of Nkx6.1-expressing vector. The Nkx6.1-expressing vector (pcDNA3.1 + Nkx6.1) was made as shown in this cartoon.
Fig. 2
RT-PCR of transcription factors mRNA. K-cells were found to express transcription factors which were all present in mouse islets except Nkx6.1.
Fig. 3
RT-PCR of insulin 1 mRNA. Insulin1 band was weakly detected in K-cells after 7 day-treatment with 10 mM nicotinamide and 10 pM exendin-4 in serum-free medium. 1, Mouse islets; 1-1, K-cells; 2, K-cells, 7 days in serum-free medium; 3, K-cells, 7 days in serum-free medium with 10 mM nicotinamide; 4, K-cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4.
Fig. 4
RT-PCR of insulin 1 and Nkx6.1 mRNA. Insulin1 band was clearly detected in Kn4-cells after 7 day-treatment with 10 mM nicotinamide and 10 pM exendin-4 in serum-free medium. 1, Rat islets; 1-1, Mouse islets; 2, K-cells, 7 days in serum-free medium; 3, K_pcDNA-cells, 7 days in serum-free medium; 4, Kn4-cells, 7 days in serum-free medium; 5, K-cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4; 6, K_pcDNA-cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4; 7, Kn-4 cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4.
Fig. 5
Fluorescent microscopy for insulin immunocytochemistry in K-cells and transdifferentiated Kn4-cells. Some of differentiated Kn4-cells were found to be insulin-positive (A, Insulin staining in Ins-1 cells, ×200; B, Insulin staining in K-cells, ×200; C, Insulin staining in differentiated Kn4-cells, ×400; D, GFP expression, ×400).
Fig. 6
Confocal microscopy for C-peptide immunocytochemistry in transdifferentiated Kn4-cells. Some of differentiated Kn4-cells were found to be C-peptide-positive (A, Nuclei staining with DAPI; B, GFP expression; C, C-peptide staining D, Merged image, ×400).
Fig. 7
Intracellular content of insulin. Insulin content was significantly higher in transdifferentiated Kn4-cells as
compared with K-cells. *P < 0.05.
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