Transplantation of insulin-producing cells from umbilical cord mesenchymal stem cells for the treatment of streptozotocin-induced diabetic rats

Abstract

Background: Although diabetes mellitus (DM) can be treated with islet transplantation, a scarcity of donors limits the utility of this technique. This study investigated whether human mesenchymal stem cells (MSCs) from umbilical cord could be induced efficiently to differentiate into insulin-producing cells. Secondly, we evaluated the effect of portal vein transplantation of these differentiated cells in the treatment of streptozotocin-induced diabetes in rats.

Methods: MSCs from human umbilical cord were induced in three stages to differentiate into insulin-producing cells and evaluated by immunocytochemistry, reverse transcriptase, and real-time PCR, and ELISA. Differentiated cells were transplanted into the liver of diabetic rats using a Port-A catheter via the portal vein. Blood glucose levels were monitored weekly.

Results: Human nuclei and C-peptide were detected in the rat liver by immunohistochemistry. Pancreatic β-cell development-related genes were expressed in the differentiated cells. C-peptide release was increased after glucose challenge in vitro. Furthermore, after transplantation of differentiated cells into the diabetic rats, blood sugar level decreased. Insulin-producing cells containing human C-peptide and human nuclei were located in the liver.

Conclusion: Thus, a Port-A catheter can be used to transplant differentiated insulin-producing cells from human MSCs into the portal vein to alleviate hyperglycemia among diabetic rats.

Figure 1

Diffenentiation scheme for generating insulin-producing cells from human umbilical cord mesenchymal stem cells.

Figure 2

Reverse transcriptase and real-time PCR analysis of the expression of pancreatic β-cell development-related genes and insulin production-related genes.(A) Reverse transcriptase -PCR results for non-differentiated cells (MSCs, lane 1 and 2), differentiated cells (IPCs, lane 3 and 4). (B) Real-time PCR analysis of the expression of Pax4, Pax1 and Insulin. Results are expressed as the mean ± SEM for 4 experiments. *, P ≪ 0.05 compared to the non-differentiated cells.

Figure 3

Measurement of spontaneous C-peptide secretion showing that the mesenchymal stem cells of Wharton’s jelly differentiate into insulin-producing cells. The expression of C-peptide was greater among differentiated IPCs versus undifferentiated MSCs (Figure 3A). The level of C-peptide expression increased several-fold from MSCs cultured in SFM-A, SFM-B and finally SFM-C (Figure 3B). *, P ≪ 0.05 compared to the non-differentiated cells in A and compared to SFM-A in B. †,P ≪ 0.05 compared to SFM-B.

Figure 4

Immunocytochemical staining for C-peptide. The MSCs were induced into islet-like cell clusters (ICAs) which contain C-peptide labeling cells, indicating that the induced MSCs could aggregate and form functional pancreatic endocrine cells. Bar = 50 μm.

Figure 5

Glucose challenge test for C-peptide release in response to low (5.5 mM) or high (25 mM) glucose concentrations of differentiated cells. (*, P ≪ 0.05 compared to 5.5 mM glucose). Results are expressed as the mean ± SEM for 4 experiments.

Figure 6

(A) Changes in blood glucose levels in STZ-induced diabetic rats after transplantation of insulin-producing cells into the portal vein via the Port-A catheter (study group), non-transplanted STZ-induced diabetic rats (STZ group), and normal non-diabetic rats. (*, P ≪ 0.05 compared to control group; †, P ≪ 0.05 compared to STZ group). Results are presented as the mean ± SEM for 6 rats. Double-immunostaining for human C-peptide and human nuclei in the liver of the STZ-induced diabetic rats at 8 weeks after transplantation of insulin-producing cells. (B) Stained with anti-human nuclei antibody (green). (C) Stained with anti-human C-peptide antibody (red). Bars = 100 μm.