Adult bone marrow-derived cells can be induced to differentiate into insulin-producing cells under defined conditions . Stem cell regeneration is an attractive insulin replacement therapy for those with insulin dependent DM. Stem cells from the pancreas [34, 35], bone marrow , umbilical cord blood , and embryo  have previously been used in research on regeneration therapies for DM. Recently, we found that Wharton’s jelly from the human umbilical cord contains fibroblast-like cells, which are similar to MSCs . In this study, we investigated the ability of these cells to differentiate into insulin-producing cells, as well as the potential curative effects of transplanting the insulin-producing cells into the livers of diabetic rats. Simultaneously, we tested the usefulness of the modified Port-A catheter in transplantation.
Our results illustrate that human umbilical cord MSCs could be differentiated into insulin-producing cells following incubation under specific conditions . Based on current references of pancreas endocrine cell development, a combination of various factors, including activin A, sodium butyrate, growth factors in serum free media supplements were used in this study (Figure 1).
Due to the controversy surrounding insulin uptake by cells from media supplements [37, 38], we used human C-peptide to characterize insulin production by our cells. Proinsulin, the precursor of insulin, is composed of 3 segments, the A-chain, B-chain, and C-peptide. Although C-peptide is released from proinsulin, unlike the A- and B-chains, it is not taken up by the cells. Thus, levels of C-peptide can be used as a marker of insulin secretion. After exposure of MSCs to differentiation conditions, immunocytochemical staining revealed that the cells expressed both insulin and C-peptide.
In a prior study we demonstrated that pancreatic endocrine precursor (PEP) cells could be generated from human umbilical cord MSCs . In our in vitro studies, expression of β-cell development-related genes was examined by reverse transcriptase and real time PCR before and after induction of differentiation. After differentiation for 17 days, the insulin-producing cells expressed the following pancreatic β-cell development-related genes: Pax4 Nkx2.2 MafA NeuroD Isl-1 Glut2 and insulin. Additionally, our C-peptide secretion assays revealed that the differentiated cells generated in vitro displayed functional characteristics of insulin-producing cells. After the cells were transplanted into the NOD mice via a retro-orbital vein, blood sugar levels tended to decrease .
In this study, Wharton’s jelly was induced to differentiate into islet-like cell aggregates. After differentiation for 10 days, the insulin-producing cells expressed the following pancreatic β-cell development-related genes: Pax4, Pax1 and insulin. Additionally, we found greater expression of C-peptide in differentiated versus undifferentiated MSCs (Figure 1A). After the cells were transplanted into the STZ induced rats via a portal, we found that blood sugar levels tended to decrease in comparison to STZ rats receiving sham transplantation. This method of developing insulin-producing cells was more effective, inexpensive and less time consuming.
In the current study, Wharton’s jelly tissue was used as opposed to pancreatic stem cells, as the former contains much greater quantities of stem cells than the pancreatic duct. Specifically, each cubic centimeter of Wharton’s jelly sample contains 1–1.5 × 104 MSCs and the number of cells increases 300-fold after seven passages, providing a plentiful supply of cells for transplantation. In addition, the use of Wharton’s jelly stem cells is preferable to embryonic stem (ES) cells, as doing so avoids the risk of teratoma formation as well as the ethical issues inherent in using ES cells.
The portal vein , renal subcapsular space , and tail vein  have been previously used as stem cell transplant locations for insulin regeneration therapies in the rat. In this study, cells were transplanted into the rat liver via the portal vein. Interestingly, we have observed that blood sugar levels tend to decrease sooner when using hepatic portal vein transplantation instead of renal subcapsular transplantation . These results are likely related to the finding that transplantation into the liver is more advantageous than renal subcapsular transplantation for diabetes therapy, as the former technique provides a larger surface area for implantation and recapitulates an orthotopic site physiologically. Specifically, secreted insulin enters the portal system (via the superior mesenteric vein) rather than the systemic venous system (via the renal vein) . In our previous study , transplantation of insulin-producing cells via the retro-orbital vein was used to treat NOD mice. Though techniquely it is easier to perform and less invasive than via portal vein, higher mortality rate in mice and less number (1 × 105) of MSCs could be used were the major obstacles. Transplantation of MSCs via retro-orbital vein may only be used for the smaller animals. It has been reported that insulin-producing cells were injected directly into liver parenchymal of STZ induced diabetic rats to lower blood glucose level . However, this transplantation method applied to clinical setting is limited. In clinical application, the Edmonton Protocol involves isolating islets from a cadaveric donor pancreas. Each recipient needs to transplant islets isolated from one to as many as three donors. The islets are infused into the patient’s portal vein, then these islets are stored at the liver to produces insulin. For clinical application to use hMSCs as a cell transplantation source for diabetic regeneration therapy, proof of usefulness of hMSCs transplantation via the portal vein in a large animal diabetic model is needed.
In order to test the function of the MSC-derived insulin-producing cells in vivo, we transplanted the differentiated cells into STZ-induced DM rats via a Port-A catheter into the portal vein. The modified Port-A catheter used in this study has two main advantages over techniques described in previous studies. First, since only a very small part of the catheter is inserted into the widest part of the portal vein, no veins are clamped permanently, in contrast to previous methods [42, 43], and the disturbance to intestinal blood flow is minimized. Additionally, the use of a port enhances catheter longevity, thereby permitting longer periods of infusion. Indeed, the reported duration of use of a Port-A catheter is 9–34 months .
Following transplantation of insulin-producing cells into diabetic rats in the current study, C-peptide was found in the transplanted cells of the liver and blood glucose levels decreased. Both of these findings suggest that the transplanted cells secreted functional insulin. Indeed, on the fourth week after transplantation, blood glucose levels decreased to approximately 250 mg/dl in the compared to 530 mg/dl in the STZ controls. Nevertheless, we believe that transplantation may slow down the appearance of symptoms of DM rather than cure the disease.