Transplantation of Insulin-Producing Clusters Derived From Adult Bone Marrow Stem Cells to Treat Diabetes in Rats

Janna Serbo

Paper Summary and Review

Reference:

Gabr, Mahmoud M, Mohamed M Sobh, et al. "Transplantation of Insulin-Producing Clusters Derived From Adult Bone Marrow Stem Cells to Treat Diabetes in Rats." Experimental and Clinical Transplantation 6, no. 3 (2008).

http://www.ectrx.org/forms/ectrxcontentshow.php?year=2008&volume=6&issue=3&supplement=0&makale_no=0&spage_number=236&content_type=FULL%20TEXT

Introduction:

Islets of Langerhans are cell clusters in the pancreas that contain many types of cells, including alpha, beta, and delta cells. Specifically, beta cells produce the hormone insulin and directly secrete insulin into the blood. Insulin is a hormone that controls blood glucose levels, allowing cells to absorb glucose. The cells store glucose as glycogen, a polysaccharide of glucose, to use as a short term energy supply. This way, cells do not use fat stores as an energy source until it is necessary. Diabetes Mellitus Type One, or juvenile diabetes, is an autoimmune disease in which the body attacks the patient’s pancreatic, insulin-producing beta cells.

Summary:

Transplantation of Insulin-Producing Clusters Derived From Adult Bone Marrow Stem Cells to Treat Diabetes in Rats from Experimental and Clinical Transplantation describes adult bone marrow stem cell experiments in rats as a mean to explore Diabetes treatment (Gabr, et al. 2008). Adult bone marrow stem cells derived from Sprague-Dawley rats where isolated and differentiated into glucose regulating, insulin producing cells. These cells were grown into clusters to mimics the Islets of Langerhans (beta cells) that exist in a normal, healthy pancreas. Diabetes was induced in rats by destroying their pancreatic beta cells with streptozotocin, a toxic chemical to beta cells. The differentiate bone marrow cells were transplanted into the diabetic rats by injection into the gonads. The gonads were pulled into the abdominal cavity and sutured onto the abdominal wall to create a “pancreatic like” sack. The injected cells were tested and observed for insulin production and glucose regulation. Insulin was indeed produced, and cells absorbed glucose from the blood to store as an energy source, as would happen in a non-diabetic individual.

Figure 5. (A) Changes in the glucose concentrations among the 4 studied groups: group control (sham experiment), group acellular (diabetic rats treated by culture media), group undifferentiated (diabetic rats treated by undifferentiated hematopoietic-rich stem cells), and group differentiated (diabetic rates treated by differentiated clusters). (B) Changes in the insulin levels in clusters of rats that had undergone a transplant. Blood glucose began to decrease within 2 to 3 days. Throughout the observation period, the determined blood glucose levelswere significantly lower (P < .001) and serum insulin levels were significantly higher (P < .001) in animals in group differentiated than they were in animals in group acellular and group undifferentiated. Following orchidectomy, these rats became hyperglycemic again with a steep reduction in the serum insulin levels.

This paper used a number of experimental methods to test the differentiation and insulin production ability of bone marrow derived cells (see paper for all images). Cells were characterized through protein imaging by using immunostaining. Monoclonal, rat, anti-insulin antibody was used to target and stain insulin-producing differentiate cells. The immunoreactive cells were observed with a Vectastain Elite ABC Kit using tetrachloride as a chromogen. This selected the bone marrow cells that differentiated properly and confirmed that the differentiated cells were producing insulin.

Figure 1. Morphologic changes of hematopoietic-rich stem cells during differentiation. (A) Undifferentiated hematopoietic-rich stem cells, 1 day after isolation (magnification ×200). (B) Hematopoietic-rich stem cells formed isletlike clusters by dimethyl sulfoxide treatment for 3 days; this was followed by treatment with high-glucose and pancreatic extract for 7 days (magnification ×40). (C) Collected isletlike clusters after treatment with nicotinamide and exendin-4 for 7 days (magnification ×100). D) Ditizone staining of isletlike clusters. The clusters distinctly stained crimson red by DTZ (magnification ×400).

Also, differentiated cells were characterized through the analysis of biochemical components using ELISA (Enzyme Linked Immunosorbent Assay). Intracellular C-peptide is a byproduct of the insulin process, showing that cells are producing insulin. An ELISA kit was used to see if cells were secreting Rat C-peptide. This confirmed that undifferentiated cells were not producing insulin and differentiated cells were producing insulin. Furthermore, cells were characterized by gene expression using reverse transcription polymerase chain reaction (RT-PCR). RT-PCR is when RNA is “reverse” transcribed into DNA. Then, the DNA is replicated and amplified by PCR heating and cooling cycles. This was done to check that islet related genes were expressed in differentiated cells, proving that the function of the differentiated cells was similar to the Islet of Langerhans cells. An additional test to check for differentiation was done by staining the cells with DTZ (diphenylthiocarbazone). The cells that show up red will be cells containing zinc, which plays an important role in storage, secretion, synthesis of insulin. This confirms which cells have differentiated correctly.

Shortfalls:

While this paper is very promising, there are a few general problems that were not address. The cells tested were rat adult bone marrow stem cells, making this an impractical treatment alternative. Xenogeneic treatments involve risks of pathogen transfer and immune compatibility complications. This paper was good for a proof of concept, but further research would have to be done specifically for human stem cells and for clinical trials. Also, this treatment is not fully effective for Diabetes Type one patients. The implanted cells would be autologous because they were derived from the adult subject. However in the case of diabetes type one, an autologous source of stem cells still has immune compatibility issues. The body attacks both autologous and allogeneic sources because the disease is autoimmune. Autologous beta-like cells would still be recognized as foreign material by the body. The gonads were used as an encasing, but an impractical one used to keep the cell clusters together for easy removal. Human patients would likely be unwilling to have their gonads injected and sutured into the abdomen. Other protective encapsulation methods would need to be explored to immunoisolate injected cells. Also, future research must still explore problems such as the ability to successfully culture large numbers of islet-like cells in vitro, to provide stable and consistent culture conditions, and to optimize an area for injection and transplantation of differentiated cells.