Winning Abstracts from the 2007 Medical Student Abstract Competition: Curing Diabetes and Solving the Stem Cell Dilemma: In Vitro Differentiation of Mouse Amniotic Fluid Cells into Pancreatic Islet-like Cells
James Knutson, Wake Forest University Bowman Gray School of Medicine, 2008
Blindness, end-stage renal disease, amputation, and cardiovascular disease are just a few of the well-documented effects of diabetes, a disease that affects about 20 million Americans. Embryonic stem cells may hold a cure, but research is limited by ethical concerns. A line of amniotic fluid cells expressing the stem cell marker CD117 might be a pluripotent alternative to embryonic stem cells.
Using stem cell differentiation protocols and pancreatic embryology, we hypothesize that amniotic fluid cells can differentiate into pancreatic islet-like cells. We also hypothesize that these cells can restore glycemic control in a diabetic mouse model.
Mouse amniotic fluid cells expressing CD117 were isolated and transduced with pdx-1, an important gene in pancreatic islet cell development. Western blot and RT-PCR of Pax6 (a gene controlled by PDX-1) were performed to examine PDX-1 protein expression and functionality. Control cells were cultured in Chang media; differentiated cells received serum-free culture media with basic fibroblast growth factor (bFGF). Cell morphology and insulin/glucagon production were examined. Finally, an in vivo study was performed by injecting diabetic mice with pdx-1-transduced cells and measuring blood glucose levels.
Western blot demonstrated expression of PDX-1, and RT-PCR of Pax6 showed an increase in Pax6 levels.
Control cells showed no changes in morphology and did not stain for insulin or glucagon. Differentiated cell morphology resembled pancreatic islet cells; differentiated cell aggregates stained centrally for insulin and peripherally for glucagon.
Diabetic mice receiving pdx-1-transfected cells exhibited glycemic control similar to non-diabetic mice, while diabetic mice receiving non-transduced cells lacked glycemic control.
Amniotic fluid cell differentiation into pancreatic islet-like cells was demonstrated genetically, morphologically, and functionally. In vivo studies with these cells demonstrated correction of diabetes in mice. These results suggest a possible cure for diabetes and support the possibility of using amniotic fluid cells as an embryonic stem cell alternative.
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