M.Sc Thesis

M.Sc StudentShulman Margarita
SubjectDifferentiation of Human Embryonic Stem Cells into Insulin-
Producing Cells and their Transplantation into a
Diabetic Mouse
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Joseph Itskovitz
DR. Hani Segev
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Type I diabetes mellitus is caused by an immune destruction of the insulin-producing b cells. The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of b cells for transplantation. The unique feature of ES cells is their ability to be indefinitely cultured in an undifferentiated state and to differentiate into cells representative of all three body lineages: ectoderm, mesoderm and endoderm. Differentiation of hES cells can be induced by removing the cells from their feeder layer and growing them in suspension. Growth in suspension leads to the formation of embryoid bodies (EBs).

In this thesis human embryonic stem cells were demonstrated to differentiate into insulin-producing clusters and their transplantation into a diabetic mouse.

The differentiation protocol consisted of several steps. EBs were first cultured and plated in insulin-transferrin-selenium-fibronectin medium, followed by medium supplemented with N2, B27, and basic fibroblast growth factor (bFGF). Next, the glucose concentration in the medium was lowered, bFGF was withdrawn, and nicotinamide was added. Dissociating the cells and growing them in suspension resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability.

Reverse transcription-polymerase chain reaction detected an enhanced expression of pancreatic genes in the differentiated cells. Immunofluorescence and in situ hybridization analyses revealed a high percentage of insulin-expressing cells in the clusters. In addition to insulin, most cells also coexprssed glucagon or somatostatin, indicating a similarity to immature endocrine pancreatic cells.

To investigate whether the insulin-producing cells could rescue mice with diabetes, we transplanted the induced cells into the left renal capsules of streptozotocin-treated diabetic mice. After transplantation, the survival probability of cell-transplanted mice was not higher than that of the control mice (not operated or operated with blood clot without insulin-producing cells). The blood glucose of cell-treated mice was not reduced. We also weren't able to observe tumor formation in the kidney of mice transplanted with induced cells.

Despite failure of in vivo studies and lack of normalize the blood glucose levels of diabetic mice, after improving our protocol we observed secretory granules resembling insulin-containing granules. Developing hES cells to form mature β-cell-like structures will enable the use of these cells for future cell therapy of type I diabetes.