Phenotypic analysis of human stem cells and their differentiation potential towards pancreatic cells

Type 1 diabetes is an autoimmune disorder in which the immune system specifically destroys the insulin-producing β-cells of the pancreas, resulting in a loss of glycemic control. There is no cure for the disease, and patients have the difficult task of managing their blood glucose levels by daily injection of insulin, and monitoring of carbohydrate intake, levels of exercise, and stress. The field of stem cells gives hope to type 1 diabetes patients as stem cells are plastic and can be differentiated into the pancreatic lineage to form insulin-producing β-cell-like cells for transplantation to restore the endocrine function of the pancreas. There are many different stem cell populations described in the published literature that can be easily obtained from the blood, tissues, and organs of adults. They include peripheral blood-insulin producing cells (PB-IPCs) that can be readily isolated due to their unique attachment to hydrophobic surfaces, monocytes that can be dedifferentiated into programmable cells of monocytic origin (PCMOs) and fibroblast-resembling-macrophages (f-MOs), and adipose stem cells (ASCs). There are also other populations of stem cells that can be ethically obtained after childbirth from neonatal tissues that are normally discarded, and include human amniotic stem cells (HASCs) and cord blood hematopoietic stem cells (CB-HSCs). It has been reported in the published literature that each of the above stem cell populations except f-MOs can be induced to form insulin-producing cells. However, in most cases there has been no independent validation of the published data. The aim of this project was to study the phenotype of the above stem cells and determine which stem cell population would be an ideal source from which to derive insulin-producing cells. The published cell differentiation protocols were to be validated, and novel ways of improving upon the protocols were to be examined with the ultimate aim to produce cells that closely resembled pancreatic β-cells. In this study, the above stem cell populations were either produced from the blood of human donors, or purchased directly from commercial companies. They were phenotyped for stem cell, pancreatic cell, and other cell lineage markers. The stem cells were subjected to a variety of published and novel pancreatic differentiation protocols, and examined for the expression of markers that specify different stages of pancreatic differentiation. The ability to transdifferentiate several of the stem cell populations into other cell lineages including the endothelial, neuronal, and lymphocytic lineages was also examined. Phenotyping of the stem cell populations revealed that some undifferentiated stem cells already expressed certain markers of pancreatic progenitor cells. Monocytes dedifferentiated into PCMOs expressed SOX-17 and PDX-1, in addition to the monocytic markers CD14 and CD68. A subset of monocytes dedifferentiated into f-MOs expressed PDX-1, while the remaining cells expressed CD14 indicating the presence of two different cell populations. Therefore it appeared that the monocyte-derived stem cell populations are in an intermediate state of transdifferentiation expressing multiple cell lineage markers. Undifferentiated HASCs expressed PDX-1 and SOX-17 whilst also expressing the stem cell markers Nestin and Oct-4. Several CB-HSCs expressed SOX-17, GPR40, and Nestin. ASCs expressed SOX-17, in addition to the characteristic ASC stem cell markers CD29 and CD90. The expression of pancreatic markers by undifferentiated HASCs, CB-HSCs, and ASCs indicates they could have the potential to differentiate towards the pancreatic lineage. PB-IPCs expressed the pancreatic progenitor cell markers PDX-1, CXCR4, HNF6, and SOX-17 following culture in the absence of additional growth factors other than foetal bovine serum. They highly expressed the dendritic cell marker CD11c and appeared