Seung-Hyun Hong

Skeletal Myogenic Differentiation of Mesenchymal Stem Cells Isolated from Human Umbilical Cord Blood

Human umbilical cord blood (UCB) has been regarded as an alternative source for cell transplantation and cell therapy because of its hematopoietic and nonhematopoietic (mesenchymal) potential. Although there has been debate about whether mesenchymal stem cells (MSCs) are invariably present in UCB, several reports showed that MSC‐like cells could be consistently derived from human UCB and, moreover, could differentiate into various cells of a mesodermal origin. However, it remains unclear whether these UCB‐derived MSCs are also capable of differentiating into skeletal muscle cells. In this study, we isolated MSCs from human UCB and induced them to differentiate into skeletal muscle cells. During cell culture expansion, UCB‐derived mononuclear cells gave rise to adherent layers of fibroblast‐like cells expressing MSC‐related antigens such as SH2, SH3, α‐smooth muscle actin, CD13, CD29, and CD49e. More important, when these UCB‐derived MSCs were incubated in promyogenic conditions for up to 6 weeks, they expressed myogenic markers in accordance with myogenic differentiation pattern. Both flow cytometric and reverse transcriptase–polymerase reaction analyses showed that two early myogenic markers, MyoD and myogenin, were expressed after 3 days of incubation but not after 2 weeks. At week 6, more than half of UCB‐derived MSCs expressed myosin heavy chain, a late myogenic marker. Our results demonstrate that UCB‐derived MSCs possess a potential of skeletal myogenic differentiation and also imply that these cells could be a suitable source for skeletal muscle repair and a useful tool of muscle‐related tissue engineering.

Differential Gene Expression Profiling of Human Umbilical Cord Blood–Derived Mesenchymal Stem Cells by DNA Microarray

Mesenchymal stem cells (MSCs) retain both self‐renewal and multilineage differentiation capabilities. Despite wide therapeutic potential, many aspects of human MSCs, particularly the molecular parameters to define the stemness, remain largely unknown. Using high‐density oligonucleotide micro‐arrays, we obtained the differential gene expression profile between a fraction of mononuclear cells of human umbilical cord blood (UCB) and its MSC subpopulation. Of particular interest was a subset of 47 genes preferentially expressed at 50‐fold or higher in MSCs, which could be regarded as a molecular foundation of human MSCs. This subset contains numerous genes encoding collagens, other extracellular matrix or related proteins, cytokines or growth factors, and cytoskeleton‐associated proteins but very few genes for membrane and nuclear proteins. In addition, a direct comparison of this microarray‐generated transcriptome with the published serial analysis of gene expression data suggests that a molecular context of UCB‐derived MSCs is more or less similar to that of bone marrow–derived cells. Altogether, our results will provide a basis for studies on molecular mechanisms controlling core properties of human MSCs.

Rosiglitazone stimulates the release and synthesis of insulin by enhancing GLUT-2, glucokinase and BETA2/NeuroD expression

Peroxisome proliferator-activated receptor (PPAR)-gamma is a member of the nuclear receptor superfamily, and its ligands, the thiazolidinediones, might directly stimulate insulin release and insulin synthesis in pancreatic beta-cells. In the present study, we examined the effects of rosiglitazone (RGZ) on insulin release and synthesis in pancreatic beta-cell (INS-1). Insulin release and synthesis were stimulated by treatment with RGZ for 24h. RGZ upregulated the expressions of GLUT-2 and glucokinase (GCK). Moreover, it was found that RGZ increased the expression of BETA2/NeuroD gene which could regulate insulin gene expression. These results suggest that RGZ could stimulate the release and synthesis of insulin through the upregulation of GLUT-2, GCK, and BETA2/NeuroD gene expression.

Effects of activin A on pancreatic ductal cells in streptozotocin-induced diabetic rats.

Background. The shortage of islets for transplantation has led to find alternative insulin producing cells. Pancreatic progenitor cells in the duct have the potential to grow and differentiate into endocrine cells. In this study, we examined whether activin A can promote the expansion and/or differentiation of ductal cells into insulin-producing cells. Methods. Pancreatic ductal cells were treated with activin A for differentiation into endocrine cells, and transplanted into the renal subcapsular space of streptozotocin (STZ)-induced diabetic rats. The identity of the endocrine cells was confirmed by immunostaining and analysis of the expression of transcription factors and endocrine genes by reverse-transcriptase polymerase chain reaction. Results. Activin A treatment significantly increased the DNA synthesis and the expression of insulin I, insulin II, PDX-1, Nkx 6.1, Glut-2, Pax-4, Pax-6, and Ngn-3. De novo synthesis of insulin in activin A-treated ductal cells was observed by the immunocytochemical detection of C-peptide and the differentiated ductal cells secreted significantly increased amount of insulin compared to nontreated ductal cells in response to glucose stimulation. When activin A-treated ductal cells were transplanted on STZ-induced diabetic rats, blood glucose levels were normalized and the removal of the transplanted kidney resulted in return to hyperglycemia. Conclusions. The pancreatic ductal cells could be efficiently differentiated into insulin secreting cells by activin A treatment in vitro and normalize hyperglycemia in vivo.

Activin A, exendin-4, and glucose stimulate differentiation of human pancreatic ductal cells

Islet transplantation is one treatment option for diabetes mellitus. However, novel sources of pancreatic islets or insulin-producing cells are required because the amount of donor tissue available is severely limited. Pancreatic ductal cells are an alternative source of β-cells because they have the potential to differentiate into insulin-producing cells. We investigated whether treatment of human pancreatic ductal cells with activin A (ActA) and exendin-4 (EX-4) stimulated transdifferentiation of the cells, both in vitro and in vivo. We treated human pancreatic ductal cells with ActA and EX-4 in high-glucose media to induce differentiation into insulin-producing cells and transplanted the cells into streptozotocin-induced diabetic nude mice. Co-treatment of mice with ActA and EX-4 promoted cell proliferation, induced expression of pancreatic β-cell-specific markers, and caused glucose-induced insulin secretion compared with the ActA or EX-4 mono-treatment groups respectively. When pancreatic ductal cells treated with ActA and EX-4 in high-glucose media were transplanted into diabetic nude mice, their blood glucose levels normalized and insulin was detected in the graft. These findings suggest that pancreatic ductal cells have a potential to replace pancreatic islets for the treatment of diabetes mellitus when the ductal cells are co-treated with ActA, EX-4, and glucose to promote their differentiation into functional insulin-producing cells.