Results from this study demonstrated that after induction, cells gradually became round and gathered into islet-like cell clusters. Insulin immunofluorescence staining tests of induced cells revealed positive results. Moreover, induced cells can secrete a small amount of insulin and respond to glucose stimulation to a certain degree.
At present, three strategies can be used to induce stem cells to differentiate into insulin-secreting cells: genetically engineered cells, change of cell microenvironment, and a combination of transgene and in vitro induction. In the present study, we used the second strategy by adding a number of cytokines, matrix, nutrient substance, or chemical substances contributing to cell growth or differentiation.
EGF, as a growth factor to stimulate the growth of a variety of epidermal cells and epithelial cells, participates in the repair of injury. EGF signaling plays a role through the c-erbB receptors. β-mercaptoethanol as an antioxidant, can promote the synthesis of glutathione, and get rid of oxygen free radicals. A small amount of β-mercaptoethanol can promote cell culture when some cells are difficult to be cultured. β-mercaptoethanol has been also considered to a part replace serum and is often used as nerve cells induction agent. Several years ago, our team used retinoic acid combined with β-mercaptoethanol to successfully induce the differentiation of mouse fetal liver-derived MSCs into nerve cells. In this study, we used retinoic acid combined with β-mercaptoethanol to induce UCBMSCs into pancreatic islet β-like cells. Results demonstrated that after 7 days, the cells became three- dimensional, but we are not clear regarding what had taken place in the cells. The precise mechanism remains further investigation.
In addition, activtin A has been reported to be a transforming growth factor superfamily of cytokines[9-10], and plays an important role in cell proliferation, differentiation, and apoptosis. Activin A and its receptor exist in a variety of cells, such as the α and δ islet cells. Activin A combined with hepatocyte growth factor or betacellulin can induce the differentiation of cell line AR42J into insulin-producing cells and can induce human fetal pancreatic endocrine cell differentiation. All these findings support that activin A plays an important role in the development of β cells. So after the first step of induction, in the second step, we added 10 μg/L hepatocyte growth factor and 3 μg / L activin A to serum-free culture. The, we found that the process of cell differentiation was accelerated. After 10 days of induction, cells showed that cell soma became small, and cytoplasm was decentralized towards the periphery. By 35 days, the number of cell clusters increased to 70-100. Moreover, during the process of induction, we found that serum concentration was higher than 5%, the cells were proliferated, and it is difficult to perform directed differentiation. For this reason, we used serum-free medium, or low concentrations of serum. Zalzman et al [11-12] discussed the effect of serum on the induction of pancreatic duodenal homobox 1-transfected human fetal liver cells and concluded that serum-free culture was necessary for islet cell differentiation and serum completely inhibits the formation of cultivated human islet buds. This may be due to the complex composition of serum, containing a variety of growth factors or differentiation factors, which are difficult to be accurately controlled during the process of cell differentiation.
Taken together, results from this study primarily confirm the UCBMSCs differentiation into isletβ-like cells, which provides sufficient seed cells for islet cell transplantation. However, problems exist, for example, low differentiation efficiency and immature differentiated cells. In future experimental studies, we consider to use transgene method combined with in vitro induction to accelerate the progression of cell induction, enhance differentiation efficacy, and then promote the maturation of differentiated cells.
