The Research Of Small Molecules That Can Be Used To Manipulate Stem Cell Fates Toward Desired Outcomes, Especially To Corneal Epithelial-like Cells

Objective: Stem cells are becoming a hot spot of basic medicine and clinical research because of their wide self-renewal and differentiation potential in recent years. A number of small molecules that can be used to control stem cells self-renewal, lineage differentiation, reprogramming and regeneration could regulate stem cells fate quickly and reversibly. The aim of this assay is to discuss the method, in which embryonic stem cells differentiated into corneal epithelial-like cells.To further reveal the corresponding molecular mechanism in the process of induction, we design two group of small molecular compound to induce embryonic stem cells into corneal epithelial-like cells.To explore more safe and effective inducing way,we make use of differernt detection approaches to confirm that sample cells are corneal epithelial-like cells. If small molecules could induce human embryonic stem cell differentiation to corneal epithelial-like cells in vivo, corneal cells transplantation therapy will have unlimited source of corneal epithelium. More patients will be treated effectively and timely.Methods: We make use of AggreWellTM plates to produce uniform embryoid bodies when embryonic stem cells attach to 80% of cell aggregates. EBs recapitulate many aspects of cell differentiation during early embryogenesis, which are important in the differentiation of hESCs into different cell types in vitro. After the four-day induction period in suspension culture , cell aggregates were plated down onto matrigel coated well-plates for further differentiation in adherent culture using different corneal epithelium medium SHEM、UM、DKSFM. Cell outgrowths from the attached aggregates were observed at the day 10 time point.To study the effects of induction medium on different stage differentiation, expression of several makers/genes/protein was studied using immunocytochemistry、reverse transcription-polymerase chain reaction、western blot. The expression of the undifferentiated stem cell markers/genes/proteins are OCT4、PAX6. The expression of the corneal epithelial progenitor markers/genes/proteins are K15 and corneal epithelial markers/genes/proteins are K12、K3. Exploring the characteristics of different small molecules and induction medium to seek the optimal induction efficiency way.Results: 1 .Cell induced by small molecules and mediums outgrowths from EBs has the characteristics of corneal epithelial.2.The results of immunocytochemistry suggest the expression of the undifferentiated stem cell markers/genes/proteins are down-regulated and the expression of corneal epithelial progenitor/corneal epithelial are up-regulated. 3.The relative level of K3、K12 expression is highest at 20 day time piont in SHEM condition with the small molecule compounds IWP-2、SB505124. The relative level of K3、K12 expression is highest at 20 day time piont in UM condition with the small molecule compounds IWP-2、SB505124 and DAPT. The relative level of K3、K12 expression is highest at 30 day time piont in SHEM condition with the small molecule compounds IWP-2、SB505124 and DAPT.Conclusion: 1.The small molecule compouds IWP-2、SB505124 and DAPT could induce embryonic stem cells into corneal epithelial-like cells.2.The expression of the undifferentiated stem cell markers/genes/proteins are down-regulated and the expression of corneal epithelial progenitor/corneal epithelial are up-regulated. 3.The inductive efficiency of IWP-2、SB505124 is higher than IWP-2、SB505124 and DAPT with SHEM. The inductive efficiency of IWP-2、SB505124 is lower than IWP-2、SB505124 and DAPT with UM.There is no difference between this two sets of small molecule compouds with DKSFM.4.The inductive efficiency is highest for DKSFM at the day 30 time point, but there is no difference between different time points for SHEM、UM.