Establishment And Identification Of A Chemically Induced Pluripotent Stem Cells Lineage Reprogrammed From Fsp-Tdtomato Mouse

Background: The establishment of somatic cell reprogramming technology,including nuclear transfer technology,cell fusion technology and transduction by defined factors,promotes the development of regenerative medicine research.However,some technical problems and potential safety issues,such as complicated operation,low induction efficiency,insertion or integration of exogenous genes and the potential reactivation of virus severely limit their further development and application.Compared with those reprogramming technology,small molecules induction reprogramming possess several advantages in terms of easy synthesis and reservation,reversible function,standardization and safe cantrol.Safety issues associated with transcription factors or viruses may be avoided with the use of chemicaly induced pluripotent stem cells(CiPSCs),thus promoting their clinical application.Objective: Previously,we had successfully developed and standardized an induction method using small-molecule compound,with simple operation,uniform induction conditions,and clear constituents.This study was performed in order to verify that the CiPSCs were indeed reprogrammed from mouse embryonic fibroblasts(MEFs),and further explore the underlying mechanisms.Method: FSP-tdTomato mice were used to construct a fluorescent protein-tracking system of MEFs,for revealing the process of CiPSC reprogramming.CiPSCs were identified by morphological analysis,mRNA and protein expression of pluripotency genes,as well as teratoma formation experiments.Results: Results showed that after 40-day treatment of tdTomato-MEFs with small-molecule compounds,the cells were presented with prominent nucleoli,high core-to-cytoplasmic ratio,round shape,group and mass arrangement,and high expression of pluripotency gene.These cells could differentiate into three germ layer tissues in vivo.Conclusion: As indicated by the above results,tdTomato-MEFs could be reprogrammed into CiPSCs,a lineage that possesses pluripotency similar to mouse embryonic stem cells(mESCs),with the use of small-molecule compounds.The establishment of CiPSC lineage,tracked by fluorescent protein,would benefit further studies exploring its underlying mechanisms.With continuous expression of fluorescent proteins during cellular differentiation,this cell lineage could be used for tracking CiPSC transplantation and differentiation into functional cells.