Fast Chemical Reprogramming To Pluripotency

Cellular plasticity is a fundamental scientific question.Pluripotent stem cells can differentiate into specific types of functional cells in vitro,thus providing an important system for studying cell fate determination.Besides,pluripotent stem cells have a wide range of applications in disease modeling,drug screening,and regenerative medicine.The most typical type of pluripotent stem cell is embryonic stem cell,which is obtained by isolating from the inner cell mass of blastocyst and can be expanded infinitely in vitro.Additionally,somatic cells can be reprogrammed into induced pluripotent stem cells through the overexpression of transcriptional factors or the use of small molecules,a technique called as somatic cell reprogramming.Induced pluripotent stem cells are molecularly and developmentally equivalent to embryonic stem cells and do not depend on donor embryos,avoiding ethical controversies.Compared to transcriptional factors,small molecules possess several advantages,including ease of manipulation and nongenomic insertion.Chemical reprogramming using pure small molecule combinations is an excellent system for studying cellular plasticity and has broad prospects in clinical applications.However,the currently reported mouse chemical reprogramming systems still have scope for improvement in terms of rate.Besides,the molecular trajectory of chemical reprogramming differs significantly from transcriptional factor-induced reprogramming,indicating the specificity of the chemical reprogramming mechanism.Nevertheless,the precise molecular mechanism underlying chemical reprogramming remains to be fully elucidated.To address the issues of low efficiency and unclear mechanisms in chemical reprogramming,I focused on modifying the mouse chemical reprogramming system and further exploring its mechanisms.A range of strategies were employed during the optimization process,including small molecule library screening,concentration testing,treatment duration testing,and single small molecule removal experiments.After screening approximately 21,000 conditions,a mouse fast chemical reprogramming(FCR)system was developed,which significantly accelerated the dynamic of reversing cell fate,reducing the original protocol from approximately 40 days to 7-12 days.Furthermore,I demonstrated that the FCR system could be applied to mouse fibroblasts from various genetic backgrounds and tissue sources.Through rigorous molecular and functional validation,I confirmed that the induced pluripotent stem cells obtained from the FCR system possessed bona fide pluripotency.Additionally,a few of FCR small molecules could also promote mouse or human transcriptional factor-induced reprogramming.In sum,we first propose and complete the new concept of FCR.Mechanistically,by using RNA-seq and single cell RNA-seq(scRNA-seq),I demonstrated that FCR involves the erasure of somatic cell characteristics,transient activation of genes associated with extraembryonic endoderm and mesenchymal-toepithelium transition and establishment of pluripotency.At the late stage of FCR,small molecules promote reprogramming towards pluripotency by inhibiting cell fates of trophectoderm and primitive endoderm and supporting a cell fate of epiblast.The late stage of FCR is characterized by a unique diapause-like state,during which cells significantly reduce their rates of DNA and protein synthesis.Blocking cells entering the diapause-like state decreased the reprogramming efficiency,highlighting the importance of the diapause-like state in FCR.Thus,FCR is a valuable model to study the diapause state.In summary,this study for the first time proposes the concept of FCR,has successfully established a FCR system,and effectively addresses the critical issue of the time-consuming problem of chemical reprogramming.Through the utilization of RNA-seq and scRNA-seq,the transcriptional landscape of FCR has been comprehensively characterized,uncovering new mechanisms of cell fate transition.Thus,FCR holds great potentials for a diverse range of applications in various fields,including fundamental research,anti-aging,anti-tumor,and regenerative medicine.