Investigation Of Inducing Methods And Molecular Mechanism Of Mouse Embryonic Stem Cells Differentiating To Embryonic Sertoli-like Cells

Embryonic Sertoli cells(eSCs)are the first male-specific cell type generated during the embryonic developmental process,which play a key role in male determination.eSCs are capable of secreting multiple nutrients to support other cells' proliferation or development,such as spermatogonial stem cells(SSCs).Therefore,the research on eSCs' derivation benefits to reveal mechanism of gonadogenesis and sex determination,and to provide guidance for future clinical application.However,it's hard to isolate a big number of eSCs from male embryo by conventional tissue separation methods,resulting in barriers for the fundamental theory research and experimental application of eSCs.Thus,it is significant to establish a novel approach to induce eSCs in vitro.Recently,a new approach produced intermediate mesoderm(IM)derived cells from mouse embryonic stem cells(ESCs)by using some supplements including retinoic acid and Activin A,and then induced the IM derived cells into embryonic Sertoli-like cells(eSLCs)via some recombinant protein factors including follicle stimulating hormone(FSH).However,the differentiation fate and molecular mechanisms in this approach were still unclear.Moreover,another research successfully created eSLCs reprogrammed from fibroblasts by overexpression of 5 factors,which indicated that these development related factors,Wt1,Gata4,Sf1,Sox9 and Dmrt1,could be significant to the generation of eSCs.Thus,based on these approaches of inducing eSLCs,and established theory of differentiation fate and molecular mechanism of derivation of eSCs,this thesis work established a novel approach of inducing eSLCs.First,IM derived cells were induced from mouse ESCs,and then induced into eSLCs via sequential expression regulation of 6 development related factors,Wt1,Gata4,Sf1,Sry,Sox9 and Dmrtl.A differentiation model from mouse ESCs to eSLCs was established.The main results of this study are as follows:1.Establishment of culture and inducing methods of mouse ESCs.To obtain many mouse ESCs of great differentiation potential,some different culture methods were compared.Results showed the aggregation and proliferation capability of mouse ESCs was improved in TM4 cells derived conditioned medium on mouse embryonic fibroblasts(MEF)feeder cells.For establishment of an efficient inducing method,mouse ESCs were induced to IM derived cells by established methods,and then induced by 6 development related factors,Wt1,Gata4,Sf1,Sry,Sox9 and Dmrt1,through two methods,directly transcriptional expression regulation or supplementation of recombinant protein factors.The results showed that the potential progenitor cells of eSCs including coelomic epithelial-like cells(CK18+)and gonad derived cells(AMH+),were induced more efficiently by regulating target genes' expression via lentiviral transduction.2.A differentiation model from mouse ESCs to eSLCs was established.To reproduce a developmental fate from mouse ESCs to eSCs,the 6 factors,Wt1,Gata4,Sf1,Sry,Sox9 and Dmrt1,were lentivirally transduced into mouse ESCs and sequentially overexpressed via activating tetracycline promoter or light-switch promoter according to the in vivo derivation and development of male gonad.For 14-d induction,some suspected eSLCs(FSHR+/AMH+)were induced.The characteristic of development from mouse ESCs to eSLCs were determined in cellular morphology,expression of specific biomarkers and transcriptional expression of marker genes.The following results were obtained:In 0-8.5 d,IM derived cells were induced from mouse ESCs;In 9.5-11.5 d.coelomic epithelial somatic-like cells were induced from IM derived cells;In 11.5-12.5 d,some coelomic epithelial somatic-like cells differentiated into SF1-positive precursor-like cells(SPLCs),and then into SF1-positive gonadal precursor-like cells(SGPLCs);After 12.5 d,eSLCs were induced from SGPLCs;In 12.5-14.5 d,some eSLCs formed ring-like structures;After 14.5 d,these ring-like structures developed into tubular-like structures,being similar to the structure of seminiferous tubule(ST).These results indicated the developmental fate and characteristic in the differentiation model from mouse ESCs to eSLCs was similar to the in vivo developmental process.3.The roles of the 6 development related factors were preliminarily determined in altering the fate of differentiation from mouse ESCs to eSLCs.The inducing function of the 6 development related factors,Wt1,Gata4,Sf1,Sry,Sox9 and Dmrtl,to mouse ESCs was determined through different combination of expression in differentiation process from mouse ESCs to eSLCs.Via analysis of the cell specific biomarkers,it indicated the expression of Wt1 and Gata4 played important roles in generation of coelomic epithelial somatic-like cells;The expression of Sf1 was related to the generation of(SPLCs)and SF1-positive gonadal precursor-like cells(SGPLCs);Sry and Sox9 had similar function in male determination of SGPLCs;The expression of Dmrt1 had an influence on the cell number of induced eSLCs.4.Molecular mechanism of different developmental phases was investigated based on the differentiation model from mouse ESCs to eSLCs.To investigate the mechanism in the developmental processes from mouse ESCs to eSLCs,the processes were divided into three phases:(1)The derivation and differentiation of coelomic epithelial somatic-like cells,SPLCs and SGPLCs;(2)The differentiation of SGPLCs into eSLCs.(3)Maintenance and development of eSLCs.The molecular mechanism was researched via overexpression of target factors by lentivirally transduction or suppression by CRISPR/Cas9 knock out(KO)technique.Results indicated:(1)Overexpression of Wt1 improved the transcriptional expression of Gata4,Sf1 and other coelomic epithelium development related genes.Upregulation of Gata4 activated the expression of Lhx9.Wt1 and Gata4 potentially played important roles in derivation and differentiation of coelomic epithelial somatic-like cells and SPLCs;(2)The expression of Sfl activated the expression of Amh,Amhr2,Insl3,Dax1,and facilitated the differentiation from coelomic epithelial somatic-like cells into SPLCs.In this developmental stage,Sf1 potentially played a role in epithelial-mesenchymal transformation(EMT),cell migration and aggregation.The individual overexpression of Sf1 did not activate the onset of Sry or Sox9.However,the expression deficiency of Sf1 caused failure of male determination;(3)Sry and Sox9 are the key male determining factors.The expression of Sry activated the expression of Sox9,however,the expression of Sox9 did not activate the onset of Sry.By regulating the expression of Sry or Sox9,some similar male determination related downstream factors were activated,and then SGPLCs were likely to develop into eSLCs.In absence of the expression of Sry and Sox9,SGPLCs were likely to go through female determination and developed into ovarian supporting-like cells(AMH+/WNT4+);(4)Expression of Dmrt1 activated the onset of Amh,Ptgds,Fgf9,Gdnf,and inhibited the expression of female determining factors,Wnt4 and Foxl2.Dmrt1 potentially play a role in facilitating male development,maintenance of the expression of the specific biomarkers of eSLCs,promoting the formation of the structure of ST,and preventing the transition of eSLCs into ovarian supporting-like cells.5.The induced eSLCs were functionally maturated and verified in their physiological characteristics and spermatogenic supporting function.To verify whether these induced eSLCs possess a similar potential function and characteristic with natural eSCs,these eSLCs developed and functionally maturated via a condition medium containing supplements including testosterone,sorted by flow cytometry and applied to mice testicular transplantation test and coculture with SSCs.Results indicated that these induced Sertoli-like cells(SLCs)can grow dispersedly along seminiferous tubules without obvious tumorigenicity which showed a similar result as allogenetic mouse derived mature Sertoli cells in the positive control group.These results indicated that these induced SLCs had similar physiological characteristic with natural mature Sertoli cells;In coculture test,these induced SLCs facilitated the SSCs developing into sperm-like cells.It indicated these induced SLCs had a certain spermatogenic supporting function.In conclusion,this study established a differentiation model from mouse ESCs to eSLCs,and investigated relevant molecular mechanism based on this model.Moreover,this work established a theory foundation and technical platform which will surely be helpful to the future studies of embryonic developmental biology,sex determination and clinical application.