Mapping Developmental Paths Of Monkey Primordial Germ-like Cell Differentiation From Pluripotent Stem Cells By Single Cell RNA Sequencing Analysis

Originated in the early stages and go through a series of complex developmental process,the germ cell lineage eventually produces fully mature gametes(sperm and oocytes).In mammals,the germ cell lineage is induced into pluripotent precursor cells through signaling molecules early in development.Primordial germs(PGCs)refer to one of the basic groups of germ cell lineages,and then undergoes highly complex development after embryonic and postnatal birth,and eventually differentiates into oocytes or sperm according to individual's sex.The key events that occur during germ cell development include epigenetic reprogramming and meiotic reorganization,which respectively create a new generation of epigenetic and genetic diversity.It is worth noting that researchers have been using mouse and human pluripotent stem cells(PSCs)to restate these developmental pathways.These studies have clearly analyzed the key species differences in the developmental mechanism of mouse and human germ cells,highlighting the other breastfeeding the necessity of establishing an in vitro germ cell development system in animals(such as non-human primates).With further researches,including those involving non-human primate models,human gametogenesis may be completely reconstructed by PSCs,which will profoundly promote our understanding of human germ cell development and infertility.The purposes of this study are: 1)The induction of the primordial germ-like cells(PGCLCs)from primate pluripotent stem cells(PSCs)provides a powerful system to study the cellular and molecular mechanism underlying germline specification,which are difficult to study in vivo.2)A comprehensive single cell RNA sequencing(scRNA-seq)analysis of the developmental trajectory of PGCLCs differentiation.3)dissects the required niche of PGCLCs differentiation.The results showed: we applied the embryoid body(EB)differentiation system to induce PGCLCs specification from male and female monkey na?ve-state PSCs and then performed high throughput scRNA-seq analysis of approximately 40000 of na?ve cells and cells within EBs.We found that EBs provided a niche for PGCLCs differentiation by secreting growth factors critical for PGCLCs specification,such as BPM2,BMP4 and WNT3.Moreover,the developmental trajectory of PGCLCs was reconstituted and gene expression dynamics were revealed.Our study outlines the roadmap of PGCLCs specification from PSCs and provides insights that will improve the differentiation efficiency of PGCLCs from PSCs.