Project description:We sequenced embryoid bodies at two time points (18h, 96h) following a differentiation protocol to induce Primordial Germ Cell-like Cells (PGCLC) in a TFAP2A KO line and parental line
Project description:Current human primordial germ cell like cells (hPGCLCs) differentiation methods from pluripotent stem cells (PSCs) are inefficient, and it is challenging to generate sufficient hPGCLCs to optimize the next steps to achieve in vitro gametogenesis. We present a new differentiation method that uses diluted basement membrane extract (BME) and low BMP4 concentration to induce efficient hPGCLC differentiation, in scalable 2D cell culture. We show that BME overlay potentiates BMP/SMAD signaling, induced lumenogenesis and increases expression of key hPGCLC progenitor markers such as TFAP2A, EOMES, GATA3 and CDX2. These findings highlight the importance of factors in the BME in hPGCLC differentiation, and demonstrate the potential of the BME-overlay method as a new model for interrogating the formation of PGCs and amnion in humans.
Project description:As germ cell precursor, primordial germ cells (PGCs) are widely used in transgenic animal production, regenerative medicine and other fields. However, the regulation mechanism of chicken PGCs is not incomplete, which leads to the insufficient amount of chicken PGCs obtained in vitro, which seriously affects the specific application of PGCs. During PGC formation (differentiation from ESCs to PGCs), some proteins have inconsistent changes in transcription level and protein abundance. Mediating proteasome degradation is one of the most important roles of protein ubiquitination, and enrichment analysis of transcriptome and proteome also suggests an important role of ubiquitination in the process of PGCs. In order to explore the important functions and potential targets of ubiquitination, we collected chicken ESCs and PGCs cells for label free ubiquitomics analysis. This study preliminarily analyzed how ubiquitination regulates the formation of chicken PGCs, providing a theoretical basis for the subsequent research and specific application of PGCs.
Project description:Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as high-throughput genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpressing 4 transcriptional factors – Nanog and three PGC master regulators Prdm1, Prdm14 and Tfap2c - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. The overexpression of Nanog enhances the PGC regulatory network and suppresses differentiation of somatic lineages, enabling a significant improvement in the efficiency of PGCLC production. Transcriptomic analysis reveals that differentiated PGCLCs exhibit similarities to in vivo PGCs and are more advanced compared to cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture, and could differentiate into spermatogonium-like cells in vitro. Importantly, the ability to produce PGCLC differentiation at scale enables biochemical and functional genomic screens to dissect mechanisms of germ cell development and infertility.
Project description:Transgenic StellaGFP ESCs were used to derive primordial germ cells during embryoid body (EB) differentiation, and microarry analysis used to compared FACS sorted Stella-positive cells of day 7 Ebs with the parental ESCs and Stella-negative cells of day 7 Ebs. Keywords: in vitro, primordial germ cells, embryonic stem cells, stella
Project description:Primordial germ cells (PGCs), the embryonic precursors of eggs and sperm, are a unique model for identifying and studying regulatory mechanisms in singly migrating cells. From their time of specification to eventual colonization of the gonad, mouse PGCs traverse through and interact with many different cell types, including epithelial cells and mesenchymal tissues. Work in drosophila and zebrafish have identified many genes and signaling pathways involved in PGC migration, but little is known about this process in mammals. We have generated a point mutation in the Ror2 gene that we know disrupts primordial germ cell migration in the developing mouse embryo. We used microarray analysis to determine if this defect is mediated through genome-wide or pathway-specific transcriptional changes. We analyzed primordial germ cells (PGCs) from 4 wild-type (WT) and 4 Ror2Y324C/Y324C mutant embryos using Oct4-DPE-EGFP. PGCs were collected during their active migratory state at embryonic day 9.5 (somite range 20-25).
Project description:In vitro gametogenesis is the process of making germline cells from human pluripotent stem cells. The foundation of this model is the quality of the first progenitors called primordial germ cells (PGCs), which in vivo are specified during the peri-implantation window of human development. Here, we show using human embryo attachment culture that hPGC specification begins at day 12 post fertilization. Using single cell RNA-sequencing of hPGC-like cells (hPGCLCs) differentiated from pluripotent stem cells we discovered that hPGCLC specification involves resetting pluripotency towards a transitional state with shared characteristics between naïve and primed pluripotency followed by differentiation into lineage primed TFAP2A+ progenitors. Applying the germline trajectory to TFAP2C mutants reveals that TFAP2C functions in the TFAP2A+ progenitors upstream of PRDM1 to regulate the expression of SOX17. This serves to protect hPGCLCs from crossing the Weismann’s barrier to adopt somatic cell fates and therefore is an essential mechanism for successfully initiating in vitro gametogenesis.