Rapid Human Oogonia-like Cell Specification via Combinatorial Transcription Factor-Directed Differentiation [BiSulfite-seq]
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PROVIDER: PRJNA1143036 | ENA |
REPOSITORIES: ENA
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Project description:Rapid Human Oogonia-like Cell Specification via Combinatorial Transcription Factor-Directed Differentiation [barcode]
| PRJNA1142963 | ENA
Project description:Rapid Human Oogonia-like Cell Specification via Combinatorial Transcription Factor-Directed Differentiation [RNA-seq]
| PRJNA1141981 | ENA
Project description:The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive modeling and medicine. Methods to recapitulate advanced germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described graph theory pipeline and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX whose combinatorial overexpression drives DDX4+ induced oogonia-like cell (iOLC) formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We additionally demonstrate a method of post-isolation, feeder-free expansion of DDX4+ iOLCs that shows retained cell identity in vitro. We additionally identify DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their broad similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research.
2024-08-01 | GSE273772 | GEO
Project description:The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive modeling and medicine. Methods to recapitulate advanced germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described graph theory pipeline and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX whose combinatorial overexpression drives DDX4+ induced oogonia-like cell (iOLC) formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We additionally demonstrate a method of post-isolation, feeder-free expansion of DDX4+ iOLCs that shows retained cell identity in vitro. We additionally identify DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their broad similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research.
2024-08-01 | GSE273748 | GEO
Project description:The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive modeling and medicine. Methods to recapitulate advanced germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described graph theory pipeline and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX whose combinatorial overexpression drives DDX4+ induced oogonia-like cell (iOLC) formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We additionally demonstrate a method of post-isolation, feeder-free expansion of DDX4+ iOLCs that shows retained cell identity in vitro. We additionally identify DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their broad similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research.
2024-07-31 | GSE273465 | GEO
Project description: Not available
| S-BSST1527 | biostudies-other
Project description:The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.
2021-02-16 | GSE154690 | GEO
Project description:Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the epigenetic mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of early populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole genome bisulfite sequencing, chromatin immunoprecipitation-sequencing and RNA-Sequencing reveals unique events associated with specification towards each lineage. While we observe expected dynamics such as loss of DNA methylation and gain of H3K4me1 at distal putative regulatory elements, we frequently found a germ layer specific switch to H3K27me3 at sites of high DNA methylation in the undifferentiated state. By carefully dissecting these initial events, we may be able to devise more faithful differentiation strategies and gain novel insights in to the robust rewiring of regulatory programs during cellular transitions. 10 Samples in total, 5 in replicate. To better understand the interplay of epigenetic dynamics and transcription factor binding upon in vitro specification of human embryonic stem cells we profiled OCT4, SOX2 and NANOG in hESC and the endoderm master regulatory factor FOXA2 in in vitro derived endoderm cells (dEN). To gain further insights into the relation of DNA methylation and TF binding, we carried out ChIP-bisulfite sequencing for FOXA2 in dEN. Lastly, we were interested in the fate of genes bound by FOXA2 in dEN upon further differentiation and therefore differentiated dEN further towards a hepatocyte like state and performed RNA-Seq.
2013-05-01 | E-GEOD-46130 | biostudies-arrayexpress
Project description:Illumina-based whole genome bisulfite sequencing libraries for 4 RNA-directed DNA methylation mutants Examining DNA methylation levels in leaf tissue of RNA-directed DNA methylation mutants
2012-05-26 | E-GEOD-36143 | biostudies-arrayexpress
Project description:Illumina-based whole genome bisulfite sequencing libraries for 4 RNA-directed DNA methylation mutants
2012-05-26 | GSE36143 | GEO