Project description:We performed RNA-Seq analyses on 15 human fetal samples at 53-137 days of development, 9 female and 5 male, and identified the transcriptional changes during the transition of human cKIT+ primordial germ cells (PGCs), the precursors of gametes, to the generation of Advanced Germline Cells. Comparing the transcriptional profile of PGCs to that of H1 and UCLA1 hESCs identifies differences between the two cell types and pinpoints molecules that can be used in the development of in vitro germ cell differentiation protocols starting from human pluripotent stem cells. RNA-Seq of cKIT+ cells analyzed from 6 biological samples for testes and 9 samples for ovaries from 53-137 days. 2 biological replicates of TRA-1-81+ cells sorted from H1 and UCLA1 hESCs. WGBS of cKIT+ cells analyzed from 4 biological samples of ovaries and 1 biological sample of testes at 57-137 days of development.
Project description:Generation of research quality, clinically relevant cell types in vitro from human pluripotent stem cells (hPSCs) requires detailed understanding of the equivalent cell types in humans. Here we analyzed 130 human fetal samples at 6-20 weeks of development and identified the stages in which human cKIT+ primordial germ cells (PGCs), the precursors of gametes, undergo whole genome epigenetic reprogramming and ultimately initiation of imprint erasure with loss of both 5mC and 5-hydroxy-mC at differentially methylated regions. Using five alternate in vitro differentiation strategies combined with a single-cell microfluidic analysis, high throughput RNA sequencing and a bona fide human cKIT+ PGC signature, we show that hPSC differentiation generates a rare cKIT+ PGC subtype found in both the human fetal gonad and mouse embryo. Taken together, our study creates a resource of human germ line ontogeny that is absolutely essential for future studies aimed at interpreting in vitro differentiation of the human germ line. cKIT+ cells analyzed from 2 biological samples for testes and 2 samples for ovaries at 16 and 16.5 weeks. 3 biological replicates of TRA-1-60+ cells sorted from H1 hESCs
Project description:We designed this experiment to investigate the transcriptional changes in gonads as a result of sex transformation. Here we performed transcriptional profiling of the ovary transformed into testis from the tra loss of function (XX_tra_lof), testis transformed into ovary from the tra gain of function (XY_tra_gof) and ovary transformed into testis in dsxM gain of function (XX_DsxM_gof/lof) Drosophila melanogaster third instar larvae in biological quadruplicates. In addition, as controls we sequenced ovaries and testes from the female and male wildtype larvae respectively. We constructed polyA+ libraries of the gonads, cleaned off the fatbody and performed 50 bp, stranded single-end RNA-Seq.
Project description:We performed WGBS analyses on 6 human fetal samples at 53-137 days of development, 4 female and 2 male. We show that methylation reprogramming in the human germline is global yet incomplete with exons, 3’UTRs and human-specific transposons remaining methylated. Whole Genome Bisulfite-Seq of cKIT+ cells analyzed from 4 biological samples for fetal ovaries from 57-113 days of development and 2 samples for fetal testes at 59 and 137 days of development.
Project description:We performed RNA-Seq analyses on 15 human fetal samples at 53-137 days of development, 9 female and 5 male, and identified the transcriptional changes during the transition of human cKIT+ primordial germ cells (PGCs), the precursors of gametes, to the generation of Advanced Germline Cells. Comparing the transcriptional profile of PGCs to that of H1 and UCLA1 hESCs identifies differences between the two cell types and pinpoints molecules that can be used in the development of in vitro germ cell differentiation protocols starting from human pluripotent stem cells.
Project description:We developed a mRNA transfection-based method for the efficient generation of primordial germ cell like cells (PGCLCs) in marmoset. Single cell RNA-seq analyses (10X) confirmed that the induced marmoset PGCLCs show transcriptome profile similar to in vivo PGCs. For comparison we sequenced iPSCs, E74 ovaries, E82 ovaries, NB ovaries, E87 testes, and day 22 testes.
Project description:Oscillatory gene expression is fundamental to mammalian development, but technologies to monitor expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applications to a number of data sets, include a single-cell RNA-seq data set of human embroyonic stem cells (hESCs), demonstrate advantages of the approach and also identify a potential artifact in the Fluidigm C1 platform. Total 213 H1 single cells and 247 H1-Fucci single cells were sequenced. The 213 H1 cells were used to evaluate Oscope in identifying oscillatory genes. The H1-Fucci cells were used to confirm the cell cycle gene cluster identified by Oscope in the H1 hESCs.
Project description:Human induced Pluripotent Stem cells (iPSCs) can be generated by enforced-expression of OCT3/4, SOX2, KLF4 and c-MYC (OSKM) via intermediate state that is labeled with a surface antigen TRA-1-60. However the event during conversion of somatic cells to TRA-1-60 positive (+) fate by OSKM is still unclear. Here we show that TRA-1-60 (+) cells emerged from ESRG (+) cells with proliferation independent manner. Overcoming the proliferation pausing phase is crucial for further progression of reprogramming. Inactivation of RB pathway is associated with the expansion of early TRA-1-60 (+) cells. Indeed, extended RB activation drastically decreased the proportion of TRA-1-60 (+) cells with marked premature senescence. An RNA-binding protein LIN41 promotes the RB inactivation and facilitates reprogramming efficiency in early stage via post-transcriptional suppression of cycline dependent kinase inhibitor p21. This LIN41/p21/RB axis was invalidated in immortalized cells which showed a resistance to oncogene-induced senescence. Thus, LIN41- mediated RB inactivation promotes to bail out of transient quiescence as a major blockade in initial phase of reprogramming.
