Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset be expanded feeders with doubling time around 24 h. WNT inhibition stabilises process. transcriptome reset diverges markedly from that primed PSCs shares features inner cell mass (ICM)....

The plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols the differentiation retinal organoids from embryonic have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we developed a protocol efficient generation large, 3D-stratified that does not require evagination optic-vesicle-like structures, so far limited organoid yield. Analysis gene expression in...

The activation of the embryonic genome marks first major wave transcription in developing organism. Zygotic (ZGA) mouse 2-cell embryos and 8-cell humans is crucial for development. Here, we report discovery human 8-cell-like cells (8CLCs) among naive stem cells, which transcriptionally resemble embryo. They express ZGA markers, including ZSCAN4 LEUTX, transposable elements, such as HERVL MLT2A1. 8CLCs show reduced SOX2 levels can be identified using TPRX1 H3.Y marker proteins vitro....

In primates, the amnion emerges through cavitation of epiblast during implantation, whereas in other species it does so later at gastrulation by folding ectoderm. How mechanisms amniogenesis diversified evolution remains unknown. Unexpectedly, single-cell analysis primate embryos uncovered two transcriptionally and temporally distinct waves. To study this, we employed naive-to-primed transition human pluripotent stem cells (hPSCs) to model peri-implantation development. Partially primed...

Transgenesis is a cornerstone of molecular biology. The ability to integrate specifically engineered piece DNA into the genome living system fundamental our efforts understand life and exploit its implications for medicine, nanotechnology bioprospecting. However, transgenesis has been hampered by position effects multi-copy integration problems, which are mainly due use small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned bacterial artificial chromosomes...

Human naïve pluripotent stem cells (PSCs) share features with the pre-implantation epiblast. They therefore provide an unmatched opportunity for characterising developmental programme of pluripotency in Homo sapiens Here, we confirm that PSCs do not respond directly to germ layer induction, but must first acquire competence. Capacitation multi-lineage differentiation occurs without exogenous growth factor stimulation and is facilitated by inhibition Wnt signalling. Whole-transcriptome...

In contrast to conventional human pluripotent stem cells (hPSCs) that are related post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive established by resetting hPSCs, or derived from dissociated inner cell masses. Here we investigate conditions for transgene-free reprogramming somatic pluripotency. We find Wnt inhibition promotes RNA-mediated induction demonstrate application independent fibroblast cultures and endothelial progenitor cells. show...

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation skeletal tissues. However the identity BM MSC subpopulations is poorly defined mainly due to absence specific markers allowing in situ localization those and isolation pure cell types. Here, we aimed at characterization surface mouse MSCs their subsets distinct differentiation potential. Using conditionally immortalized performed screening 176 antibodies...

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or replacement therapy. Type I diabetes is considered major target applications due to the shortage primary human beta cells. Several protocols have been reported generating pancreatic progenitors by vitro differentiation Here we first assessed one these on panel lines capacity engender glucose sensitive insulin-producing after...

Developmental timing differs strikingly between mammals. All embryonic and some placental lineages emerge from the pluripotent epiblast in a temporally defined sequence, lasting about two weeks human embryo, but only days mice. Moreover, order of lineage segregation gene expression differ species. We used stem cells to recapitulate this window development vitro. Simultaneous profiling chromatin accessibility single revealed robust, autonomous switch cell states during process. reconstructed...

Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some the limitations conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated transcriptionally closer postimplantation than This prompted us comprehensively study...

Abstract Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because transgene accurately recapitulates expression endogenous gene. However, method demanding and integrity copy number BAC difficult control. Here, we describe a simpler pronuclear injection that relies on transposition introduce full‐length BACs into mouse genome. The backbone hPAX6‐GFP reporter was retrofitted with PiggyBac transposon inverted terminal repeats...

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form CGD (X-CGD), which due mutations in CYBB (gp91phox) gene, a component oxidase, accounts about two-thirds cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox...

Abstract Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, can be achieved by the reversible expression immortalizing proteins. Here, we describe use conditional based on modified tetracycline‐regulated system SV40 large T‐antigen in embryonic stem (ES) cells mice. The relies codon improved reverse tetracycline transactivator (irtTA) fused to ligand‐binding domain (LBD) androgen receptor (irtTA‐ABD) or mutated...

ABSTRACT Gene duplication events can drive evolution by providing genetic material for new gene functions, and they create opportunities diverse developmental strategies to emerge between species. To study the contribution of duplicated genes human early development, we examined function NANOGP1, a tandem duplicate transcription factor NANOG. We found that NANOGP1 NANOG have overlapping but distinct expression profiles, with high restricted epiblast cells naïve-state pluripotent stem cells....

Naïve human pluripotent stem cells (hPSC) resemble the embryonic epiblast at an earlier time-point in development than conventional, 'primed' hPSC. We present a comprehensive miRNA profiling of naïve-to-primed transition hPSC, process recapitulating aspects early vivo embryogenesis. identify miR-143-3p and miR-22-3p as markers naïve state miR-363-5p, several members miR-17 family, miR-302 family primed markers. uncover that miR-371-373 are highly expressed MiR-371-373 homologs mouse miR-290...

Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural variations, combinations a format suitable downstream analysis. Here, we describe strategy targeted isolation large regions (∼35 kb) from genomes that also applicable any interest. method relies on recombineering fish out target fosmid clones pools...

Bone, cartilage, and marrow adipocytes are generated by skeletal progenitors, but the relationships between lineages mechanisms controlling their differentiation poorly understood. We established mouse clonal progenitors with distinct properties analyzed transcriptome. Unipotent osteogenic adipogenic cells expressed specific transcriptional programs, whereas bipotent clones combined expression of those genes did not show a unique signature. tested potential regulators lineage commitment...

ABSTRACT In contrast to conventional human pluripotent stem cells (hPSC) that are related post-implantation embryo stages, naïve hPSC exhibit features of pre-implantation epiblast. Naïve established by resetting hPSC, or derived from dissociated inner cell masses. Here we investigate conditions for transgene-free reprogramming somatic pluripotency. We find tankyrase inhibition promotes RNA-mediated induction demonstrate application independent fibroblast cultures and endothelial progenitor...

SUMMARY Much attention has focussed on conversion of human pluripotent stem cells (PSC) to a more naive developmental status. Here we provide method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset be expanded feeders with doubling time around 24 hours. WNT inhibition stabilises the process. transcriptome reset diverges markedly from primed shares features inner cell mass (ICM)....