CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells

The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naive state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naive stem cells. Editing efficiencies of respectively 1.3-8.4% and 3.819% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naive hESCs were successfully differentiated to neural progenitor cells. As a proofof- principle of our workflow, two monoclonal genome-edited naive hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naive hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning

Influence of activin A supplementation during human embryonic stem cell derivation on germ cell differentiation potential

Human embryonic stem cells (hESCs) are more similar to primed mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro

Direct comparison of distinct naive pluripotent states in human embryonic stem cells

Until recently, human embryonic stem cells (hESCs) were shown to exist in a state of primed pluripotency, while mouse embryonic stem cells (mESCs) display a naive or primed pluripotent state. Here we show the rapid conversion of in-house-derived primed hESCs on mouse embryonic feeder layer (MEF) to a naive state within 5-6 days in naive conversion media (NCM-MEF), 6-10 days in naive human stem cell media (NHSM-MEF) and 14-20 days using the reverse-toggle protocol (RT-MEF). We further observe enhanced unbiased lineage-specific differentiation potential of naive hESCs converted in NCM-MEF, however, all naive hESCs fail to differentiate towards functional cell types. RNA-seq analysis reveals a divergent role of PI3K/AKT/mTORC signalling, specifically of the mTORC2 subunit, in the different naive hESCs. Overall, we demonstrate a direct evaluation of several naive culture conditions performed in the same laboratory, thereby contributing to an unbiased, more in-depth understanding of different naive hESCs

Elucidating the role of coding and noncoding genes in neuronal development using human embryonic stem cells and the CRISPR/Cas9 system

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Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation