Sharat Warrier

Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage‐specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency‐associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene‐free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long‐term single‐cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation. Stem Cells 2015;33:2686–2698

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.

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.

Establishment and Characterization of Naïve Pluripotency in Human Embryonic Stem Cells.

Mouse embryonic stem cells are known to represent the naïve state of pluripotency, while human embryonic stem cells typically represented the primed state of pluripotency, characterized by a higher drift toward differentiation and some other disadvantages. Here we describe an efficient method for rapid, transgene free induction of the naïve pluripotent state in human by applying a novel combination of small molecules and growth factors in the culture medium (2i, LIF, basic fibroblast growth factor, ascorbic acid, and forskolin). Conversion of primed human embryonic stem cells towards the naive pluripotent state should be confirmed by a detailed characterization of the cells, as described in this chapter.

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 naïve state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naïve stem cells. Editing efficiencies of respectively 1.3–8.4% and 3.8–19% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naïve hESCs were successfully differentiated to neural progenitor cells. As a proof-of-principle of our workflow, two monoclonal genome-edited naïve 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 naïve hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning.