Reconciling the different roles of Gsk3β in “na?ve” and “primed” pluripotent stem cells

Signaling pathways orchestrated by PI3K/Akt, Raf/Mek/Erk and Wnt/β-catenin are known to play key roles in the self-renewal and differentiation of pluripotent stem cells. The serine/threonine protein kinase Gsk3β has roles in all three pathways, making its exact function difficult to decipher. Consequently, conflicting reports have implicated Gsk3β in promoting self-renewal, while others suggest that it performs roles in the activation of differentiation pathways. Different thresholds of Gsk3β activity also have different biological effects on pluripotent cells, making this situation even more complex. Here, we describe a further level of complexity that is most apparent when comparing “naïve” murine and “primed” human pluripotent stem cells. In naïve cells, Gsk3β activity is restrained by PI3K/Akt, but when released from inhibitory signals it antagonizes self-renewal pathways by targeting pluripotency factors such as Myc and Nanog. This situation also applies in primed cells, but, in addition, a separate pool of Gsk3β is required to suppress canonical Wnt signaling. These observations suggest that different Gsk3β-protein complexes shift the balance between naïve and primed pluripotent cells and identify fundamental differences in their cell signaling. Altogether, these findings have important implications for the mechanisms underpinning the establishment of different pluripotent cell states and for the control of self-renewal and differentiation.     

INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.     

Search for nave human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.     

Fate by RNA methylation: m6A steers stem cell pluripotency

The N⁶-methyladenosine (m⁶A) modification of mRNA has a crucial function in regulating pluripotency in murine stem cells: it facilitates resolution of naïve pluripotency towards differentiation.     

A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency

A short G1 phase is a characteristic feature of mouse embryonic stem cells (ESCs). To determine if there is a causal relationship between G1 phase restriction and pluripotency, we made use of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) reporter system to FACS-sort ESCs in the different cell cycle phases. Hence, the G1 phase cells appeared to be more susceptible to differentiation, particularly when ESCs self-renewed in the naïve state of pluripotency. Transitions from ground to naïve, then from naïve to primed states of pluripotency were associated with increased durations of the G1 phase, and cyclin E-mediated alteration of the G1/S transition altered the balance between self-renewal and differentiation. LIF withdrawal resulted in a lengthening of the G1 phase in naïve ESCs, which occurred prior to the appearance of early lineage-specific markers, and could be reversed upon LIF supplementation. We concluded that the short G1 phase observed in murine ESCs was a determinant of naïve pluripotency and was partially under the control of LIF signaling.     

Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

ObjectivesThe rats are crucial animal models for the basic medical researches. Rat embryonic stem cells (ESCs), which are widely studied, can self‐renew and exhibit pluripotency in long‐term culture, but the mechanism underlying how they exit pluripotency remains obscure. To investigate the key modulators on pluripotency exiting in rat ESCs, we perform genome‐wide screening using a unique rat haploid system.Materials and MethodsRat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. REX1 is a sensitive marker for the naïve pluripotency that is often utilized to monitor pluripotency exit, thus rat haESCs carrying a Rex1‐GFP reporter are used for genetic screening. Genome‐wide mutations are introduced into the genomes of rat Rex1‐GFP haESCs via piggyBac transposon, and differentiation‐retarded mutants are obtained after random differentiation selection. The exact mutations are elucidated by high‐throughput sequencing and bioinformatic analysis. The role of candidate mutation is validated in rat ESCs by knockout and overexpression experiments, and the phosphorylation of ERK1/2 (p‐ERK1/2) is determined by western blotting.ResultsHigh‐throughput sequencing analysis reveals numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrests the differentiation of rat ESCs by inhibiting the p‐ERK1/2, whereas overexpression of Thop1 promotes rat ESCs to exit from pluripotency.ConclusionsOur findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self‐renewal or pluripotency of rat ESCs.

Differentiation of pluripotent rat embryonic stem cells (ESCs) in vitro is difficult to achieve for unknown mechanisms. Rat haploid ESCs (haESCs) have been validated as a powerful tool to target unknown functional genes and pathways based on homozygous genetic screening. Xu et al. utilized Rex1‐GFP labelled‐rat haESCs to conduct genome‐scale screening of genes modulating pluripotency exiting. Validation experiments showed that Thop1 (one of the screened out genes) played very important roles in the random differentiation of rat ESCs in vitro via modulating phosphorylation of ERK.     

Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.     

Pluripotency-associated miR-290/302 family of microRNAs promote the dismantling of naive pluripotency

The molecular mechanism controlling the dismantling of naive pluripotency is poorly understood. Here we show that microRNAs (miRNAs) have important roles during naive to primed pluripotency transition. Dgcr8^−/− embryonic stem cells (ESCs) failed to completely silence the naive pluripotency program, as well as to establish the primed pluripotency program during differentiation. miRNA profiling revealed that expression levels of a large number of miRNAs changed dynamically and rapidly during naive to primed pluripotency transition. Furthermore, a miRNA screen identified numerous miRNAs promoting naive to primed pluripotency transition. Unexpectedly, multiple miRNAs from miR-290 and miR-302 clusters, previously shown as pluripotency-promoting miRNAs, demonstrated the strongest effects in silencing naive pluripotency. Knockout of both miR-290 and miR-302 clusters but not either alone blocked the silencing of naive pluripotency program. Mechanistically, the miR-290/302 family of miRNAs may facilitate the exit of naive pluripotency in part by promoting the activity of MEK pathway and through directly repressing Akt1. Our study reveals miRNAs as an important class of regulators potentiating ESCs to transition from naive to primed pluripotency, and uncovers context-dependent functions of the miR-290/302 family of miRNAs at different developmental stages.     

Live isolation of naïve ESCs via distinct glucose metabolism and stored glycogen

Naïve and primed pluripotent stem cells recapitulate the peri- and post-implantation development, respectively. Thus, investigation of distinct traits between each pluripotent stem cell type would shed light on early embryonic processes. Herein, by screening a fluorescent probe library, we found that intracellular glycogen led to specific reactivity to CDg4, a glycogen fluorescence sensor, in both human and mouse naïve embryonic stem cells (ESCs). The requirement of constant inhibition of Gsk3β as well as high oxidative phosphorylation (OxPHOS) in naïve compared to primed ESCs was closely associated to high level of intracellular glycogen in naïve ESCs. Both capacity of OxPHOS and stored glycogen, rescued naïve ESCs by transient inhibition of glycolysis, which selectively eliminated primed ESCs. Additionally, naïve ESCs with active OxPHOS were enriched from a mixture with primed ESCs by high reactivity to ATP-Red1, a mitochondrial ATP fluorescence probe. These results indicate the active OxPHOS and high intracellular glycogen as a novel “biomarker” delineating metabolic remodeling during the transition of naïve pluripotency.