Search

The transcription factors NANOG and POU5F1 (OCT4) play crucial roles in maintaining pluripotency in embryonic stem (ES) cells. While their functions have been well-studied, the specific interactions between NANOG and POU5F1 and their combined effects on pluripotency in ES-like and Epiblast cells remain less understood. Understanding these associations is vital for refining pluripotent stem cell characterization and advancing regenerative medicine. In this matter, we investigated the associations between NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells and how these interactions contribute to the distinct pluripotent states of these cells. In the present paper, we examined the pattern of NANOG expression by the immunocytochemical method in embryonic stem-like (ES-like) cells and compared it with its expression pattern in embryonic stem cells (ESCs). Similarly, we examined the expression pattern of POU5F1 in ES-like cells, ESCs, and epiblast cells and compared the expression pattern of these two genes with each other. On the other hand, using Fluidigm Biomark system analysis, we compared the amount of NANOG mRNA in these three cell lines and differentiated and undifferentiated Spermatogonial stem cells in several passages. Microscopic observations indicated the cytoplasmic expression of NANOG in the considered cells; moreover, they showed a similar expression pattern of NANOG with POU5F1 in the experimented cells. It has also been suggested that the more limited the cell's pluripotency, the lower the expression of these two genes. However, the decrease in NANOG expression is less than that of POU5F1. Fluidigm real-time RT-PCR analysis also confirmed these results. During the experimental process, protein-protein (PPI) network analysis shows a significant association of NANOG with other stem cell proteins, such as POU5F1. Our findings reveal distinct yet overlapping roles of NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells. The differential binding patterns and functional interactions between these factors underscore the complexity of pluripotency regulation in different stem cell states. This study provides new insights into the molecular mechanisms governing pluripotency and highlights potential targets for enhancing stem cell-based therapies., (© 2024. The Author(s).)

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cell nuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding., (© 2024. The Author(s).)

Naïve pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naïve pluripotency-specific transcription factors (TFs). Upon exiting naïve pluripotency, embryonic stem cells (ESCs) transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage choice. However, the mechanisms underlying disengagement from the naïve GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localisation of FoxO TFs in naïve ESCs. PTEN-mediated reduction of AKT activity upon exit from naïve pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for the activation of the formative pluripotency-specific GRN. Our work uncovers a pivotal role for FoxO TFs in establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition., (© 2024. The Author(s).)

Background: Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive., Results: By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes., Conclusions: Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity., (© 2024. The Author(s).)

MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency., (© 2024. The Author(s).)

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution., Competing Interests: Declaration of interests The authors declare no competing or financial interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Primordial germ cells (PGCs) are the embryonic precursors of sperm and oocytes, which transmit genetic/epigenetic information across generations. Mouse PGC and subsequent gamete development can be fully reconstituted in vitro, opening up new avenues for germ cell studies in biomedical research. However, PGCs show molecular differences between rodents and humans. Therefore, to establish an in vitro system that is closely related to humans, we studied PGC development in vivo and in vitro in the common marmoset monkey Callithrix jacchus ( cj ). Gonadal cjPGCs at embryonic day 74 express SOX17, AP2Ɣ, BLIMP1, NANOG, and OCT4A, which is reminiscent of human PGCs. We established transgene-free induced pluripotent stem cell (cjiPSC) lines from foetal and postnatal fibroblasts. These cjiPSCs, cultured in defined and feeder-free conditions, can be differentiated into precursors of mesendoderm and subsequently into cjPGC-like cells (cjPGCLCs) with a transcriptome similar to human PGCs/PGCLCs. Our results not only pave the way for studying PGC development in a non-human primate in vitro under experimentally controlled conditions, but also provide the opportunity to derive functional marmoset gametes in future studies., (© 2024 Kurlovich et al.)

Pluripotency is established in E4.5 preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of pluripotency, however, their gene expression signature only partially resembles that of developmental ground-state. Induced PRAMEL7 expression, a protein highly expressed in the ICM but lowly expressed in ESCs, reprograms developmentally advanced ESC+serum into ground-state pluripotency by inducing a gene expression signature close to developmental ground-state. However, how PRAMEL7 reprograms gene expression remains elusive. Here we show that PRAMEL7 associates with Cullin2 (CUL2) and this interaction is required to establish ground-state gene expression. PRAMEL7 recruits CUL2 to chromatin and targets regulators of repressive chromatin, including the NuRD complex, for proteasomal degradation. PRAMEL7 antagonizes NuRD-mediated repression of genes implicated in pluripotency by decreasing NuRD stability and promoter association in a CUL2-dependent manner. Our data link proteasome degradation pathways to ground-state gene expression, offering insights to generate in vitro models to reproduce the in vivo ground-state pluripotency., (© 2024. The Author(s).)

The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α +/+ ) and phosphorylation-deficient mutant eIF2α (eIF2α A/A ) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/β) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

All somatic cells develop from the epiblast, which occupies the upper layer of two-layered embryos and in most mammals is formed after the implantation stage but before gastrulation initiates. Once the epiblast is established, the epiblast cells begin to develop into various somatic cells via large-scale cell reorganization, namely, gastrulation. Different pluripotent stem cell lines representing distinct stages of embryogenesis have been established: mouse embryonic stem cells (mESCs), human embryonic stem cells (hESCs), and mouse epiblast stem cells (EpiSCs), which represent the preimplantation stage inner cell mass, an early  post-implantation stage epiblast, and a later-stage epiblast, respectively. Together, these cell lines provide excellent in vitro models of cell regulation before somatic cells develop. This chapter addresses these early developmental stages., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

In the last two decades, considerable progress has been made in the derivation of mammalian germ cells from pluripotent stem cells such as Embryonic Stem Cells (ESCs) and induced Pluripotent Stem Cells (iPSCs). The pluripotent stem cells are generally first induced into pre-gastrulating endoderm/mesoderm-like status and then specified into putative primordial germ cells (PGCs) termed PGC-like cells (PGCLCs) which possess the potential to generate oocytes and sperms. Adipose-derived mesenchymal stromal cells (ASCs) are multipotent cells, having the capacity to differentiate into cell types such as adipocytes, osteocytes and chondrocytes. Since no information is available about the capability of female human ASCs (hASCs) to generate PGCLCs, we compared protocols to produce such cells from hASCs themselves or from hASC-derived iPSCs. The results showed that, providing pre-induction into a peri-gastrulating endoderm/mesoderm-like status, hASCs can generate PGCLCs. This process, however, shows a lower efficiency than when hASC-derived iPSCs are used as starting cells. Although hASCs possess multipotency and express mesodermal genes, direct induction into PGCLCs resulted less efficient., (© 2023. The Author(s).)

Background: The heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/β-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature., Methods: Toxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds., Results: Obtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels., Conclusion: This study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs., Competing Interests: Declaration of Competing Interest None., (Published by Elsevier GmbH.)

MicroRNAs (miRNAs), small non-coding RNA molecules, regulate a wide range of critical biological processes, such as proliferation, cell cycle progression, differentiation, survival, and apoptosis, in many cell types. The regulatory functions of miRNAs in embryogenesis and stem cell properties have been extensively investigated since the early years of miRNA discovery. In this review, we will compare and discuss the impact of stem-cell-specific miRNA clusters on the maintenance and regulation of early embryonic development, pluripotency, and self-renewal of embryonic stem cells, particularly in vertebrates.

The interplay among mitogenic signaling pathways is crucial for proper embryogenesis. These pathways collaboratively act through intracellular master regulators to determine specific cell fates. Identifying the master regulators is critical to understanding embryogenesis and to developing new applications of pluripotent stem cells. In this report, we demonstrate protein kinase C (PKC) as an intrinsic master switch between embryonic and extraembryonic cell fates in the differentiation of human pluripotent stem cells (hPSCs). PKCs are essential to induce the extraembryonic lineage downstream of BMP4 and other mitogenic modulators. PKC-alpha (PKCα) suppresses BMP4-induced mesoderm differentiation, and PKC-delta (PKCδ) is required for trophoblast cell fate. PKC activation overrides mesoderm induction conditions and leads to extraembryonic fate. In contrast, PKC inhibition leads to β-catenin (CTNNB1) activation, switching cell fate from trophoblast to mesoderm lineages. This study establishes PKC as a signaling boundary directing the segregation of extraembryonic and embryonic lineages. The manipulation of intrinsic PKC activity could greatly enhance cell differentiation under mitogenic regulation in stem cell applications., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cancer continues to remain a "Black Box," as there is no consensus on how it initiates, progresses, metastasizes, or recurs. Many imponderables exist about whether somatic mutations initiate cancer, do cancer stem cells (CSCs) exist, and if yes, are they a result of de-differentiation or originate from tissue-resident stem cells; why do cancer cells express embryonic markers, and what leads to metastasis and recurrence. Currently, the detection of multiple solid cancers through liquid biopsy is based on circulating tumor cells (CTCs) or clusters, or circulating tumor DNA (ctDNA). However, quantity of starting material is usually adequate only when the tumor has grown beyond a certain size. We posit that pluripotent, endogenous, tissue-resident, very small embryonic-like stem cells (VSELs) that exist in small numbers in all adult tissues, exit from their quiescent state due to epigenetic changes in response to various insults and transform into CSCs to initiate cancer. VSELs and CSCs share properties like quiescence, pluripotency, self-renewal, immortality, plasticity, enrichment in side-population, mobilization, and resistance to oncotherapy. HrC test, developed by Epigeneres, offers the potential for early detection of cancer using a common set of VSEL/CSC specific bio-markers in peripheral blood. In addition, NGS studies on VSELs/CSCs/tissue-specific progenitors using the All Organ Biopsy (AOB) test provide exomic and transcriptomic information regarding impacted organ(s), cancer type/subtype, germline/somatic mutations, altered gene expressions, and dysregulated pathways. To conclude, HrC and AOB tests can confirm the absence of cancer and categorize the rest of subjects into low/moderate/high risk of cancer, and also monitor response to therapy, remission, and recurrence., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A comprehensive and precise definition of the pluripotency gene regulatory network (PGRN) is crucial for clarifying the regulatory mechanisms in embryonic stem cells (ESCs). Here, after a CRISPR/Cas9-based functional genomics screen and integrative analysis with other functional genomes, transcriptomes, proteomes and epigenome data, an expanded pluripotency-associated gene set is obtained, and a new PGRN with nine sub-classes is constructed. By integrating the DNA binding, epigenetic modification, chromatin conformation, and RNA expression profiles, the PGRN is resolved to six functionally independent transcriptional modules (CORE, MYC, PAF, PRC, PCGF and TBX). Spatiotemporal transcriptomics reveal activated CORE/MYC/PAF module activity and repressed PRC/PCGF/TBX module activity in both mouse ESCs (mESCs) and pluripotent cells of early embryos. Moreover, this module activity pattern is found to be shared by human ESCs (hESCs) and cancers. Thus, our results provide novel insights into elucidating the molecular basis of ESC pluripotency., (© 2023. The Author(s).)

The establishment of cellular identity is driven by transcriptional and epigenetic regulators of the chromatin proteome - the chromatome. Comprehensive analyses of the chromatome composition and dynamics can therefore greatly improve our understanding of gene regulatory mechanisms. Here, we developed an accurate mass spectrometry (MS)-based proteomic method called Chromatin Aggregation Capture (ChAC) followed by Data-Independent Acquisition (DIA) and analyzed chromatome reorganizations during major phases of pluripotency. This enabled us to generate a comprehensive atlas of proteomes, chromatomes, and chromatin affinities for the ground, formative and primed pluripotency states, and to pinpoint the specific binding and rearrangement of regulatory components. These comprehensive datasets combined with extensive analyses identified phase-specific factors like QSER1 and JADE1/2/3 and provide a detailed foundation for an in-depth understanding of mechanisms that govern the phased progression of pluripotency. The technical advances reported here can be readily applied to other models in development and disease., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pluripotency describes the ability of stem cells to differentiate into derivatives of the three germ layers. In reporting new human pluripotent stem cell lines, their clonal derivatives or the safety of differentiated derivatives for transplantation, assessment of pluripotency is essential. Historically, the ability to form teratomas in vivo containing different somatic cell types following injection into immunodeficient mice has been regarded as functional evidence of pluripotency. In addition, the teratomas formed can be analyzed for the presence of malignant cells. However, use of this assay has been subject to scrutiny for ethical reasons on animal use and due to the lack of standardization in how it is used, therefore questioning its accuracy. In vitro alternatives for assessing pluripotency have been developed such as ScoreCard and PluriTest. However, it is unknown whether this has resulted in reduced use of the teratoma assay. Here, we systematically reviewed how the teratoma assay was reported in publications between 1998 (when the first human embryonic stem cell line was described) and 2021. Our analysis of >400 publications showed that in contrast to expectations, reporting of the teratoma assay has not improved: methods are not yet standardized, and malignancy was examined in only a relatively small percentage of assays. In addition, its use has not decreased since the implementation of the ARRIVE guidelines on reduction of animal use (2010) or the introduction of ScoreCard (2015) and PluriTest (2011). The teratoma assay is still the preferred method to assess the presence of undifferentiated cells in a differentiated cell product for transplantation since the in vitro assays alone are not generally accepted by the regulatory authorities for safety assessment. This highlights the remaining need for an in vitro assay to test malignancy of stem cells.

Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitry that coordinates gene expression at multiple levels from chromatin to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis, yet the underlying molecular mechanisms remain largely unknown. Here, we identified RSL24D1 as highly expressed in both mouse and human pluripotent stem cells. RSL24D1 is associated with nuclear pre-ribosomes and is required for the biogenesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors and of components from the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact on differentiation, RSL24D1 depletion significantly alters ESC self-renewal and lineage commitment choices. Altogether, these results demonstrate that RSL24D1-dependant ribosome biogenesis is both required to sustain the expression of pluripotent transcriptional programs and to silence PRC2-regulated developmental programs, which concertedly dictate ESC homeostasis., (© 2023. The Author(s).)

We previously demonstrated gradual loss of epiblast during diapause in embryos lacking components of the LIF/IL6 receptor. Here, we explore the requirement for the downstream signalling transducer andactivator of transcription STAT3 and its target, TFCP2L1, in maintenance of naïve pluripotency. Unlike conventional markers, such as NANOG, which remains high in epiblast until implantation, both STAT3 and TFCP2L1 proteins decline during blastocyst expansion, but intensify in the embryonic region after induction of diapause, as observed visually and confirmed using our image-analysis pipeline, consistent with our previous transcriptional expression data. Embryos lacking STAT3 or TFCP2L1 underwent catastrophic loss of most of the inner cell mass during the first few days of diapause, indicating involvement of signals in addition to LIF/IL6 for sustaining naïve pluripotency in vivo. By blocking MEK/ERK signalling from the morula stage, we could derive embryonic stem cells with high efficiency from STAT3 null embryos, but not those lacking TFCP2L1, suggesting a hitherto unknown additional role for this essential STAT3 target in transition from embryo to embryonic stem cells in vitro. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Derivation of bovine embryonic stem cells from somatic cell nuclear transfer embryos enables the derivation of genetically matched pluripotent stem cell lines to valuable and well-characterized animals. In this chapter, we describe a step-by-step procedure for deriving bovine embryonic stem cells from whole blastocysts produced by somatic cell nuclear transfer. This simple method requires minimal manipulation of blastocyst-stage embryos, relies on commercially available reagents, supports trypsin passaging, and allows the generation of stable primed pluripotent stem cell lines in 3-4 weeks., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

The self-renewal and differentiation potential of embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. Expression levels of the core pluripotency factor Nanog are tightly regulated by a negative feedback autorepression loop. However, it remains unclear how ESCs perceive NANOG levels and execute autorepression. Here, we show that a dose-dependent induction of Fgfbp1 and Fgfr2 by NANOG activates autocrine-mediated ERK signaling in Nanog-high cells to trigger autorepression. pERK recruits NONO to the Nanog locus to repress transcription by preventing POL2 loading. This Nanog autorepression process establishes a self-perpetuating reciprocal NANOG-pERK regulatory circuit. We further demonstrate that this reciprocal regulatory circuit induces pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells. Collectively our data suggest that NANOG induces Fgfr2 and Fgfbp1 to activate ERK signaling in Nanog-high cells to establish a NANOG-pERK reciprocal regulatory circuit. This circuit regulates ERK signaling dynamics and Nanog autoregulation in pluripotent cells., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Pluripotent character is described as the potency of cells to differentiate into all three germ layers. The best example to reinstate the term lies in the context of embryonic stem cells (ESCs). Pluripotent ESC describes the in vitro status of those cells that originate during the complex process of embryogenesis. Pre-implantation to post-implantation development of embryo embrace cells with different levels of stemness. Currently, four states of pluripotency have been recognized, in the progressing order of "naïve," "poised," "formative," and "primed." Epigenetics act as the "conductor" in this "orchestra" of transition in pluripotent states. With a distinguishable gene expression profile, these four states associate with different epigenetic signatures, sometimes distinct while otherwise overlapping. The present review focuses on how epigenetic factors, including DNA methylation, bivalent chromatin, chromatin remodelers, chromatin/nuclear architecture, and microRNA, could dictate pluripotent states and their transition among themselves., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pluripotent cells in mouse embryos, which first emerge in the inner cell mass of the blastocyst, undergo gradual transition marked by changes in gene expression, developmental potential, polarity, and morphology as they develop from the pre-implantation until post-implantation gastrula stage. Recent studies of cultured mouse pluripotent stem cells (PSCs) have clarified the presence of intermediate pluripotent stages between the naïve pluripotent state represented by embryonic stem cells (ESCs-equivalent to the pre-implantation epiblast) and the primed pluripotent state represented by epiblast stem cells (EpiSCs-equivalent to the late post-implantation gastrula epiblast). In this review, we discuss these recent findings in light of our knowledge on peri-implantation mouse development and consider the implications of these new PSCs to understand their temporal sequence and the feasibility of using them as model system for pluripotency., (© 2022 The Authors.)

Endogenous retroviruses (ERVs) have been reported to participate in pre-implantation development of mammalian embryos. In early human embryogenesis, different ERV sub-families are activated in a highly stage-specific manner. How the specificity of ERV activation is achieved remains largely unknown. Here, we demonstrate the mechanism of how LTR7Ys, the human morula-blastocyst-specific HERVH long terminal repeats, are activated by the naive pluripotency transcription network. We find that KLF5 interacts with and rewires NANOG to bind and regulate LTR7Ys; in contrast, the primed-specific LTR7s are preferentially bound by NANOG in the absence of KLF5. The specific activation of LTR7Ys by KLF5 and NANOG in pluripotent stem cells contributes to human-specific naive pluripotency regulation. KLF5-LTR7Y axis also promotes the expression of trophectoderm genes and contributes to the expanded cell potential toward extra-embryonic lineage. Our study suggests that HERVs are activated by cell-state-specific transcription machinery and promote stage-specific transcription network and cell potency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Pluripotent, very small embryonic-like stem cells (VSELs) and the 'progenitors' endometrial stem cells (EnSCs) along with associated molecular changes in endometrial cancer, that developed seven months after neonatal exposure to estradiol in one of the sixty mice, were studied in the present study. Endocrine disruption affected both endometrium and myometrium, there was accumulation of endometrial fluid and significant hyperplasia. Disrupted endometrial-myometrial junction resulted in mobilization of myometrial cells into endometrium and epithelial and stromal cells into myometrium suggestive of adenomyosis. Markers specific for VSELs/ EnSCs (OCT-4, NANOG, SSEA-1, SCA-1, c-KIT) showed increased expression in uterine sections and marked upregulation of corresponding transcripts (Oct-4A, Oct-4, Sox-2, Nanog, Sca-1, c-Kit) was noted in RNA extracted from both uterine tissue and stem cells enriched from endometrial fluid. Hormonal receptors (ER-α, ER-β, PR, FSHR) were upregulated in both tumor sections and in endometrial fluid. ER-β and FSHR (Fshr3) expression was prominent suggesting a major role in endometrial cancer. Cancer cells showed global hypomethylation (reduced expression of 5-methyl cytosine), reduced expression of tumor suppressor gene (PTEN) and increased expression of cancer stem cells marker (CD166) which suggested dysregulation and aberrant oncogenic events. Increased expression of PCNA, Ki67, SOX-9 suggested excessive proliferation and hyperplasia which are predominant signs of endometrial cancer. Results suggest that VSELs increase in numbers and possibly transform into cancer stem cells (co-express CD166 and OCT-4) in endometrial cancer. Expression of OCT-4, CD133, ALDHA1 and CD166 in side-population cells from human endometrial cancer samples suggests a possible role of VSELs in human endometrial cancer as well., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The ectopic expression of the transcription factors OCT4, SOX2, KLF4 and MYC (OSKM) enables reprogramming of differentiated cells into pluripotent embryonic stem cells. Methods based on partial and reversible in vivo reprogramming are a promising strategy for tissue regeneration and rejuvenation. However, little is known about the barriers that impair reprogramming in an in vivo context. We report that natural killer (NK) cells significantly limit reprogramming, both in vitro and in vivo. Cells and tissues in the intermediate states of reprogramming upregulate the expression of NK-activating ligands, such as MULT1 and ICAM1. NK cells recognize and kill partially reprogrammed cells in a degranulation-dependent manner. Importantly, in vivo partial reprogramming is strongly reduced by adoptive transfer of NK cells, whereas it is significantly increased by their depletion. Notably, in the absence of NK cells, the pancreatic organoids derived from OSKM-expressing mice are remarkably large, suggesting that ablating NK surveillance favours the acquisition of progenitor-like properties. We conclude that NK cells pose an important barrier for in vivo reprogramming, and speculate that this concept may apply to other contexts of transient cellular plasticity., Competing Interests: Competing interests M.S. is shareholder of Senolytic Therapeutics, Rejuveron Senescence Therapeutics and Life Biosciences., (© 2022. Published by The Company of Biologists Ltd.)

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency., Competing Interests: Declaration of interests G.G. is a current employee of Patch Biosciences. S.E.V. is a current employee of Retro Biosciences. N.E.S. is an adviser to Vertex and Qiagen., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lima1 is an extensively studied prognostic marker of malignancy and is also considered to be a tumour suppressor, but its role in a developmental context of non-transformed cells is poorly understood. Here, we characterise the expression pattern and examined the function of Lima1 in mouse embryos and pluripotent stem cell lines. We identify that Lima1 expression is controlled by the naïve pluripotency circuit and is required for the suppression of membrane blebbing, as well as for proper mitochondrial energetics in embryonic stem cells. Moreover, forcing Lima1 expression enables primed mouse and human pluripotent stem cells to be incorporated into murine pre-implantation embryos. Thus, Lima1 is a key effector molecule that mediates the pluripotency control of membrane dynamics and cellular metabolism., (© 2022. The Author(s).)

The genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Pluripotent stem cells (PSCs) are promising tools for modern regenerative medicine applications because of their stemness properties, which include unlimited self-renewal and the ability to differentiate into all cell types in the body. Evidence suggests that a rare population of cells within a tumor, termed cancer stem cells (CSCs), exhibit stemness and phenotypic plasticity properties that are primarily responsible for resistance to chemotherapy, radiotherapy, metastasis, cancer development, and tumor relapse. Different therapeutic approaches that target CSCs have been developed for tumor eradication., Results and Discussion: In this review, we first provide an overview of different viewpoints about the origin of CSCs. Particular attention has been paid to views believe that CSCs are probably appeared through dysregulation of very small embryonic-like stem cells (VSELs) which reside in various tissues as the main candidate for tissue-specific stem cells. The expression of pluripotency markers in these two types of cells can strengthen the validity of this theory. In this regard, we discuss the common properties of CSCs and PSCs, and highlight the potential of PSCs in cancer studies, therapeutic applications, as well as educating the immune system against CSCs., Conclusion: In conclusion, the resemblance of CSCs to PSCs can provide an appropriate source of CSC-specific antigens through cultivation of PSCs which brings to light promising ideas for prophylactic and therapeutic cancer vaccine development., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Rabbit embryos develop as bilaminar discs at gastrulation as in humans and most other mammals, whereas rodents develop as egg cylinders. Primordial germ cells (PGCs) appear to originate during gastrulation according to many systematic studies on mammalian embryos. Here, we show that rabbit PGC (rbPGC) specification occurs at the posterior epiblast at the onset of gastrulation. Using newly derived rabbit pluripotent stem cells, we show robust and rapid induction of rbPGC-like cells in vitro with WNT and BMP morphogens, which reveals SOX17 as the critical regulator of rbPGC fate as in several non-rodent mammals. We posit that development as a bilaminar disc is a crucial determinant of the PGC regulators, regardless of the highly diverse development of extraembryonic tissues, including the amnion. We propose that investigations on rabbits with short gestation, large litters, and where gastrulation precedes implantation can contribute significantly to advances in early mammalian development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor. SeV-Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre-like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell-targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human-induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

To improve the recapitulative quality of human pluripotent stem cell (hPSC) differentiation, we removed exogenous haematopoietic cytokines from the defined differentiation system. Here, we show that endogenous stimuli and VEGF are sufficient to induce robust hPSC-derived haematopoiesis, intensive generation of haematopoietic progenitors, maturation of blood cells and the emergence of definitive precursor cells including those that phenotypically identical to early human embryonic haematopoietic stem cells (HSCs). Moreover, the cytokine-free system produces significantly higher numbers of haematopoietic progenitors compared to the published protocols. The removal of cytokines revealed a broad developmental potential of the early blood cells, stabilized the hPSC-derived definitive precursors and led to spontaneous activation of inflammatory signalling. Our cytokine-free protocol is simple, efficient, reproducible and applicable for embryonic stem cells (ESCs) and induced PSCs. The spectrum of recapitulative features of the novel protocol makes the cytokine-free differentiation a preferred model for studying the early human haematopoietic development., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of the pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways, including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg Effect. Until recently, metabolism has been regarded as a by-product of cell fate, however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg Effect. Both isoforms catalyze the final, rate limiting step of glycolysis, generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective of this study was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm, but also accumulate in differential subnuclear regions of mESC, mEpiLCs, and mEpiSCs as determined by a quantitative confocal microscopy employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 changes during the transition from mESCs, mEpiLCs, and mEpiSCs. Finally, we have comprehensively validated the appropriateness and power of the Pearson's correlation coefficient and Manders's overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 may assist with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative, and primed pluripotency., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polycomb repressive complex 2 (PRC2) deposits H3K27me3 on chromatin to silence transcription. PRC2 broadly interacts with RNAs. Currently, the role of the RNA-PRC2 interaction in human cardiogenesis remains elusive. Here, we found that human-specific heart brake lncRNA 1 (HBL1) interacted with two PRC2 subunits, JARID2 and EED, in human pluripotent stem cells (hPSCs). Loss of JARID2, EED or HBL1 significantly enhanced cardiac differentiation from hPSCs. HBL1 depletion disrupted genome-wide PRC2 occupancy and H3K27me3 chromatin modification on essential cardiogenic genes, and broadly enhanced cardiogenic gene transcription in undifferentiated hPSCs and later-on differentiation. In addition, ChIP-seq revealed reduced EED occupancy on 62 overlapped cardiogenic genes in HBL1-/- and JARID2-/- hPSCs, indicating that the epigenetic state of cardiogenic genes was determined by HBL1 and JARID2 at pluripotency stage. Furthermore, after cardiac development occurs, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved 'microRNA-1-JARID2' axis to modulate cardiogenic gene transcription. Overall, our findings delineate the indispensable role of HBL1 in guiding PRC2 function during early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to orchestrate human cardiogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

This study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4 , which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in fibroblast growth factor (FGF)- or leukemia inhibitory factor (LIF)-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. Porcine embryonic fibroblasts were coinfected with the p OCT4 -ΔPE-eGFP (distal enhancer [DE]-green fluorescent protein [GFP]) and p OCT4 -ΔDE-DsRed2 (proximal enhancer [PE]-red fluorescent protein [RFP]) vectors, and GFP and RFP expression were verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 DE and PE were activated in different expression patterns simultaneously as changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems confirmed that porcine iPSCs are in primed state after reprogramming in FGF2- or LIF-containing media. This work demonstrates the applicability of porcine OCT4 upstream region-derived dual fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.

A brain organoid is a self-organizing three-dimensional tissue derived from human embryonic stem cells or pluripotent stem cells and is able to simulate the architecture and functionality of the human brain. Brain organoid generation methods are abundant and continue to improve, and now, an in vivo vascularized brain organoid has been encouragingly reported. The combination of brain organoids with immune-staining and single-cell sequencing technology facilitates our understanding of brain organoids, including the structural organization and the diversity of cell types. Recent publications have reported that brain organoids can mimic the dynamic spatiotemporal process of early brain development, model various human brain disorders, and serve as an effective preclinical platform to test and guide personalized treatment. In this review, we introduce the current state of brain organoid differentiation strategies, summarize current progress and applications in the medical domain, and discuss the challenges and prospects of this promising technology.

Pluripotent stem cells (PSCs) have the ability of self-renewal that can retain the characteristics of the mother cell, and of pluripotency that can differentiate into several body types. PSCs typically include embryonic stem cells (ESCs) derived from the inner cell mass of the preimplantation embryo, and epiblast stem cells (EpiSCs) derived from the epiblast of postimplantation embryo. Although PSCs are able to be used by differentiation into endothelial cells as a potential treatment for vascular diseases, human ESCs and induced PSCs (iPSCs) are followed by ethical and safety issues. Pigs are anatomically and physiologically similar to humans. Therefore, the goal of this study was to establish an efficient protocol that differentiates porcine EpiSCs (pEpiSCs) into the endothelial cells for applying the treatment of human vascular diseases. As a result, alkaline phosphatase (AP)-negative (-) pEpiSCs cultured in endothelial cell growth basal medium-2 (EBM-2) differentiation medium in association with 50 ng/mL of vascular endothelial growth factor (VEGF) for 8 days were changed morphologically like the feature of endothelial cells, and expression of pluripotency-associated markers (OCT-3/4, NANOG, SOX2, and C-MYC) in porcine differentiated cells was significantly decreased ( p  < 0.05). Additionally, when pEpiSCs were cultured in EBM-2 + 50 ng/mL of VEGF, porcine differentiated cells represented a common endothelial cell marker positive (CD31+) but monocytes and lymphocytes marker negative (CD45-). Therefore, these results indicated that pEpiSCs cultured in EBM-2 + 50 ng/mL of VEGF culture condition were efficiently differentiated into endothelial cells for the treatment of blood vessel diseases.

The regulation of stem cell fate is poorly understood. Genetic studies in Caenorhabditis elegans lead to the hypothesis that a conserved cytoplasmic double-negative feedback loop consisting of the RNA-binding protein Trim71 and the let-7 microRNA controls the pluripotency and differentiation of stem cells. Although let-7-microRNA-mediated inhibition of Trim71 promotes differentiation, whether and how Trim71 regulates pluripotency and inhibits the let-7 microRNA are still unknown. Here, we show that Trim71 represses Ago2 mRNA translation in mouse embryonic stem cells. Blocking this repression leads to a specific post-transcriptional increase of mature let-7 microRNAs, resulting in let-7-dependent stemness defects and accelerated differentiation in the stem cells. These results not only support the Trim71-let-7-microRNA bi-stable switch model in controlling stem cell fate, but also reveal that repressing the conserved pro-differentiation let-7 microRNAs at the mature microRNA level by Ago2 availability is critical to maintaining pluripotency., Competing Interests: QL, XC, MN, SZ, WH No competing interests declared, (© 2021, Liu et al.)

The regulatory circuitry underlying embryonic stem (ES) cell self-renewal is well defined, but how this circuitry is disintegrated to enable lineage specification is unclear. RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and preliminary data suggest that they might regulate ES cell fate. By combining bioinformatic analyses with functional screening, we identified seven RBPs played important roles for the exit from pluripotency of ES cells. We characterized hnRNPLL, which mainly functions as a global regulator of alternative splicing in ES cells. Specifically, hnRNPLL promotes multiple ES cell-preferred exon skipping events during the onset of ES cell differentiation. hnRNPLL depletion thus leads to sustained expression of ES cell-preferred isoforms, resulting in a differentiation deficiency that causes developmental defects and growth impairment in hnRNPLL-KO mice. In particular, hnRNPLL-mediated alternative splicing of two transcription factors, Bptf and Tbx3, is important for pluripotency exit. These data uncover the critical role of RBPs in pluripotency exit and suggest the application of targeting RBPs in controlling ES cell fate., (© 2020 The Authors.)

Pluripotent embryonic stem cells (ESCs) are derived from early embryos and can differentiate into any type of cells in living organisms. Induced pluripotent stem cells (iPSCs) resemble ESCs, both of which serve as excellent sources to study early embryonic development and realize cell replacement therapies for age-related degenerative diseases and other cell dysfunction-related illnesses. To achieve these valuable applications, comprehensively understanding of the mechanisms underlying pluripotency maintenance and acquisition is critical. Ubiquitination modifies proteins with Ubiquitin (Ub) at the post-translational level to monitor protein stability and activity. It is extensively involved in pluripotency-specific regulatory networks in ESCs and iPSCs. Ubiquitination is achieved by sequential actions of the Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub ligase E3. Compared with E1s and E2s, E3s are most abundant, responsible for substrate selectivity and functional diversity. In this review, we focus on E3 ligases to discuss recent progresses in understanding how they regulate pluripotency and somatic cell reprogramming through ubiquitinating core ESC regulators.

To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.

The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2 - stem cell comparable with placenta-derived TS cells-they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2 + stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2 - stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The heterochromatin protein 1 (HP1) family members are canonical effectors and propagators of gene repression mediated by histone H3 lysine 9 (H3K9) methylation. HP1γ exhibits an increased interaction with active transcription elongation-associated factors in embryonic stem cells (ESCs) compared to somatic cells. However, whether this association has a functional consequence remains elusive. Here we find that genic HP1γ colocalizes and enhances enrichment of transcription elongation-associated H3K36me3 rather than H3K9me3. Unexpectedly, sustained H3K36me3 deposition is dependent on HP1γ. HP1γ-deleted ESCs display reduced H3K36me3 enrichment, concomitant with decreased expression at shared genes which function to maintain cellular homeostasis. Both the H3K9me3-binding chromodomain and histone binding ability of HP1γ are dispensable for maintaining H3K36me3 levels. Instead, the chromoshadow together with the hinge domain of HP1γ that confer protein and nucleic acid-binding ability are sufficient because they retain the ability to interact with NSD1, an H3K36 methyltransferase. HP1γ-deleted ESCs have a slower self-renewal rate and an impaired ability to differentiate towards cardiac mesoderm. Our findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell-like cells (hPGCLCs) derived from human-induced pluripotent stem cells (hiPSCs) can be propagated to at least ~10 6 -fold over a period of 4 months under a defined condition in vitro. During expansion, hPGCLCs maintain an early hPGC-like transcriptome and preserve their genome-wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to ~50-fold) and persist only around 1 week, yet undergo cell-autonomous genome-wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic-reconstituted ovaries, expanded hPGCLCs initiate genome-wide DNA demethylation and differentiate into oogonia/gonocyte-like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage., (© 2020 The Authors.)