Pluripotency state of mouse ES cells determines their contribution to self-organized layer formation by mesh closure on microstructured adhesion-limiting substrates.

Assembly of pluripotent stem cells to initiate self-organized tissue formation on engineered scaffolds is an important process in stem cell engineering. Pluripotent stem cells are known to exist in diverse pluripotency states, with heterogeneous subpopulations exhibiting differential gene expression levels, but how such diverse pluripotency states orchestrate tissue formation is still an unrevealed question. In this study, using microstructured adhesion-limiting substrates, we aimed to clarify the contribution to self-organized layer formation by mouse embryonic stem cells in different pluripotency states: ground and naïve state. We found that while ground state cells as well as sorted REX1-high expression cells formed discontinuous cell layers with limited lateral spread, naïve state cells could successfully self-organize to form a continuous layer by progressive mesh closure within 3 days. Using sequential immunofluorescence microscopy to examine the mesh closure process, we found that KRT8+ cells were particularly localized around unfilled holes, occasionally bridging the holes in a manner suggestive of their role in the closure process. These results highlight that compared with ground state cells, naïve state cells possess a higher capability to contribute to self-organized layer formation by mesh closure. Thus, this study provides insights with implications for the application of stem cells in scaffold-based tissue engineering.

Mettl14 is required for mouse postimplantation development by facilitating epiblast maturation.

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification of mammalian messenger and noncoding RNAs mediated by specific m6A writer, reader, and eraser proteins. As an m6A writer, the methyltransferase-like 3-methyltransferase-like 14 (METTL14)-Wilms tumor 1-associated protein complex dynamically regulates m6A modification and plays important roles in diverse biologic processes. However, our knowledge about the complete functions of this RNA methyltransferase complex, the contributions of each component to the methylation, and their effects on different biologic pathways are still limited. By using both in vivo and in vitro models, we here report that METTL14 is indispensable for postimplantation embryonic development by facilitating the conversion from naive to primed state of the epiblast. Depletion of Mettl14 leads to conspicuous embryonic growth retardation from embryonic d 6.5, mainly as a result of resistance to differentiation, which further leads to embryonic lethality early in gestation. Our data highlight the critical function of METTL14 as an m6A modification regulator in orchestrating early mouse embryogenesis.-Meng, T.-G., Lu, X., Guo, L., Hou, G.-M., Ma, X.-S., Li, Q.-N., Huang, L., Fan, L.-H., Zhao, Z.-H., Ou, X.-H., OuYang, Y.-C., Schatten, H., Li, L., Wang, Z.-B., Sun, Q.-Y. Mettl14 is required for mouse postimplantation development by facilitating epiblast maturation.

Stat3 activation is critical for pluripotency maintenance.

Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic transcription with many important functions, including regulation of cell proliferation, differentiation, survival, angiogenesis, and immune response. Besides, it plays critical roles in regulating the pluripotency. With the ability of self-renewal and differentiation, embryonic stem (ES) cells provide an unlimited source for cell transplantation. ES cells can maintain its undifferentiated state with leukemia inhibitory factor, the role which is achieved by the activation of the Stat3 pathway. Moreover, Stat3 activation is necessary for the naïve state maintenance of the ES cells and somatic stem cells reprogramming. This study presents an overview of the critical roles of Stat3 activation in the pluripotency maintenance of ES cells, somatic cell reprogramming, and naïve-primed pluripotent states conversion of ES cells.

Glycans define the stemness of naïve and primed pluripotent stem cells.

Cell surface glycans are tissue-specific and developmentally regulated. They function as essential modulators in cell-cell interactions, cell-extracellular matrix interactions, and ligand-receptor interactions, binding to various ligands, including Wnt, fibroblast growth factors, and bone morphogenetic proteins. Embryonic stem (ES) cells, originally derived from the inner cell mass of blastocysts, have the essential characteristics of pluripotency and self-renewal. Recently, it has been proposed that mouse and human conventional ES cells are present in different developmental stages, namely pre-implantation blastocyst and post-implantation blastocyst stages, also called the naïve state and the primed state, respectively. They therefore require different extrinsic signals for the maintenance of self-renewal and pluripotency, and also appear to require different surface glycans. Understanding of molecular mechanisms involving glycans in self-renewal and pluripotency of ES cells is increasingly important for potential clinical applications, as well as for basic research. This review focuses on the roles of glycans in the two different states of pluripotent stem cells, namely the naïve state and the primed state, and the transition between these two states.

Revealing cell populations catching the early stages of human embryo development in naive pluripotent stem cell cultures.

Naive human pluripotent stem cells (hPSCs) are defined as the in vitro counterpart of the human preimplantation embryo's epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period.

Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells.

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3-4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.

Label-free and non-destructive identification of naïve and primed embryonic stem cells based on differences in cellular metabolism.

Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging. This study reports an electrochemical method that enables simple, label-free, and non-destructive detection of naïve embryonic stem cells (ESCs) derived from mouse ESCs, based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)-glycolysis and oxidative phosphorylation (OXPHOS)-were blocked, and it was found that mitochondrial energy generation is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve-primed switchable metastable ESCs. The mouse PSCs derived from doxycycline-inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non-invasive and label-free manner. Thus, it can be useful in the development of stem cell-derived therapeutics.

An expedition in the jungle of pluripotent stem cells of non-human primates.

For nearly three decades, more than 80 embryonic stem cell lines and more than 100 induced pluripotent stem cell lines have been derived from New World monkeys, Old World monkeys, and great apes. In this comprehensive review, we examine these cell lines originating from marmoset, cynomolgus macaque, rhesus macaque, pig-tailed macaque, Japanese macaque, African green monkey, baboon, chimpanzee, bonobo, gorilla, and orangutan. We outline the methodologies implemented for their establishment, the culture protocols for their long-term maintenance, and their basic molecular characterization. Further, we spotlight any cell lines that express fluorescent reporters. Additionally, we compare these cell lines with human pluripotent stem cell lines, and we discuss cell lines reprogrammed into a pluripotent naive state, detailing the processes used to attain this. Last, we present the findings from the application of these cell lines in two emerging fields: intra- and interspecies embryonic chimeras and blastoids.

KLF17 promotes human naive pluripotency through repressing MAPK3 and ZIC2.

The pluripotent state of embryonic stem cells (ESCs) is regulated by a sophisticated network of transcription factors. High expression of KLF17 has recently been identified as a hallmark of naive state of human ESCs (hESCs). However, the functional role of KLF17 in naive state is not clear. Here, by employing various gain and loss-of-function approaches, we demonstrate that KLF17 is essential for the maintenance of naive state and promotes the primed to naive state transition in hESCs. Mechanistically, we identify MAPK3 and ZIC2 as two direct targets repressed by KLF17. Overexpression of MAPK3 or ZIC2 partially blocks KLF17 from promoting the naive pluripotency. Furthermore, we find that human and mouse homologs of KLF17 retain conserved functions in promoting naive pluripotency of both species. Finally, we show that Klf17 may be essential for early embryo development in mouse. These findings demonstrate the important and conserved function of KLF17 in promoting naive pluripotency and reveal two essential transcriptional targets of KLF17 that underlie its function.

Two Small Molecule Inhibitors Promote Reprogramming of Guangxi Bama Mini-Pig Mesenchymal Stem Cells Into Naive-Like State Induced Pluripotent Stem Cells.

Past researches have shown that pluripotency maintenance of naive and primed-state pluripotent stem cells (PSCs) depends on different signaling pathways, and naive-state PSCs possess the ability to produce chimeras when they are introduced into a blastocyst. Considering porcine is an attractive model for preclinical studies, many researches about pig induced pluripotent stem cells (piPSCs) have been reported. Some cytokines and small molecule compounds could transform primed piPSCs into naive state. However, there are no suitable culture conditions for generation of naive-state piPSCs with high efficiency; other small molecule compounds need further exploration. In this study, we investigated whether p38 MAPK and JNK signal pathway inhibitor SB203580 and SP600125 could be of benefit for acquiring naive-state piPSCs. By comparing reprogramming efficiencies under conditions of different donor cells and culture environment, we found that porcine bone marrow mesenchymal stem cells (PBMSCs) have higher efficiency on piPSC induction, and the culture condition of CHIR99021+PD0325901(2i)+Lif+bFGF is more suitable for subculturing of piPSCs. Our results also indicate that SB203580 and SP600125 could promote reprogramming of PBMSCs into naive-like state piPSCs. These results provide guidance for choosing donor cells, culture conditions, and research of different state iPSCs during the process of reprogramming pig somatic cells.

Human ES and iPS cells display less drug resistance than differentiated cells, and naïve-state induction further decreases drug resistance.

Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

microRNA regulation of pluripotent state transition.

microRNAs (miRNAs) play essential roles in mouse embryonic stem cells (ESCs) and early embryo development. The exact mechanism by which miRNAs regulate cell fate transition during embryo development is still not clear. Recent studies have identified and captured various pluripotent stem cells (PSCs) that share similar characteristics with cells from different stages of pre- and post-implantation embryos. These PSCs provide valuable models to understand miRNA functions in early mammalian development. In this short review, we will summarize recent work towards understanding the function and mechanism of miRNAs in regulating the transition or conversion between different pluripotent states. In addition, we will highlight unresolved questions and key future directions related to miRNAs in pluripotent state transition. Studies in these areas will further our understanding of miRNA functions in early embryo development, and may lead to practical means to control human PSCs for clinical applications in regenerative medicine.

Dppa3 is critical for Lin28a-regulated ES cells naïve-primed state conversion.

Lin28a is a pluripotent factor that promotes somatic cell reprogramming. Unlike other pluripotent factors, Lin28a expression is transient and accumulated in primed embryonic stem (ES) cells, but its exact function and mechanism in the conversion of ES cells from naïve to primed state remain unclear. Here, we present evidence for Dppa3, a protein originally known for its role in germ cell development, as a downstream target of Lin28a in naïve-primed conversion. Using rescue experiment, we demonstrate that Dppa3 functions predominantly downstream of Lin28a during naïve-primed state conversion. Higher level of Lin28a prevents let-7 maturation and results in Dnmt3a/b (target of let-7) upregulation, which in turn induces hypermethylation of the Dppa3 promoter. Dppa3 demarcates naïve versus primed pluripotency states. These results emphasize that Lin28a plays an important role during the naïve-primed state conversion of ES cells, which is partially mediated by a Lin28a-let-7-Dnmt3a/b-Dppa3 axis.

Unique molecular events during reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) at naïve state.

Derivation of human naïve cells in the ground state of pluripotency provides promising avenues for developmental biology studies and therapeutic manipulations. However, the molecular mechanisms involved in the establishment and maintenance of human naïve pluripotency remain poorly understood. Using the human inducible reprogramming system together with the 5iLAF naïve induction strategy, integrative analysis of transcriptional and epigenetic dynamics across the transition from human fibroblasts to naïve iPSCs revealed ordered waves of gene network activation sharing signatures with those found during embryonic development from late embryogenesis to pre-implantation stages. More importantly, Transcriptional analysis showed a significant transient reactivation of transcripts with 8-cell-stage-like characteristics in the late stage of reprogramming, suggesting transient activation of gene network with human zygotic genome activation (ZGA)-like signatures during the establishment of naïve pluripotency. Together, Dissecting the naïve reprogramming dynamics by integrative analysis improves the understanding of the molecular features involved in the generation of naïve pluripotency directly from somatic cells.

LincU Preserves Naïve Pluripotency by Restricting ERK Activity in Embryonic Stem Cells.

Although the functional roles of long noncoding RNAs (lncRNAs) have been increasingly identified, few lncRNAs that control the naïve state of embryonic stem cells (ESCs) are known. Here, we report a naïve-state-associated lncRNA, LincU, which is intrinsically activated by Nanog in mESCs. LincU-deficient mESCs exhibit a primed-like pluripotent state and potentiate the transition from the naïve state to the primed state, whereas ectopic LincU expression maintains mESCs in the naïve state. Mechanistically, we demonstrate that LincU binds and stabilizes the DUSP9 protein, an ERK-specific phosphatase, and then constitutively inhibits the ERK1/2 signaling pathway, which critically contributes to maintenance of the naïve state. Importantly, we reveal the functional role of LincU to be evolutionarily conserved in human. Therefore, our findings unveil LincU as a conserved lncRNA that intrinsically restricts MAPK/ERK activity and maintains the naïve state of ESCs.

Glycans in stem cell regulation: from Drosophila tissue stem cells to mammalian pluripotent stem cells.

Cell surface glycans, which are tissue-specific and developmentally regulated, work as essential modulators in ligand-receptor interactions, binding to various signal ligands including Wnt, Hedgehog, fibroblast growth factors, epidermal growth factors, and bone morphogenetic proteins, as well as in cell-cell interactions and cell-extracellular matrix interactions. These signals are essential for the stemness and differentiation of various kinds of stem cells. In addition, the intracellular O-linked N-acetylglucosamine, a form of glycosylation found only on nuclear or cytoplasmic proteins, regulates core transcription factors of stemness and phosphorylation of downstream signal components. Therefore, various kinds of glycans regulate the stem cell status; the structures of many of which are evolutionarily conserved from Drosophila to mammals. Understanding the molecular mechanisms of glycans in stemness and differentiation is increasingly important for innovative clinical applications, as well as for basic research. This Review focuses on the roles of glycans in Drosophila tissue stem cells and mammalian pluripotent stem cells.

Lineage-Specific Differentiation Is Influenced by State of Human Pluripotency.

Human pluripotent stem cells (hPSCs) have been reported in naive and primed states. However, the ability to generate mature cell types remains the imperative property for utility of hPSCs. Here, we reveal that the naive state enhances self-renewal while restricting lineage differentiation in vitro to neural default fate. Molecular analyses indicate expression of multiple lineage-associated transcripts in naive hPSCs that failed to predict biased functional differentiation capacity. Naive hPSCs can be converted to primed state over long-term serial passage that permits recovery of multi-germ layer differentiation. Suppression of OCT4 but not NANOG allows immediate recovery directly from naive state. To this end, we identified chemical inhibitors of OCT4 that restore naive hPSC differentiation. Our study reveals unique cell-fate restrictions in human pluripotent states and provides an approach to overcome these barriers that harness both efficient naive hPSC growth while maintaining in vitro differentiation essential for hPSC applications.

SMARCAD1 Contributes to the Regulation of Naive Pluripotency by Interacting with Histone Citrullination.

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R26Cit peaks and SMARCAD1 peaks. These data suggest a model in which SMARCAD1 regulates naive pluripotency by interacting with H3R26Cit and suppressing heterochromatin formation.

Dissecting the variation in transcriptional circuits between naive and primed pluripotent states.

Naive and primed pluripotent states are very similar to each other, but subtle differences exist in their maintenance and differentiation programmes. Transcription factors (TFs) play a key role towards maintaining pluripotency and cellular reprogramming. However, TF expression dynamics and regulatory mechanisms in naive and primed pluripotent states are poorly understood. Here, we performed a comprehensive transcriptional analysis of both states, which revealed a gene expression pattern in mESCs (naive state) that appear to be distinct from mEpiSCs (primed state). We screened 10 TFs essential for maintenance, self-renewal and differentiation, of which the TFs- Notch3, Meis1, Gli3 and Srf can act as novel markers distinguishing the two states. Furthermore, a detailed bioinformatic analysis (involving these TFs) elucidated essential transcriptional circuits between the naive and primed pluripotent states.

Naïve Induced Pluripotent Stem Cells Generated From ß-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9.

UNLABELLED: Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with ß-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. SIGNIFICANCE: In the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with ß-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved single-cell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as ß-thalassemia.