Sox2, a key factor in the regulation of pluripotency and neural differentiation

Sex determining region Y-box 2(Sox2), a member of the SoxB1 transcription factor family, is an important transcriptional regulator in pluripotent stem cells(PSCs). Together with octamer-binding transcription factor 4 and Nanog, they co-operatively control gene expression in PSCs and maintain their pluripotency. Furthermore, Sox2 plays an essential role in somatic cell reprogram-ming, reversing the epigenetic configuration of differ-entiated cells back to a pluripotent embryonic state. In addition to its role in regulation of pluripotency, Sox2 is also a critical factor for directing the differentiation of PSCs to neural progenitors and for maintaining the properties of neural progenitor stem cells. Here, we review recent findings concerning the involvement of Sox2 in pluripotency, somatic cell reprogramming and neural differentiation as well as the molecular mecha-nisms underlying these roles.

LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells

LIN28A,an RNA-binding protein,plays an important role in porcine induced pluripotent stem cells(piPSCs).However,the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear.Here,we explored the function of LIN28A in piPSCs based on its overexpression and knockdown.We performed total RNA sequencing(RNA-seq)of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction(qRT-PCR),western blot analysis,and immunofluorescence staining.Results indicated that piPSC proliferation ability decreased following LIN28A knockdown.Furthermore,when LIN28A expression in the shLIN28A2 group was lower(by 20%)than that in the negative control knockdown group(shNC),the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells.Results also showed that LIN28A overexpression inhibited the expression of DUSP(dual-specificity phosphatases)family phosphatases and activated the mitogen-activated protein kinase(MAPK)signaling pathway.Thus,LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs.Our study provides a new resource for exploring the functions of LIN28A in piPSCs.

Acquisition of pluripotency in the chick embryo occurs during intrauterine embryonic development via a unique transcriptional network

Background: Acquisition of pluripotency by transcriptional regulatory factors is an initial developmental event that is required for regulation of cell fate and lineage specification during early embryonic development. The evolutionarily conserved core transcriptional factors regulating the pluripotency network in fishes, amphibians, and mammals have been elucidated. There are also species-specific maternally inherited transcriptional factors and their intricate transcriptional networks important in the acquisition of pluripotency. In avian species, however, the core transcriptional network that governs the acquisition of pluripotency during early embryonic development is not well understood.Results: We found that chicken NANOG(c NANOG) was expressed in the stages between the pre-ovulatory follicle and oocyte and was continuously detected in Eyal-Giladi and Kochav stage I(EGK.I) to X. However, c POUV was not expressed during fol iculogenesis, but began to be detectable between EGK.V and VI. Unexpectedly, c SOX2 could not be detected during fol iculogenesis and intrauterine embryonic development. Instead of c SOX2, c SOX3 was maternally inherited and continuously expressed during chicken intrauterine development. In addition, we found that the pluripotency-related genes such as c ENS-1, c KIT, c LIN28 A, c MYC, c PRDM14, and c SALL4 began to be dramatical y upregulated between EGK.VI and VII.Conclusion: These results suggest that chickens have a unique pluripotent circuitry since maternally inherited c NANOG and c SOX3 may play an important role in the initial acquisition of pluripotency. Moreover, the acquisition of pluripotency in chicken embryos occurs at around EGK.VI to VI I.

Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells

Stem cell pluripotency and differentiation are global processes regulated by several pathways that have been studied intensively over recent years. Nitric oxide(NO) is an important molecule that affects gene expression at the level of transcription and translation and regulates cell survival and proliferation in diverse cell types. In embryonic stem cells NO has a dual role, controlling differentiation and survival, but the molecular mechanisms by which it modulates these functions are not completely defined. NO is a physiological regulator of cell respiration through the inhibition of cytochrome c oxidase. Many researchers have been examining the role that NO plays in other aspects of metabolism such as the cellular bioenergetics state, the hypoxia response and the relationship of these areas to stem cell stemness.

In vitro induced pluripotency from urine-derived cells in porcine

BACKGROUND The generation of induced pluripotent stem cells(iPSC)has been a game-changer in translational and regenerative medicine;however,their large-scale applicability is still hampered by the scarcity of accessible,safe,and reproducible protocols.The porcine model is a large biomedical model that enables translational applications,including gene editing,long term in vivo and offspring analysis;therefore,suitable for both medicine and animal production.AIM To reprogramme in vitro into pluripotency,and herein urine-derived cells(UDCs)were isolated from porcine urine.METHODS The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4(OCT4),SRY-box2(SOX2),Kruppel-like factor 4(KLF4),and C-MYC,and cultured with basic fibroblast growth factor(bFGF)supplementation.To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase(AP),OCT4,SOX2,NANOG,TRA181 and SSEA 1 detection.Endogenous transcripts related to the pluripotency(OCT4,SOX2 and NANOG)were analyzed via reverse transcription quantitative realtime polymerase chain reaction in different time points during the culture,and all three lineages formed embryoid bodies(EBs)when cultured in suspension without bFGF supplementation.RESULTS The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming.Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors(mOSKM),but not from human factors(hOSKM).Three clonal lineages were isolated and further cultured for at least 28 passages,all the lineages were positive for AP detection,the OCT4,SOX2,NANOG markers,albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1,similar results were observed in the abundance of the endogenous transcripts related to pluripotent state.All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts.CONCLUSION For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotentlike state,enabling new numerous applications in both human or veterinary regenerative medicine.

Low complexity domains, condensates, and stem cell pluripotency

Biological reactions require self-assembly of factors in the complex cellular milieu.Recent evidence indicates that intrinsically disordered,low-complexity sequence domains(LCDs)found in regulatory factors mediate diverse cellular processes from gene expression to DNA repair to signal transduction,by enriching specific biomolecules in membraneless compartments or hubs that may undergo liquidliquid phase separation(LLPS).In this review,we discuss how embryonic stem cells take advantage of LCD-driven interactions to promote cell-specific transcription,DNA damage response,and DNA repair.We propose that LCDmediated interactions play key roles in stem cell maintenance and safeguarding genome integrity.

Mechanochemical mechanisms of embryonic stem cell pluripotency and differentiation

Embryonic stem (ES) cell biology is attracting much attention in cell biology because of their pluripotent behaviors and potential therapeutic applications. However,what maintains ES cell pluripotency and what triggers ES cell

Effect of Down-regulated Expression of cPouV and cNanog on Pluripotency of Chicken(Gallus gallus) Embryonic Stem Cells(cESCs)

To explore the pluripotency maintenance and update the functional influence of pluripotency genes cNanog and cPouV in chicken （ C,a/lus gallus） embry- onic stem cells （ cESCs）, the stable RNAi vectors pSuper-cNanog and pSuper-cPouV constructed previously were used to transfect cESCs. The mRNA levels of two target genes were detected with real- time PCR. These two genes were down-regulated since the 48^th and the down-reg-lation continued with the extension of time, the interference efficiency reached 65% at 96^th hour （P 〈0.05）. With the down-regulation of cNanog or cPouV gene, cESCs showed differentiation and prolifera- tion rate of these cells slowed down, the domed colony of these cells disappeared gradually when the edge of colony became irregular. At 96^th hour after transfection, the alkline phosphatase （AKP） and stage-specific embryonic antigen-1 （ SSEA-1 ） were not be detected in cNanog gene-knecked out eESCs, but it was done in that with cPouV gene -knocked out. The cPouV-suppressing cESCs were again transfected with pSuper-cNanog, the pluripotency markers AKP and SSEA-1 were both not found expressing at the 48^th hour. The results showed that cPouV and cNartog genes played an important role in maintaining pluripotency and self- renewal in cESCs, and cNanog gene was dominant. To sum up, our results may provide insights into the molecular regulation mechanism of avian during development.

Pluripotency of induced pluripotent stem cells

Recent studies have demonstrated that differentiated somatic cells from various mammalian species can be reprogrammed into induced pluripotent stem （iPS） cells by the ectopic expression of four transcription factors that are highly expressed in embryonic stem （ES） cells. The generation of patient-specific iPS cells directly from somatic cells without using oocytes or embryos holds great promise for curing numerous diseases that are currently unresponsive to traditional clinical approaches. However, some recent studies have argued that various iPS cell lines may still retain certain epigenetic memories that are inherited from the somatic cells. Such observations have raised concerns regarding the safety and efficacy of using iPS cell derivatives for clinical applications. Recently, our study demonstrated full pluripotency of mouse iPS cells by tetraploid complementation, indicating that it is possible to obtain fully reprogrammed iPS cells directly from differentiated somatic cells. Therefore, we propose in this review that further comprehensive studies of both mouse and human iPS cells are required so that additional information will be available for evaluating the quality of human iPS cells.

Human adult pluripotency: Facts and questions

Cellular reprogramming and induced pluripotent stem cell(IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine.While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium-and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues(such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.

Reprogramming of germ cells into pluripotency

Primordial germ cells(PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells

While Mek1/2 and Gsk3βinhibition("2i")supports the maintenance of murine embryonic stem cells(EsCs)in a homogenous naive state,prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential.Additionally,2i fails to support derivation and culture of fully potent female ESCs.Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin(AlbuMAx)undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture.Mechanisticaily,lipids in AlbuMAx impact intracellular metabolism including nucleotide biosynthesis,lipid biogenesis,and TCA cycle intermediates,with enhanced expression of DNMT3s that prevent DNA hypomethylation.Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation,which also alleviates X chromosome loss in female ESCs.Importantly,both male and female"all-ESc"mice can be generated from de novo derived ESCs using AlbuMAXbased media.Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

Trim28 citrullination maintains mouse embryonic stem cell pluripotency via regulating Nanog and Klf4 transcription

Protein citrullination,including histone H1 and H3 citrullination,is important for transcriptional regulation,DNA damage response,and pluripotency of embryonic stem cells(ESCs).Tripartite motif containing 28(Trim28),an embryonic development regulator involved in ESC self-renewal,has recently been identified as a novel substrate for citrullination by Padi4.However,the physiological functions of Trim28 citrullination and its role in regulating the chromatin structure and gene transcription of ESCs remain unknown.In this paper,we show that Trim28 is specifically citrullinated in mouse ESCs(m ESCs),and that the loss of Trim28 citrullination induces loss of pluripotency.Mechanistically,Trim28 citrullination enhances the interaction of Trim28with Smarcad1 and prevents chromatin condensation.Additionally,Trim28 citrullination regulates m ESC pluripotency by promoting transcription of Nanog and Klf4 which it does by increasing the enrichment of H3K27ac and H3K4me3 and decreasing the enrichment of H3K9me3 in the transcriptional regulatory region.Thus,our findings suggest that Trim28citrullination is the key for the epigenetic activation of pluripotency genes and pluripotency maintenance of ESCs.Together,these results uncover a role Trim28 citrullination plays in pluripotency regulation and provide novel insight into how citrullination of proteins other than histones regulates chromatin compaction.

Pluripotency and its layers of complexity

Pluripotency is depicted by a self-renewing state that can competently differentiate to form the three germ layers.Different stages of early murine development can be captured on a petri dish,delineating a spectrum of pluripotent states,ranging from embryonic stem cells,embryonic germ cells to epiblast stem cells.Anomalous cell populations displaying signs of pluripotency have also been uncovered,from the isolation of embryonic carcinoma cells to the derivation of induced pluripotent stem cells.Gaining insight into the molecular circuitry within these cell types enlightens us about the significance and contribution of each stage,hence deepening our understanding of vertebrate development.In this review,we aim to describe experimental milestones that led to the understanding of embryonic development and the conception of pluripotency.We also discuss attempts at exploring the realm of pluripotency with the identification of pluripotent stem cells within mouse teratocarcinomas and embryos,and the generation of pluripotent cells through nuclear reprogramming.In conclusion,we illustrate pluripotent cells derived from other organisms,including human derivatives,and describe current paradigms in the comprehension of human pluripotency.

The MORC2 p.S87L mutation reduces proliferation of pluripotent stem cells derived from a patient with the spinal muscular atrophy-like phenotype by inhibiting proliferation-related signaling pathways

Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.

Mechanism and methods to induce pluripotency

Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body.They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure.However,non-autologous cells will cause immune rejection.Induced pluripotent stem cell(iPSC)technology can convert somatic cells to the pluripotent state,and therefore offers a solution to this problem.Since the first generation of iPSCs,there has been an explosion of relevant research,from which we have learned much about the genetic networks and epigenetic landscape of pluripotency,as well as how to manipulate genes,epigenetics,and microRNAs to obtain iPSCs.In this review,we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency.We also highlight new problems emerging from iPSCs.Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine.

LncRNA Platr22 promotes super-enhancer activity and stem cell pluripotency

Super-enhancers(SEs)comprise large clusters of enhancers,which are co-occupied by multiple lineage-specific and master tran-scription factors,and play pivotal roles in regulating gene expression and cell fate determination.However,it is still largely un-known whether and how SEs are regulated by the noncoding portion of the genome.Here,through genome-wide analysis,wefound that tpng noncoding RNA(IncRNA)genes preferentially lie next to SEs.In mouse embryonic stem cells(mESCs),depletionof$E-associated IlncRNA transcripts dysregulated the activity of their nearby SEs.Specifically,we revealed a critical regulatoryrole of the IncRNA gene Platr22 in modulating the activity of a nearby SE and the expression of the nearby pluripotency regulatorZFP281.Through these regulatory events,Platr22 contributes to pluripotency maintenance and proper differentiation of mESCs.Mechanistically,Platr22 transcripts coat chromatin near the SE region and interact with DDX5 and hnRNP-L.DDX5 further recruitsp300 and other factors related to active transcription.We propose that these factors assemble into a transcription hub,thus pro-moting an open and active epigenetic chromatin state.0ur study highlights an unanticipated role for a class of lncRNAs in epige-netically controlling the activity and vulnerability to perturbation of nearby SEs for cell fate determination.

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(h ESCs).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 h ESCs.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.

Core pluripotency factors promote glycolysis of human embryonic stem cells by activating GLUT1 enhancer

Human embryonic stem cells (hESCs) depend on glycolysis for energy and substrates for biosynthesis. To understand the mechanisms governing the metabolism of hESCs, we investigated the transcriptional regulation of glucose transporter 1 (GLUT1, SLC2A1), a key glycolytic gene to maintain pluripotency. By combining the genome-wide data of bin ding sites of the core pluripotency factors (SOX2, OCT4, NANOG, denoted SON), chromosomal interaction and histone modification in hESCs, we identified a potential enhancer of the GLUT1 gene in hESCs, de noted GLUT1 enhancer (GE) element GE interacts with the promoter of GLUT1, and the deletion of GE significantly reduces the expression of GLUT1, glucose uptake and glycolysis of hESCs, confirming that GE is an enhancer of GLUT1 in hESCs. In addition, the mutation of SON binding motifs within GE reduced the expression of GLUT1 as well as the interaction between GE and GLUT1 promoter, indicating that the binding of SON to GE is important for its activity. Therefore, SON promotes glucose uptake and glycolysis in hESCs by inducing GLUT1 expression through directly activating the enhancer of GLUT1.

Induced Pluripotency for Translational Research

The advent of induced pluripotent stem cells （iPSCs） has revolutionized the concept of cellular reprogramming and potentially will solve the immunological compatibility issues that have so far hindered the application of human pluripotent stem cells in regenerative medicine. Recent findings showed that pluripotency is defined by a state of balanced lineage potency, which can be artificially instated through various procedures, including the conventional Yamanaka strategy. As a type of pluripotent stem cell, iPSCs are subject to the usual concerns over purity of differen- tiated derivatives and risks of tumor formation when used for cell-based therapy, though they pro- vide certain advantages in translational research, especially in the areas of personalized medicine, disease modeling and drug screening, iPSC-based technology, human embryonic stem cells （hESCs） and direct lineage conversion each will play distinct roles in specific aspects of translational medi- cine, and continue yielding surprises for scientists and the public.