Stem cell pluripotency and transcription factor Oct4

Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells.

Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog

Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family.The rest ofnanog contains no apparent homology to any known proteins characterized so far.It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes.To test this hypothesis,we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs.Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities.Despite the fact that it contains no apparent transactivation motifs,the C-terminal domain is about 7 times as active as the N-terminal one.This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.

Nanog and transcriptional networks in embryonic stem cell pluripotency

Several extrinsic signals such as LIF, BMP and Wnt can support the self-renewal and pluripotency of embryonic stem （ES） cells through regulating the ＂pluripotent genes.＂ A unique homeobox transcription factor, Nanog, is one of the key downstream effectors of these signals. Elevated level of Nanog can maintain the mouse ES cell self-renewal independent of LIF and enable human ES cell growth without feeder cells. In addition to the external signal pathways, intrinsic transcription factors such as FoxD3, P53 and Oct4 are also involved in regulating the expression of Nanog. Functionally, Nanog works together with other key pluripotent factors such as Oct4 and Sox2 to control a set of target genes that have important functions in ES cell pluripotency. These key factors form a regulatory network to support or limit each other＇s expression level, which maintains the properties of ES cells.

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.

Four recombinant pluripotency transcriptional factors containing a protein transduction domain maintained the in vitro pluripotency of chicken embryonic stem cells

Long-term in vitro maintenance of embryonic stem cell (ESC) pluripotency enables the pluripotency and differentiation of ESCs in animals to be investigated. The ability to successfully maintain and differentiate chicken embryonic stem cells (cESCs) would provide a useful tool for avian biology research and would be a resource directly applicable to agricultural production. In this study, endogenous chicken pluripotency transcription factors, POUV, Sox-2, Nanog and Lin28 were cloned and expressed as recombinant proteins containing a nine consecutive arginine protein transduction domain (PTD). cESCs were cultured with these recombinant proteins to maintain cESC pluripotency in vitro. Cultured cESCs exhibited typical characteristics of pluripotency, even after six generations of rapid doubling, including positive staining for stage-specific embryonic antigen I, and strong staining for alkaline phosphatase. Expression levels of the pluripotency markers, POUV, Nanog, C-Myc, Sox-2 and Lin28 were the same as in uncultured stage X blastoderm cells, and most significantly, the formation of embryoid bodies (EBs) by 6th generation cESCs confirmed the ability of these cultured cESCs to differentiate into cells of all three embryonic germ layers. Thus, transcription factors could be translocated through the cell membrane into the intracellular space of cESCs by using a PTD of nine consecutive arginines and the pluripotency of cESCs could be maintained in vitro for at least six generations.

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.

TRPC3 is required for the survival, pluripotency and neural differentiation of mouse embryonic stem cells(mESCs)

Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells(mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout(KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes.Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.

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.

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.

Germ layer formation during Xenopus embryogenesis: the balance between pluripotency and differentiation

The African clawed frog, Xenopus laevis, has long been a model animal for the studies in the fields of animal cloning, developmental biology, biochemistry, cell biology, and physiology. With the aid of Xenopus, major molecular mechanisms that are involved in embryonic development have been understood. Germ layer formation is the first event of embryonic cellular differentiation, which is induced by a few key maternal factors and subsequently by zygotic signals. Meanwhile, another type of signals, the pluripotency factors in ES cells, which maintain the undifferentiated state, are also present during early embryonic cells. In this review, the functions of the pluripotency factors during Xenopus germ layer formation and the regulatory relationship between the signals that promote differentiation and pluripotency factors are discussed.