[Expression and significance analysis of GDF3 in testicular cancer based on TCGA and GTEx databases].

OBJECTIVE: To investigate the expression and significance of GDF3 in testicular cancer through bioinformatics analysis. METHODS: Using the TCGA and GTEx databases, differential expression analysis and pan-cancer analysis were performed to identify the target gene GDF3, and the clinical relevance of GDF3 in testicular cancer was analyzed using the UALCAN database. Based on the R packages "org.Hs.eg.db" and "clusterProfiler," gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to explore the potential functions of GDF3 in testicular cancer. The correlation of GDF3 with immune chemokines and immune inhibitors in testicular cancer was investigated using the TISIDB database. RESULTS: The GDF3 was significantly upregulated in testicular cancer (P<0.001) and closely associated with clinical staging (P<0.05) and tumor subtypes (P<0.001). The immune-related analysis revealed that GDF3 was strongly correlated with immune chemokines CCL26 (rho=0.599, P<0.001), CCL7 (rho=0.525, P<0.001), immune inhibitor ADORA2A (rho=0.723, P<0.001), and PVRL2 (rho=0.585, P<0.001). CONCLUSION: The GDF3 is closely related to the occurrence, development, and immune microenvironment of testicular cancer.

Brd4 modulates diet-induced obesity via PPARγ-dependent Gdf3 expression in adipose tissue macrophages.

Macrophage-mediated inflammatory response has been implicated in the pathogenesis of obesity and insulin resistance. Brd4 has emerged as a key regulator in the innate immune response. However, the role of Brd4 in obesity-associated inflammation and insulin resistance remains uncharacterized. Here, we demonstrated that myeloid lineage-specific Brd4 knockout (Brd4-CKO) mice were protected from high-fat diet-induced (HFD-induced) obesity with less fat accumulation, higher energy expenditure, and increased lipolysis in adipose tissue. Brd4-CKO mice fed a HFD also displayed reduced local and systemic inflammation with improved insulin sensitivity. RNA-Seq of adipose tissue macrophages (ATMs) from HFD-fed WT and Brd4-CKO mice revealed that expression of antilipolytic factor Gdf3 was significantly decreased in ATMs of Brd4-CKO mice. We also found that Brd4 bound to the promoter and enhancers of Gdf3 to facilitate PPARγ-dependent Gdf3 expression in macrophages. Furthermore, Brd4-mediated expression of Gdf3 acted as a paracrine signal targeting adipocytes to suppress the expression of lipases and the associated lipolysis in cultured cells and mice. Controlling the expression of Gdf3 in ATMs could be one of the mechanisms by which Brd4 modulates lipid metabolism and diet-induced obesity. This study suggests that Brd4 could be a potential therapeutic target for obesity and insulin resistance.

Administration of GDF3 Into Septic Mice Improves Survival via Enhancing LXRα-Mediated Macrophage Phagocytosis.

The defective eradication of invading pathogens is a major cause of death in sepsis. As professional phagocytic cells, macrophages actively engulf/kill microorganisms and play essential roles in innate immune response against pathogens. Growth differentiation factor 3 (GDF3) was previously implicated as an important modulator of inflammatory response upon acute sterile injury. In this study, administration of recombinant GDF3 protein (rGDF3) either before or after CLP surgery remarkably improved mouse survival, along with significant reductions in bacterial load, plasma pro-inflammatory cytokine levels, and organ damage. Notably, our in vitro experiments revealed that rGDF3 treatment substantially promoted macrophage phagocytosis and intracellular killing of bacteria in a dose-dependent manner. Mechanistically, RNA-seq analysis results showed that CD5L, known to be regulated by liver X receptor α (LXRα), was the most significantly upregulated gene in rGDF3-treated macrophages. Furthermore, we observed that rGDF3 could promote LXRα nuclear translocation and thereby, augmented phagocytosis activity in macrophages, which was similar as LXRα agonist GW3965 did. By contrast, pre-treating macrophages with LXRα antagonist GSK2033 abolished beneficial effects of rGDF3 in macrophages. In addition, rGDF3 treatment failed to enhance bacteria uptake and killing in LXRα-knockout (KO) macrophages. Taken together, these results uncover that GDF3 may represent a novel mediator for controlling bacterial infection.

Obesity-Linked PPARγ S273 Phosphorylation Promotes Insulin Resistance through Growth Differentiation Factor 3.

The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn't been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling in vitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3.

GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype.

Macrophages are critical for regulation of inflammatory response during endotoxemia and septic shock. However, the mediators underlying their regulatory function remain obscure. Growth differentiation factor 3 (GDF3), a member of transforming growth factor beta (TGF-ß) superfamily, has been implicated in inflammatory response. Nonetheless, the role of GDF3 in macrophage-regulated endotoxemia/sepsis is unknown. Here, we show that serum GDF3 levels in septic patients are elevated and strongly correlate with severity of sepsis and 28-day mortality. Interestingly, macrophages treated with recombinant GDF3 protein (rGDF3) exhibit greatly reduced production of pro-inflammatory cytokines, comparing to controls upon endotoxin challenge. Moreover, acute administration of rGDF3 to endotoxin-treated mice suppresses macrophage infiltration to the heart, attenuates systemic and cardiac inflammation with less pro-inflammatory macrophages (M1) and more anti-inflammatory macrophages (M2), as well as prolongs mouse survival. Mechanistically, GDF3 is able to activate Smad2/Smad3 phosphorylation, and consequently inhibits the expression of nod-like receptor protein-3 (NLRP3) in macrophages. Accordingly, blockade of Smad2/Smad3 phosphorylation with SB431542 significantly offsets rGDF3-mediated anti-inflammatory effects. Taken together, this study uncovers that GDF3, as a novel sepsis-associated factor, may have a dual role in the pathophysiology of sepsis. Acute administration of rGDF3 into endotoxic shock mice could increase survival outcome and improve cardiac function through anti-inflammatory response by suppression of M1 macrophage phenotype. However, constitutive high levels of GDF3 in human sepsis patients are associated with lethality, suggesting that GDF3 may promote macrophage polarization toward M2 phenotype which could lead to immunosuppression.

Derivation of notochordal cells from human embryonic stem cells reveals unique regulatory networks by single cell-transcriptomics.

Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.

Phylogenetic evidence for independent origins of GDF1 and GDF3 genes in anurans and mammals.

Growth differentiation factors 1 (GDF1) and 3 (GDF3) are members of the transforming growth factor superfamily (TGF-ß) that is involved in fundamental early-developmental processes that are conserved across vertebrates. The evolutionary history of these genes is still under debate due to ambiguous definitions of homologous relationships among vertebrates. Thus, the goal of this study was to unravel the evolution of the GDF1 and GDF3 genes of vertebrates, emphasizing the understanding of homologous relationships and their evolutionary origin. Our results revealed that the GDF1 and GDF3 genes found in anurans and mammals are the products of independent duplication events of an ancestral gene in the ancestor of each of these lineages. The main implication of this result is that the GDF1 and GDF3 genes of anurans and mammals are not 1:1 orthologs. In other words, genes that participate in fundamental processes during early development have been reinvented two independent times during the evolutionary history of tetrapods.

Insulin Regulates Lipolysis and Fat Mass by Upregulating Growth/Differentiation Factor 3 in Adipose Tissue Macrophages.

Previous genetic studies in mice have shown that functional loss of activin receptor-like kinase 7 (ALK7), a type I transforming growth factor-ß receptor, increases lipolysis to resist fat accumulation in adipocytes. Although growth/differentiation factor 3 (GDF3) has been suggested to function as a ligand of ALK7 under nutrient-excess conditions, it is unknown how GDF3 production is regulated. Here, we show that a physiologically low level of insulin converts CD11c- adipose tissue macrophages (ATMs) into GDF3-producing CD11c+ macrophages ex vivo and directs ALK7-dependent accumulation of fat in vivo. Depletion of ATMs by clodronate upregulates adipose lipases and reduces fat mass in ALK7-intact obese mice, but not in their ALK7-deficient counterparts. Furthermore, depletion of ATMs or transplantation of GDF3-deficient bone marrow negates the in vivo effects of insulin on both lipolysis and fat accumulation in ALK7-intact mice. The GDF3-ALK7 axis between ATMs and adipocytes represents a previously unrecognized mechanism by which insulin regulates both fat metabolism and mass.

Functional and In Silico Assessment of GDF3 Gene Variants in a Chinese Congenital Scoliosis Population.

BACKGROUND The present study aimed to evaluate the pathogenicity of 5 [i]GDF3[/i] gene variations using functional and [i]in silico[/i] assessment approaches in a Chinese congenital scoliosis population. MATERIAL AND METHODS We selected 13 patients carrying 5 variants from a congenital scoliosis cohort. The PCR products of samples were verified by Sanger sequencing. The data and sequence alignment were analyzed using Chromas and ClustalW. SIFT and PolyPhen-2 were used to predict the functional effects of each missense and amino acid substitutions. SWISS-MODEL server and Swiss-PdbViewer were used to analyze conformational changes of GDF3 structure. DUET, UCSF Chimera, and Ligplot software were used to further explore the protein stability, side chains, and hydrophobic interaction changes, respectively. Luciferase reporter gene and Western blot assays were used to perform functional assessments for every variant from the molecular level. RESULTS Of the 13 patients, the S212L variant reoccurred in 9 patients. The rest of the patients carried 1 missense mutation each. The variants of R84L and R84C were predicted as probably damaging [i]loci[/i]. S212L, N215S, A251T were predicted as benign [i]loci[/i]. In functional assays, R84L, S212L, and A251T display inhibitory effects on functional assays. N251S mutation showed a negative effect in protein expression assays but not in luciferase reporter gene assays. The variant of R84C displayed no negative effects on 2 functional assays. CONCLUSIONS Our results suggest that the 4 of the 5 variants in [i]GDF3[/i] gene contribute different pathogenicity in congenital scoliosis, which may provide molecular evidence for clinical genetic testing.

Involvement of Bone Morphogenetic Proteins (BMP) in the Regulation of Ovarian Function.

Primordial germ cells migrate to the fetal gonads and proliferate during gestation to generate a fixed complement of primordial follicles, the so-called ovarian reserve. Primordial follicles comprise an oocyte arrested at the diplotene stage of meiosis, surrounded by a layer of pregranulosa cells. Activation of primordial follicles to grow beyond this arrested stage is of particular interest because, once activated, they are subjected to regulatory mechanisms involved in growth, selection, maturation, and ultimately, ovulation or atresia. The vast majority of follicles succumb to atresia and are permanently lost from the quiescent or growing pool of follicles. The bone morphogenetic proteins (BMPs), together with other intraovarian growth factors, are intimately involved in regulation of follicle recruitment, dominant follicle selection, ovulation, and atresia. Activation of primordial follicles appears to be a continuous process, and the number of small antral follicles at the beginning of the menstrual cycle provides an indirect indication of ovarian reserve. Continued antral follicle development during the follicular phase of the menstrual cycle is driven by follicle stimulating hormone (FSH) and luteinizing hormone (LH) in conjunction with many intraovarian growth factors and inhibitors interrelated in a complex web of regulatory balance. The BMP signaling system has a major intraovarian role in many species, including the human, in the generation of transcription factors that influence proliferation, steroidogenesis, cell differentiation, and maturation prior to ovulation, as well as formation of corpora lutea after ovulation. At the anterior pituitary level, BMPs also contribute to the regulation of gonadotrophin production.

Comparative characteristics of laryngeal-resident mesenchymal stromal cell populations isolated from distinct sites in the rat larynx.

BACKGROUND: Although tissue-resident mesenchymal stromal cells (MSCs) in the larynx have been described, their distinct characteristics and roles have not been thoroughly explored. Therefore, we investigated stem cell characteristics and regenerative potentials of single clonal populations isolated from rat epiglottic mucosa (EM), lamina propria (LP), and macula flava (MF) to determine whether they comprised laryngeal tissue-resident stem cells. METHODS: Single clonal laryngeal cells were isolated following microdissection of the EM, LP, and MF from the rat larynx. Several clonal populations from the three laryngeal subsites were selected and expanded in vitro. We compared the stem cell characteristics of self-renewal and differentiation potential, as well as the cell surface phenotypes and gene expression profiles, of laryngeal MSC-like cells to that of bone marrow MSCs (BM-MSCs). We also investigated the regenerative potential of the laryngeal cells in a radiation-induced laryngeal injury animal model. RESULTS: Self-renewing, clonal cell populations were obtained from rat EM, LP, and MF. EM-derived and LP-derived clonal cells had fibroblast-like features, while MF-resident clonal cells had stellate cell morphology and lipid droplets containing vitamin A. All laryngeal clonal cell populations had MSC-like cell surface marker expression (CD29, CD44, CD73, and CD90) and the potential to differentiate into bone and cartilage cell lineages; EM-derived and MF-derived cells, but not LP-derived cells, were also able to differentiate into adipocytes. Clonal cells isolated from the laryngeal subsites exhibited differential extracellular matrix-related gene expression. We found that the mesenchymal and stellate cell-related genes desmin and nestin were enriched in laryngeal MSC-like cells relative to BM-MSCs (P < 0.001). Growth differentiation factor 3 (GDF3) and glial fibrillary acidic protein (GFAP) transcript and protein levels were higher in MF-derived cells than in other laryngeal populations (P < 0.001). At 4 weeks after transplantation, laryngeal MF-derived and EM-derived cells contributed to laryngeal epithelial and/or glandular regeneration in response to radiation injury. CONCLUSIONS: These results suggest that cell populations with MSC characteristics reside in the EM, LP, and MF of the larynx. Laryngeal MSC-like cells contribute to regeneration of the larynx following injury; further investigation is needed to clarify the differential roles of the populations in laryngeal tissue regeneration, as well as the clinical implications for the treatment of laryngeal disease.

Inflammasome-driven catecholamine catabolism in macrophages blunts lipolysis during ageing.

Catecholamine-induced lipolysis, the first step in the generation of energy substrates by the hydrolysis of triglycerides, declines with age. The defect in the mobilization of free fatty acids in the elderly is accompanied by increased visceral adiposity, lower exercise capacity, failure to maintain core body temperature during cold stress, and reduced ability to survive starvation. Although catecholamine signalling in adipocytes is normal in the elderly, how lipolysis is impaired in ageing remains unknown. Here we show that adipose tissue macrophages regulate the age-related reduction in adipocyte lipolysis in mice by lowering the bioavailability of noradrenaline. Unexpectedly, unbiased whole-transcriptome analyses of adipose macrophages revealed that ageing upregulates genes that control catecholamine degradation in an NLRP3 inflammasome-dependent manner. Deletion of NLRP3 in ageing restored catecholamine-induced lipolysis by downregulating growth differentiation factor-3 (GDF3) and monoamine oxidase A (MAOA) that is known to degrade noradrenaline. Consistent with this, deletion of GDF3 in inflammasome-activated macrophages improved lipolysis by decreasing levels of MAOA and caspase-1. Furthermore, inhibition of MAOA reversed the age-related reduction in noradrenaline concentration in adipose tissue, and restored lipolysis with increased levels of the key lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Our study reveals that targeting neuro-immunometabolic signalling between the sympathetic nervous system and macrophages may offer new approaches to mitigate chronic inflammation-induced metabolic impairment and functional decline.

Identification and characterization of secreted factors that are upregulated during somatic cell reprogramming.

The process of cell reprogramming has been characterized considerably since the successful generation of induced pluripotent stem cells. However, the importance of cell-cell communications for cellular reprogramming remains largely unknown. Secreted factors, which are expressed and secreted during reprogramming, may influence the reprogramming efficiency. Here, we have identified Sostdc1, Glb1l2, Fetub, Dpp4, Gdf3, Trh, and Tdgf1 as prominently upregulated secreted factors during reprogramming. Our detailed analysis reveals that these seven factors may be categorized into four groups based on their expression patterns in relation to the reprogramming stages. Remarkably, knockdown of Sostdc1, which is the most prominently upregulated factor and which is expressed earlier than the other six factors, results in reduced reprogramming efficiency, suggesting its involvement in the reprogramming process.

Growth and Differentiation Factor 3 Is Transcriptionally Regulated by OCT4 in Human Embryonic Carcinoma Cells.

Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor ß ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.

Two girls with short stature, short neck, vertebral anomalies, Sprengel deformity and intellectual disability.

Here, we report two unrelated girls with prenatal onset short stature, short neck, cervical vertebral anomalies, Sprengel deformity, and mild intellectual disability. The association of these features first suggested a syndromic form of Klippel-Feil anomaly. We therefore analyzed the three known disease causing genes and the candidate gene PAX1. However, direct sequencing of GDF6, GDF3, PAX1, and MEOX1 failed to identify any mutation. To our knowledge, the phenotype we report has not been described previously, leading us to speculate that this condition may represent a new syndrome.

A novel variation of GDF3 in Chinese Han children with a broad phenotypic spectrum of non-syndromic CHDs.

BACKGROUND: The GDF3 gene plays a fundamental role in embryonic morphogenesis. Recent studies have indicated that GDF3 plays a previously unrecognised role in cardiovascular system development. Non-syndromic CHDs might be a clinically isolated manifestation of GDF3 mutations. The purpose of the present study was to identify potential pathological mutations in the GDF3 gene in Chinese children with non-syndromic CHDs, and to gain insight into the aetiology of non-syndromic CHDs. METHODS: A total of 200 non-syndromic CHDs patients and 202 normal control patients were sampled. There were two exons of the human GDF3 gene amplified using polymerase chain reaction. The polymerase chain reaction products were purified and directly sequenced. RESULTS: One missense mutation (c.C635T, p.Ser212 Leu, phenotype: isolated muscular ventricular septal defect) was found that has not been reported previously. CONCLUSIONS: To the best of our knowledge, this is the first study to investigate the role of the GDF3 gene in non-syndromic CHDs. Our results expand the spectrum of mutations associated with CHDs and first suggest the potentially disease-related GDF3 gene variant in the pathogenesis of CHDs.

Growth and differentiation factor 3 induces expression of genes related to differentiation in a model of cancer stem cells and protects them from retinoic acid-induced apoptosis.

Misexpression of growth factors, particularly those related to stem cell-like phenotype, is often observed in several cancer types. It has been found to influence parameters of disease progression like cell proliferation, differentiation, maintenance of undifferentiated phenotype and modulation of the immune system. GDF3 is a TGFB family member associated with pluripotency and differentiation during embryonic development that has been previously reported to be re-expressed in a number of cancer types. However, its role in tumor development and progression has not been clarified yet. In this study we decipher the role of GDF3 in an in vitro model of cancer stem cells, NCCIT cells. By classical approach to study protein function combined with high-throughput technique for transcriptome analysis and differentiation assays we evaluated GDF3 as a potential therapeutic target. We observed that GDF3 robustly induces a panel of genes related to differentiation, including several potent tumor suppressors, without impacting the proliferative capacity. Moreover, we report for the first time the protective effect of GDF3 against retinoic acid-induced apoptosis in cells with stem cell-like properties. Our study implies that blocking of GDF3 combined with retinoic acid-treatment of solid cancers is a compelling direction for further investigations, which can lead to re-design of cancer differentiation therapies.

[Expression of TGFbeta family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems].

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases ofpluripotency, we examined the expression of TGFbeta family factors (ActivinA, Nodal, Leftyl, TGFbeta1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFbeta1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFbeta1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3BMP/Smad1/5/8 endogenous branches of TGFbeta signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFbeta family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smadl/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primary states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.