Nanog Transcription Research Articles

Characterization of the stemness potency of mammospheres isolated from the breast cancer cell lines.

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24- cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.

Expression of pluripotency-related genes is highly dependent on trichostatin A-assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos.

The present study sought to examine whether trichostatin A (TSA)-assisted epigenetic transformation of porcine bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) affects the transcriptional activities of pluripotency-related genes (Oct4, Nanog, c-Myc, Sox2 and Rex1), multipotent stemness-related gene (Nestin) and anti-apoptotic/anti-senescence-related gene (Survivin). Epigenetically transformed or non-transformed BM-MSCs that had been transcriptionally profiled by qRT-PCR and had been analysed for different stages of apoptosis progression provided a source of nuclear donor cells for the in vitro production of cloned pig embryos. TSA-mediated epigenomic modulation has been found to enhance the multipotency extent, stemness and intracellular anti-ageing properties of porcine BM-MSCs. This has been confirmed by the relative abundances for Nanog, c-Myc Rex1, Sox2 and Survivin mRNAs in TSA-exposed BM-MSCs that turned out to be significantly higher than those of TSA-unexposed BM-MSCs. Additionally, TSA-assisted epigenomic modulation of BM-MSCs did not impact the caspase-8 activity, Bax protein expression and the incidence of TUNEL-positive cells. In conclusion, the considerably elevated quantitative profiles of Sox2, Rex1, c-Myc, Nanog and Survivin mRNA transcripts seem to trigger improved reprogrammability of TSA-treated BM-MSC nuclei in cloned pig embryos that thereby displayed remarkably increased blastocyst formation rates as compared to those noticed for embryos derived from TSA-untreated BM-MSCs.

Dynamical Reorganization of Transcriptional Events Governs Robust Nanog Heterogeneity.

Nanog maintains the pluripotency of embryonic stem cells (ESCs), while demonstrating high expression heterogeneity. Intriguingly, the overall heterogeneity at the Nanog mRNA level under various culture conditions gets precisely partitioned into intrinsic and extrinsic fluctuations. However, the dynamical origin of such a robust transcriptional noise regulation still remains illusive. Herein, we propose a new stochastic simulation strategy that efficiently reconciles the strict apportioning of fluctuations observed in Nanog transcription, while predicting possible experimental scenarios to avoid such an exact noise segregation. Importantly, our model analyses reveal that different culture conditions essentially preserve the robust Nanog expression heterogeneity by altering the dynamics of transcriptional events. In the future, these insights will be useful to systematically maneuver cell-fate decision-making events of ESCs.

Study of SOX2, OCT4 and NANOG Genes Expression in Peripheral Blood of Patients with Non-small Cell Lung Cancer (NSCLC)

Introduction: lung cancer is a disease that is characterized by an uncontrolled growth of the cell in the lung tissue. This cancer is the most common cause of cancer death worldwide. The SOX2, OCT4 and NANOG transcription factor are the main regulators of maintaining vitality state and self-renewal in embryonic stem cells. The aim of this study was to evaluate the genes expression of SOX2 in peripheral blood of patients with non-small cell lung cancer (NSCLC). Material and Methods: in this research, the expression of SOX2, OCT4 and NANOG transcription factors were investigated in peripheral blood of 30 patients with NSCLC. For this purpose, after RNA extraction from patients' blood by TRIzol then cDNA synthesis, the expression of the genes was analyzed by Real Time PCR technique and using specific primers for the genes. Results: after reviewing the results by Graph Pad software and t-test, SOX2, OCT4 and NANOG expression in patient samples was significantly higher than normal samples. Discussion: the expression of the genes studied in this research in the patients with lung cancer was significantly higher than normal. Regarding the gene expression profile, it seems that the gene is a potential biomarker for the diagnosis of lung cancer.

Inhibition of pancreatic cancer stem cell characteristics by α-Mangostin: Molecular mechanisms involving Sonic hedgehog and Nanog.

The current investigation was intended to elucidate the molecular mechanism of α‐Mangostin in the regulation of pancreatic cancer stem cell (CSC) characteristics. Here, we demonstrate that α‐Mangostin inhibited cell proliferation in pancreatic CSCs and cancer cell lines while it showed no effect on human pancreatic normal ductal epithelial cells. Also, α‐Mangostin inhibited colony formation and induced apoptosis in these cells. Further, α‐Mangostin inhibited the self‐renewal capacity of CSCs isolated from human primary tumours and KrasG12D mice. Furthermore, α‐Mangostin inhibited the invasive and metastatic ability of pancreatic CSCs by suppressing the epithelial‐to‐mesenchymal transition (EMT) via up‐regulation of E‐cadherin and down‐regulation of mesenchymal phenotype by inhibiting N‐cadherin, Snail and Slug expression. Interestingly, the pluripotency maintaining factors and CSC markers were inhibited by α‐Mangostin thus suggesting that α‐Mangostin can target CSCs to inhibit pancreatic cancer effectively. Gli signalling plays a crucial role in the self‐renewal and pluripotency of CSCs. α‐Mangostin inhibited the Gli transcription and the expression of Gli target genes (Nanog, Oct4, c‐Myc, Sox‐2 and KLF4) in CSCs. Using ChIP assay, we demonstrated that Nanog could directly bind to promoters of Cdk2, Cdk6, FGF4, c‐Myc and α‐Mangostin inhibited Nanog binding to these promoters. Conversely, the inhibitory effects of the α‐Mangostin on CSC proliferation and Gli or Nanog transcription and their targets were abrogated by either enforced activation of sonic hedgehog (Shh) or by the overexpression of Nanog. Taken together, our studies suggest that α‐Mangostin may act as Gli inhibitor and establishes the pre‐clinical significance of α‐Mangostin for the prevention and treatment of pancreatic cancer.

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12–14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.

Evaluation of stemness marker expression in bovine ovarian granulosa cells.

The objective of this study was to assess the stemness marker expressions (Oct4, Nanog, and Sox2) of granulosa cells (GCs) collected from bovine ovarian follicles and in vitro expansion. The single bovine ovarian follicles were isolated and categorized into 4 groups according to their diameter including group A (<2 mm), group B (2-3 mm), group C (3-4 mm), and group D (>4 mm). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunostaining were applied to evaluate the stemness marker expression of bovine GCs from ovarian follicles. We also estimated the stemness marker transcript expressions of GCs during in vitro expression by qRT-PCR. qRT-PCR analysis demonstrated that fresh GCs from bovine ovarian follicles expressed the stemness markers (Oct4, Nanog, Sox2). These markers were down-regulated during antral stage follicular development. We also estimated stemness marker transcript expressions of GCs which were isolated and in vitro expanded from ovarian follicles of group A. The qRT-PCR results showed that Oct4 and Sox2 transcript expressions were reduced during in vitro expansion while Nanog transcript was not expressed.

Peroxisome Proliferator-Activated Receptor α Agonist and Its Target Nanog Cooperate to Induce Pluripotency.

The pharmaceutical compounds that modulate pluripotent stem cell (PSC) identity and function are increasingly adopted to generate qualified PSCs and their derivatives, which have promising potential in regenerative medicine, in pursuit of more accuracy and safety and less cost. Here, we demonstrate the peroxisome proliferator-activated receptor α (PPARα) agonist as a novel enhancer of pluripotency acquisition and induced pluripotent stem cell (iPSC) generation. We found that PPARα agonist, examined and selected Food and Drug Administration (FDA) -approved compound libraries, increase the expression of pluripotency-associated genes, such as Nanog, Nr5A2, Oct4, and Rex1, during the reprogramming process and facilitate iPSC generation by enhancing their reprogramming efficiency. A reprogramming-promoting effect of PPARα occurred via the upregulation of Nanog, which is essential for the induction and maintenance of pluripotency. Through bioinformatic analysis, we identified putative peroxisome proliferator responsive elements (PPREs) located within the promoter region of the Nanog gene. We also determined that PPARα can activate Nanog transcription by specific binding to putative PPREs. Taken together, our findings suggest that PPARα is an important regulator of PSC pluripotency and reprogramming, and PPARα agonists can be used to improve PSC technology and regenerative medicine.

Dysregulated cancer cell transdifferentiation into erythrocytes is an additional metabolic stress in hepatocellular carcinoma.

A number of human and canine hepatocellular carcinoma tissues showed clear signs of hypoxia indicated by HIF1α-activation and the presence of large clusters of cells resembling erythrocytes at different stages of nuclear elimination without any defined endothelial cell lining or blood vessel walls. Differentiated erythrocytic identity of such cells in hepatocellular carcinoma tissues was apparent from their non-nucleated and evolving basophilic to eosinophilic staining characteristics. In addition to the fully differentiated non-nucleated mesenchymal cell clusters, the onset of erythroblastic transdifferentiation was apparent from the activation of Glycophorin A, a marker of erythrocytic progenitors, in some epithelial cancer cells. Activation of canonical Wnt signalling in such tumours was apparent from the expression of Wnt2 ligand and active β-catenin translocation into the nucleus indicating Wnt signalling to be one of the key signalling pathways participating in such cell transdifferentiation. Sonic hedgehog and bone morphogenetic protein signalling along with Sulf1/Sulf2 activation was also observed in such hepatocellular carcinoma tissue samples. The presence of stem cell markers and the cell signalling pathways associated with erythropoiesis, and the detection of messenger RNAs for both α and β haemoglobins, support the assumption that hepatocellular carcinoma cells have the potential to undergo cell fate change despite this process being dysregulated as indicated by the lack of simultaneous generation of endothelial cell lining. Lack of blood vessel walls or endothelial cell lining around erythrocytic clusters was confirmed by non-detection of multiple blood vessel markers such as vWF, CD146 and smooth muscle α-actin that were clearly apparent in normal and unaffected adjacent regions of hepatocellular carcinoma livers. In addition to the activation of Glycophorin A, transdifferentiation of some hepatocellular carcinoma hepatocytes into other cell fates was further confirmed by the activation of some stem cell markers, for example, NANOG and OCT4 transcription factors, not only by reverse transcription polymerase chain reaction but also by their restricted expression in such cells at protein level.

LIN28A activates the transcription of NANOG in dairy goat male germline stem cells.

LIN28A serves as a crucial marker of dairy goat male germline stem cells (GmGSCs). In our previous study, we demonstrated that LIN28A promotes proliferation, self-renewal, and maintains the stemness of GmGSCs. Here, we found that LIN28A could activate the transcription of NANOG in a let-7g independent manner. We cloned the 5' upstream of two NANOG genes which were located on chromosome 15 ( NANOG-ch15) and chromosome 5 ( NANOG-ch5), respectively, and then examined their promoter activities and promoter methylation levels. Results showed that NANOG-ch15 is a pseudogene whereas NANOG-ch5 is active in Capra hircus. Bioinformatics analysis indicated that the 5' upstream region of NANOG-ch5 does not have typical CpG islands but contains several CG enrichment regions and several LIN28A binding sites. Deletion analysis suggested that NANOG-ch5 promoter can be activated by LIN28A directly binding to the site -210 but not by the indirect effect from the inhibition of let-7g, which is known to be downregulated by LIN28A. Mechanistically, LIN28A recruits and interacts with 5-methylcytosine-dioxygenase Ten-Eleven translocation 1 (TET1) to NANOG-ch5 gene promoter binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. These results revealed the role of LIN28A in NANOG transcriptional regulation via epigenetic DNA modifications to maintain the stemness of GmGSC.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells Using Auto-Erasable Sendai Virus Vector

Canine induced pluripotent stem cells (ciPSCs) can be used in regenerative medicine. However, there are no reports on the generation of genome integration-free and completely exogenous gene-silenced (footprint free) ciPSCs that are tolerant to enzymatic single-cell passage. In this study, we reprogrammed canine embryonic fibroblasts using the auto-erasable replication-defective and persistent Sendai virus vector, SeVdp(KOSM)302L, and generated two ciPSC lines. The ciPSCs were positive for pluripotent markers, including alkaline phosphatase activity as well as OCT3/4, SOX2, and NANOG transcripts, and NANOG, stage-specific embryonic antigen-1, and partial TRA-1-60 protein expression, even after SeVdp(KOSM)302L removal. The ciPSCs were induced to differentiate into all the three germ layers as embryoid bodies in vitro and as teratomas in vivo. Furthermore, SeVdp(KOSM)302L-free ciPSCs maintained a normal karyotype even after repeated enzymatic single-cell passaging. Therefore, to our knowledge, for the first time, we demonstrated the generation of footprint-free and high-quality ciPSCs that can be passaged at the single-cell stage using enzymatic methods. Our method for generation of ciPSCs is a good step toward the development of clinical application of ciPSCs.

Abstract 1997: Pro-inflammatory cytokine IL-6 induces the upregulation and deacetylation of FRA-1, thus promoting colon cancer stemness and aggressiveness

Abstract Colon cancer is an aggressive epithelial tumor type encompassing cancer stem cells (CSCs) that not only contribute to its grow at the primary site but also to its malignant progression, resistance to chemotherapy, and distant metastases thus underlying poor patient outcomes. We have previously reported that significant upregulation of the gene encoding for the Fos-related antigen-1 (FOSL1) upon pro-inflammatory cytokine interleukin-6 (IL-6)-driven epithelial-to-mesenchymal transition (EMT) in colon cancer. However, the underlying molecular and cellular mechanism through which inflammatory cytokines like IL6 regulate CSCs plasticity during colon cancer onset and progression development remain unclear. Here, we show that IL-6 enhanced the stemness, drug resistance and metastasis of colon cancer cells by upregulation and deacetylation of the transcription factor FRA1. Ectopic expression of FRA1 in colon cancer cells induced stem-like properties and metastasis, while its knockdown suppressed these IL-6 induced effects. IL-6 enhanced FRA1 transcription through the STAT3 pathway. Also, we show that FRA1 directly interacts with the promoter of the CSC transcriptional factor Nanog and transactivates its expression to modulate colon cancer stemness. Furthermore, deacetylation of lysine 116 (K116) in FRA1 was essential for its activity on Nanog transcription. Preliminary analyses with Trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs) I and II, but not nicotinamide (NAM), an inhibitor of the sirtuin (SIRT) family of deacetylases, showed that some member of the HDACs family might be responsible for FRA1 deacetylation. Finally, we examined the expression of IL6/STAT3-pY(705)/FRA1/k116ac/Nanog/HDACs/β-catenin in 120 paraffin-embedded human colon cancer tissues by immunohistochemistry (IHC). We found that while FRA1 was mainly localized in the nucleus of cancer cells, K116ac of FRA1 was present both in the nucleus and cytoplasm and showed a low level of Lys116 acetylation in the FRA1 nuclear fraction. Moreover, analysis of patient-derived colon cancer samples supported the clinical relevance of the IL-6/STAT3/FRA1/Nanog axis in contributing to the increased colon cancer stemness and metastasis. We propose that a transcriptional regulatory cross-talk between pro-inflammatory and cancer stemness signaling pathways drives the reprogramming of colon cancer cells to CSCs thus promoting progression towards malignancy and distant metastasis. Combinations of anti-IL6 therapies with HDACs inhibitors may offer a potential novel approach for colon cancer treatment. Keywords: Inflammation, cancer cell stemness, transcription, deacetylation, colon cancer Citation Format: Tingyang Wang, Jimin Shao. Pro-inflammatory cytokine IL-6 induces the upregulation and deacetylation of FRA-1, thus promoting colon cancer stemness and aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1997.

Probiotic Metabolites Promote Stem Cell Proliferation and Differentiation

Probiotic metabolites (PM) impart numerous health benefits in human and may have a potential to induce cell proliferation and differentiation. We tested the effects of PM released by probiotics found in yogurts on stem cell proliferation and differentiation. Five different brands of yogurts (Oikos, Stonyfield, Yoplait, Chobani and Activia) often sold in the supermarkets were tested in this study. Yogurt cultures analyzed by Gram‐staining and DNA sequencing of 16s rRNA gene identified that these yogurts are not equally supplemented and include different types and various amounts of probiotics. The probiotics were cultured and the condition media containing PMs were collected and used in our study. To evaluate the effect of PM on stem cells, human induced pluripotent stem cells (iPSC) were treated with probiotic conditioned media. We found that probiotic Lactobacillus reuteri conditioned media (LRCM) induced iPSC proliferation at lower doses but inhibited cell proliferation at higher doses. Additionally, expression of the proliferating cell nuclear antigen (PCNA) and pluripotent stem cell factors, OCT4, SOX2 and NANOG transcripts was upregulated in iPSCs treated with LRCM. iPS cells exposed to LRCM during in vitro differentiation led to enhanced expression of myocyte markers, Nkx2.5 and cTnT. Consistently, sodium butyrate, a defined bacterial metabolite (short chain fatty acid), had similar effect on iPSC proliferation and expression of pluripotent markers and myocyte markers. In summary, we have demonstrated that probiotics in different brands of yogurts are diverse in their numbers, viability and composition. Further, our results show that PMs can potentiate iPS proliferation and induce cardiomyocyte differentiation. Our findings have laid a foundation for further investigation of how probiotics and its metabolites from yogurt affect human health and disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cryosurvival of rabbit embryos obtained after superovulation with corifollitropin alfa with or without LH

The efficiency of an embryo bank depends on provision of optimal conditions for recovery, cryopreservation and transfer to a breed or strain. In this sense, increasing the number of embryos available using superovulation should improve the cryobank efficiency. However, vagueness of response to conventional protocols to control or increase ovarian response and the quality of oocytes and embryos and their cryotolerance remain a challenge. The aim of our study was to evaluate the effect of corifollitropin alpha (CTP) and a recombinant human FSH (rhFSH), alone or supplemented with rhLH, on embryo cryosurvival by in vitro development and OCT4 and NANOG mRNA abundance at blastocyst stage and offspring rate. In vitro development of vitrified embryos was not significantly affected by superstimulation with or without rhLH supplementation, resulting in similar development rates to those of the control groups (fresh and vitrified embryos from non-superstimulated donor does). Blastocysts developed from vitrified embryos showed higher levels of OCT4 transcript abundance than fresh control, while NANOG transcript abundance was only higher in the blastocysts developed from vitrified embryos after superstimulation treatment in comparison with control groups. The implantation and offspring rates at birth were negatively affected by supplementation with rhLH. Both rhFSH or CTP vitrified embryo groups showed an implantation rate similar to those of the control groups, but an offspring rate lower than control. In conclusion, embryos produced using corifollitropin alpha did not compromise the cryosurvival of vitrified embryos in the rabbit. In addition, this study points out the negative effect of rhLH supplementation in terms of offspring rate on embryo vitrification.

KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency.

SummaryCooperative action of a transcription factor complex containing OCT4, SOX2, NANOG, and KLF4 maintains the naive pluripotent state; however, less is known about the mechanisms that disrupt this complex, initiating exit from pluripotency. We show that, as embryonic stem cells (ESCs) exit pluripotency, KLF4 protein is exported from the nucleus causing rapid decline in Nanog and Klf4 transcription; as a result, KLF4 is the first pluripotency transcription factor removed from transcription-associated complexes during differentiation. KLF4 nuclear export requires ERK activation, and phosphorylation of KLF4 by ERK initiates interaction of KLF4 with nuclear export factor XPO1, leading to KLF4 export. Mutation of the ERK phosphorylation site in KLF4 (S132) blocks KLF4 nuclear export, the decline in Nanog, Klf4, and Sox2 mRNA, and differentiation. These findings demonstrate that relocalization of KLF4 to the cytoplasm is a critical first step in exit from the naive pluripotent state and initiation of ESC differentiation.

YY1 Positively Regulates Transcription by Targeting Promoters and Super-Enhancers through the BAF Complex in Embryonic Stem Cells.

SummaryYin Yang 1 (YY1) regulates early embryogenesis and adult tissue formation. However, the role of YY1 in stem cell regulation remains unclear. YY1 has a Polycomb group (PcG) protein-dependent role in mammalian cells. The PcG-independent functions of YY1 are also reported, although their underlying mechanism is still undefined. This paper reports the role of YY1 and BAF complex in the OCT4-mediated pluripotency network in mouse embryonic stem cells (mESCs). The interaction between YY1 and BAF complex promotes mESC proliferation and pluripotency. Knockdown of Yy1 or Smarca4, the core component of the BAF complex, downregulates pluripotency markers and upregulates several differentiation markers. Moreover, YY1 enriches at both promoter and super-enhancer regions to stimulate transcription. Thus, this study elucidates the role of YY1 in regulating pluripotency through its interaction with OCT4 and the BAF complex and the role of BAF complex in integrating YY1 into the core pluripotency network.

Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell-based skin tissue engineering in the future.

Design of a nanocomposite substrate inducing adult stem cell assembly and progression toward an Epiblast-like or Primitive Endoderm-like phenotype via mechanotransduction.

This work shows that the active interaction between human umbilical cord matrix stem cells and Poly (l-lactide)acid (PLLA) and PLLA/Multi Walled Carbon Nanotubes (MWCNTs) nanocomposite films results in the stem cell assembly as a spheroid conformation and affects the stem cell fate transition. We demonstrated that spheroids directly respond to a tunable surface and the bulk properties (electric, dielectric and thermal) of plain and nanocomposite PLLA films by triggering a mechanotransduction axis. This stepwise process starts from tethering of the cells' focal adhesion proteins to the surface, together with the adherens junctions between cells. Both complexes transmit traction forces to F-Actin stress fibres that link Filamin-A and Myosin-IIA proteins, generating a biological scaffold, with increased stiffening conformation from PLLA to PLLA/MWCNTs, and enable the nucleoskeleton proteins to boost chromatin reprogramming processes. Herein, the opposite expression of NANOG and GATA6 transcription factors, together with other lineage specification related proteins, steer spheroids toward an Epiblast-like or Primitive Endoderm-like lineage commitment, depending on the absence or presence of 1wt% MWCNTs, respectively. This work represents a pioneering effort to create a stem cell/material interface that can model the stem cell fate transition under growth culture conditions.

Secretome of in vitro cultured human embryos contains extracellular vesicles that are uptaken by the maternal side

Communication between embryo and maternal endometrium occurs during a specific time frame in which implantation is possible. Here we demonstrate for the first time that conditioned media from non-manipulated human embryos cultured in vitro for 3 days or up to the blastocyst stage contain extracellular vesicles (EVs) with a diameter of 50 to 200 nm and bearing the traditional microvesicle and exosome marker proteins CD63, CD9 and ALIX. The embryonic origin of these EVs has been confirmed by the presence of stemness gene transcripts and their enrichment in the non-classical HLA-G protein. NANOG and POU5F1 transcripts were shown to be contained in vesicles deriving from embryos at different stages of development. In line with a higher detection rate of the HLA-G protein in blastocysts compared to cleavage stage embryos, a significantly higher amount of HLA-G was found in vesicles accumulated in spent media from day 3 to day 5 of development compared to those isolated from the earlier stage. Uptake of dye-labeled embryo-derived EVs by human primary endometrial epithelial and stromal cells was also demonstrated with a fluorescence intensity signal significantly higher for cells treated with vesicles derived from blastocysts. Based on these findings, EV exchange may be suggested as an emerging way of communication at the maternal-fetal interface.

NANOG gene expression is regulated by the ETS transcription factor ETV4 in human embryonic carcinoma NCCIT cells

We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naïve cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (−285 to −138) of the proximal promoter. Site-directed mutagenesis of the putative EBS (−196AGGATT−191) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression.