Oct4 Transcription Research Articles

The relationship between nanoscale genome organization and gene expression in mouse embryonic stem cells during pluripotency transition.

During early development, gene expression is tightly regulated. However, how genome organization controls gene expression during the transition from naïve embryonic stem cells to epiblast stem cells is still poorly understood. Using single-molecule microscopy approaches to reach nanoscale resolution, we show that genome remodeling affects gene transcription during pluripotency transition. Specifically, after exit from the naïve pluripotency state, chromatin becomes less compacted, and the OCT4 transcription factor has lower mobility and is more bound to its cognate sites. In epiblast cells, the active transcription hallmark, H3K9ac, decreases within the Oct4 locus, correlating with reduced accessibility of OCT4 and, in turn, with reduced expression of Oct4 nascent RNAs. Despite the high variability in the distances between active pluripotency genes, distances between Nodal and Oct4 decrease during epiblast specification. In particular, highly expressed Oct4 alleles are closer to nuclear speckles during all stages of the pluripotency transition, while only a distinct group of highly expressed Nodal alleles are in close proximity to Oct4 when associated with a nuclear speckle in epiblast cells. Overall, our results provide new insights into the role of the spatiotemporal genome remodeling during mouse pluripotency transition and its correlation with the expression of key pluripotency genes.

Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80°C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

OCT4 Expression in Gliomas Is Dependent on Cell Metabolism.

The OCT4 transcription factor is necessary to maintain cell stemness in the early stages of embryogenesis and is involved in the formation of induced pluripotent stem cells, but its role in oncogenesis is not yet entirely clear. In this work, OCT4 expression was investigated in malignant gliomas. Twenty glioma cell lines and a sample of normal adult brain tissue were used. OCT4 expression was found in all studied glioma cell lines but was not detected in normal adult brain tissue. For one of these lines, OCT4 knockdown caused tumor cell death. By varying the culture conditions of these cells, we unexpectedly found that OCT4 expression increased when cells were incubated in serum-free medium, and this effect was significantly enhanced in serum-free and L-glutamine-free medium. L-glutamine and the Krebs cycle, which is slowed down in serum-free medium according to our NMR data, are sources of α-KG. Thus, our data indicate that OCT4 expression in gliomas may be regulated by the α-KG-dependent metabolic reprogramming of cells.

NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells.

NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. However, its role in mammalian development remains unclear. Here, we found that NAT10 expression positively correlates with pluripotency in vivo and in vitro. High throughput ac4C-targeted RNA immunoprecipitation sequencing (ac4C-RIP-seq), NaCNBH3-based chemical ac4C sequencing (ac4C-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays revealed noticeable ac4C modifications in transcriptome of hESCs, among which transcripts encoding core pluripotency transcription factors are favorable targets of ac4C modification. Further validation assays demonstrate that genetic inactivation of NAT10, the ac4C writer enzyme, led to ac4C level decrease on target genes, promoted the core pluripotency regulator OCT4 (POU5F1) transcript decay, and finally impaired self-renewal and promoted early differentiation in hESCs. Together, our work presented here elucidates a previously unrecognized interconnectivity between the core pluripotent transcriptional network for the maintenance of human ESC self-renewal and NAT10-catalyzed ac4C RNA epigenetic modification.

IMMUNOEXPRESSION OF STEM CELL MARKERS IN AMELOBLASTOMA

Objective Ameloblastoma (AME) is a benign, odontogenic neoplasm characterized by a locally invasive growth pattern. In the interest of understanding the aggressiveness of neoplasms, research on the role of stem cells has gained prominence. SOX-2, NANOG, and OCT4 transcription factors are important stem cell biomarkers. Study Design Immunohistochemistry was performed to verify the expression of these transcription factors in tissue samples of AME, dentigerous cyst (CD), and dental follicle (DF). In addition, indirect immunofluorescence and transcriptomic analysis were performed from a cell line derived from AME (AME-hTERT). Results The expression of SOX-2, NANOG, and OCT4 was observed in tissue samples and in the AME-hTERT cell line. Higher immunostaining of the studied proteins was observed in AME in comparison to CD and FD (p<0.001). There was also an association of OCT4 and SOX-2 proteins in AME neoplastic cells (p=0.0294). Transcriptomic analysis revealed a network of mutual interaction between the three transcription factors. Conclusion The presence of these biomarkers indicates a probable role of stem cells in the genesis and progression of AME. Ameloblastoma (AME) is a benign, odontogenic neoplasm characterized by a locally invasive growth pattern. In the interest of understanding the aggressiveness of neoplasms, research on the role of stem cells has gained prominence. SOX-2, NANOG, and OCT4 transcription factors are important stem cell biomarkers. Immunohistochemistry was performed to verify the expression of these transcription factors in tissue samples of AME, dentigerous cyst (CD), and dental follicle (DF). In addition, indirect immunofluorescence and transcriptomic analysis were performed from a cell line derived from AME (AME-hTERT). The expression of SOX-2, NANOG, and OCT4 was observed in tissue samples and in the AME-hTERT cell line. Higher immunostaining of the studied proteins was observed in AME in comparison to CD and FD (p<0.001). There was also an association of OCT4 and SOX-2 proteins in AME neoplastic cells (p=0.0294). Transcriptomic analysis revealed a network of mutual interaction between the three transcription factors. The presence of these biomarkers indicates a probable role of stem cells in the genesis and progression of AME.

Predictive and Regenerative Medicine for Humans

In human history, finding a cure for the devastating disease of cancer has been a long, ongoing battle. Recently, scientific research has shown several feasible bio-medicinal ways for the treatment of cancer. The first method is through predictive regenerative medicine, which converts (breast) cancer cells back into phenotypically benign cells using continually updated predictions from Bayesian inference models. Another common method is the rewriting of damaged coordinating genes, but this has underlying moral issues — who can authorize the right to alter human genes? To investigate these methods, this paper will discuss elementary point-set topologies, physical gene mapping, and gene sequencing. The main purpose of this paper is to establish a statistical method with the help of mathematics in our biological life science. The proposed method can be applied in the case of manipulating genetic transcription and expression with a suitable Bayesian inference model (for genetic reprogramming). The peak value of cancer cell conversion is its Bayesian regression (based on the model’s parameters) from the corresponding Bayesian inference (for the normal posterior distribution, prior and likelihood). The relevant predictive conditions can be determined from the respective model, which could be used to reveal elements that help cancer cell conversion maximization/minimization. Practically, we may apply both of the Bayesian optimization and the related attribute adjustment (to the Oct4, SoX2, Klf4 and c-Myc transcription factors) that can induce the reprogramming of cancer cells. The expected outcome is that we can biomedical-engine the cancer cells conversion. To go a further step, one may apply my proposed Net-Seizing Theory to capture the genetic mutations that underly cancer disease. It is hope that we can design the corresponding universal strategy to heal cancer. Finally, one may establish an intelligent warning mechanism for those high risked cancer candidates but NOT for the thinking control in most of the dictatorship countries.

379 Human Endogenous Retrovirus-K (HML-2) Contributes to a Unique Stem-Cell Niche in Glioblastoma

INTRODUCTION: Human Endogenous Retrovirus (HERV) are ancestral viral relics that comprise nearly 8% of the human genome. Although silenced in normal tissues, the most recently integrated provirus HERV-K (HML-2) can be pathologically reactivated in certain cancers. METHODS: We utilized a combination approach using scRNA-seq, multiplex immunofluorescence, and TEM to characterize HERV-K expression in malignant gliomas. Using CRISPR engineering and CHIP-qPCR, we downregulated HML-2 in both patient-derived glioma neurospheres and intracranial orthotopic models and identified interactions between HERV-K and pluirpotency transcription factors. RESULTS: We identified pathological expression of HML-2 in human malignant gliomas in CSF (HERV-K DNA/RPP30 = 35.2 ± 8.8 vs 23.1 ± 6.7, n = 18, p = 0.02) and tumors (HERV-K RNA/HPRT mean = 1.15 ± 0.2 vs. 0.5 ± 0.2, p = 0.01, n = 20) compared to epilepsy controls. Aberrant HML-2 expression corresponded to a unique stem-cell niche using multivoxel automated segmentation. Using a tailored scRNA-seq sequencing pipeline, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells that can drive cellular plasticity (ANOVA, p &lt; 0.001). Using CRISPR technology, we demonstrate that HML-2 is critical to maintenance of glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models (OS: 26 days vs. 18.6, p=0.0008, n=20). Downregulation of HERV-K using CRISPRi reduces the glioblastoma neurosphere formation (2-way ANOVA, p &lt; 0.0001) and HERV-K env, Polymerase, OCT4 and Nestin transcripts (2-way ANOVA p &lt; 0.001). Moreover, using Transmission Electron Microscopy, we discovered that some glioma stem-cells form immature retroviral virions in glioblastoma. Inhibiting HML-2 expression with nucleoside reverse transcriptase inhibitors reduces extracellular reverse transcriptase (One-way ANOVA, p &lt; 0.05), tumor viability (IC50 = 75.8-123.1 uM), and pluripotency (1-way ANOVA, p &lt; 0.01). CONCLUSIONS: Our results suggest that HML-2 is overexpressed in the glioblastoma stem-cell niche. Since persistence of glioblastoma stem-cells is considered responsible for treatment resistance/recurrence, HML-2 may serve as a unique therapeutic target in glioblastoma.

Generation of an induced pluripotent stem cell lines NSHDMUi001-A from patients with type 2 diabetes

Type 2 diabetes mellitus (T2DM) is common in China, and its aetiology and pathogenesis are still unclear. We reprogrammed pEP4EO2SEN2K and pEP4EO2SET2K, pCEP4-M2L was electrotransfected in T2DM patients with pEP4EO2SEN2K, and pCEP4-M2L was electrotransfected in T2DM patients expressing the OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT transcription factors to obtain induced pluripotent stem cells (iPSCs). The obtained iPSCs have been verified to have pluripotency, normal karyotype and differentiation potential; therefore, these cells can be used in the study of disease pathophysiology and drug development to create new therapeutic targets for T2DM and associated central nervous system damage.

Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation

Emerging evidence shows that the biomechanical environment is required to support cancer stem cells (CSCs), which play a crucial role in drug resistance. However, how mechanotransduction signals regulate CSCs and its clinical significance has remained unclear. Using clinical-practice ultrasound elastography for patients’ lesions and atomic force microscopy for surgical samples, we reveal that increased matrix stiffness is associated with poor responses to neoadjuvant chemotherapy, worse prognosis, and CSC enrichment in patients with breast cancer. Mechanically, TAZ activated by biomechanics enhances CSC properties via phase separation with NANOG. TAZ-NANOG phase separation, which is dependent on acidic residues in the N-terminal activation domain of NANOG, promotes the transcription of SOX2 and OCT4. Therapeutically, targeting NANOG or TAZ reduces CSCs and enhances the chemosensitivity in vivo. Collectively, this study demonstrated that the phase separation of a pluripotency transcription factor links mechanical cues in the niche to the fate of CSCs.

STEM-07. DEFINING THE ROLE OF HUMAN ENDOGENOUS RETROVIRUS-K (HML-2) IN THE GLIOBLASTOMA STEM-CELL NICHE

Abstract Human Endogenous Retrovirus (HERV) are ancestral viral relics that comprise nearly 8% of the human genome. Although silenced in normal tissues, the most recently integrated provirus HERV-K (HML-2) can be pathologically reactivated in certain cancers. Here, we report pathological expression of HML-2 transcripts in human malignant gliomas in cerebrospinal fluid (HERV-K DNA/RPP30=35.2±8.8 vs 23.1±6.7, n=18, p=0.02) and tumors (HERV-K RNA/HPRTmean=1.15±0.2 vs. 0.5±0.2, p=0.01, n=20) compared to epilepsy controls. Aberrant HML-2 expression corresponded to a unique stem-cell niche using multivoxel automated segmentation. Using a tailored single-cell RNA sequencing pipeline to detect retrotransposons, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells that can drive cellular plasticity (ANOVA, multiple-testing correction, p&amp;lt; 0.001). Using CRISPR technology, we demonstrate that HML-2 is critical to maintenance of glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models (OS: 26 days vs. 18.6, p=0.0008, n=20). Downregulation of HERV-K using CRISPRi reduces the glioblastoma neurosphere formation (2-way ANOVA, p&amp;lt; 0.0001). and HERV-K env, Polymerase, OCT4 and Nestin transcripts (2-way ANOVA p&amp;lt; 0.001). Using Chromatin Immunoprecipitation, we determined that this interaction in gliomas is likely mediated by the nuclear transcription factor OCT4 which binds to an HML-2 specific Long-Terminal Repeat (LTR5Hs). Moreover, using Transmission Electron Microscopy, we discovered that some glioma stem-cells form immature retroviral virions in glioblastoma. Inhibiting HML-2 expression with nucleoside reverse transcriptase inhibitors reduces extracellular reverse transcriptase (One-way ANOVA, p&amp;lt; 0.05), tumor viability (IC50=75.8-123.1 uM), and pluripotency (One-way ANOVA, p&amp;lt; 0.01). Our results suggest that HML-2 is overexpressed in the cancer stem-cell niche of glioblastoma. Since persistence of glioblastoma stem-cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target in glioblastoma.

Differential Expression Pattern of linc-ROR Spliced Variants in Pluripotent and Non-Pluripotent Cell Lines.

The human large intergenic non-coding RNA-regulator of reprogramming program (linc-ROR) is known as a stem cell specific linc-RNA. linc-ROR counteracts differentiation via sequestering microRNA-145 (miR-145) that targets OCT4 transcript. Despite the research on the expression and function, the exact structure of linc-ROR transcripts is not clear. Considering the contribution of alternative splicing in transcripts structures and function, identifying different spliced variants of linc-ROR is necessary for further functional analyses. We aimed to find the alternatively spliced transcripts of linc-ROR and investigate their expression pattern in stem and cancer cell lines and during neural differentiation of NT2 cells as a model for understanding linc-ROR role in stem cell and differentiation. In this experimental study, linc-ROR locus was scanned for identifying novel exons. Different primer sets were used to detect new spliced variants by reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing. Quantitative PCR (qPCR) and RT-PCR were employed to profile expression of linc-ROR transcripts in different cell lines and during neural differentiation of stem cells. We could discover 13 novel spliced variants of linc-ROR harboring unique array of exons. Our work uncovered six novel exons, some of which were the product of exonized transposable elements. Monitoring expression profile of the linc-ROR spliced variants in a panel of pluripotent and non-pluripotent cells exhibited that all transcripts were primarily expressed in pluripotent cells. Moreover, the examined linc-ROR spliced variants showed a similar downregulation during neural differentiation of NT2 cells. Altogether, our data showed despite the difference in the structure and composition of exons, various spliced variants of linc-ROR showed similar expression pattern in stem cells and through differentiation.

β-Catenin promotes long-term survival and angiogenesis of peripheral blood mesenchymal stem cells via the Oct4 signaling pathway

Stem cell therapy has been extensively studied to improve heart function following myocardial infarction; however, its therapeutic potency is limited by low rates of engraftment, survival, and differentiation. Here, we aimed to determine the roles of the β-catenin/Oct4 signaling axis in the regulation of long-term survival and angiogenesis of peripheral blood mesenchymal stem cells (PBMSCs). These cells were obtained from rat abdominal aortic blood. We showed that β-catenin promotes the self-renewal, antiapoptotic effects, and long-term survival of PBMSCs by activating the Oct4 pathway through upregulation of the expression of the antiapoptotic factors Bcl2 and survivin and the proangiogenic cytokine bFGF and suppression of the levels of the proapoptotic factors Bax and cleaved caspase-3. β-Catenin overexpression increased Oct4 expression. β-Catenin knockdown suppressed Oct4 expression in PBMSCs. However, β-catenin levels were not affected by Oct4 overexpression or knockdown. Chromatin immunoprecipitation assays proved that β-catenin directly regulates Oct4 transcription in PBMSCs. In vivo, PBMSCs overexpressing β-catenin showed high survival in infarcted hearts and resulted in better myocardial repair. Further functional analysis identified Oct4 as the direct upstream regulator of Ang1, bFGF, HGF, VEGF, Bcl2, and survivin, which cooperatively drive antiapoptosis and angiogenesis of engrafted PBMSCs. These findings revealed the regulation of β-catenin in PBMSCs by the Oct4-mediated antiapoptotic/proangiogenic signaling axis and provide a breakthrough point for improving the long-term survival and therapeutic effects of PBMSCs.

Effect of high hydrostatic pressure on the in vitro development and molecular quality of transgenic rabbit embryos derived from nano-transfected zygotes

Abstract The aim of this study was to evaluate the effect of high hydrostatic pressure (HHP) on the in vitro developmental abilities of nano-transfected rabbit zygotes, their transfection efficiency, and the molecular quality of the blastocysts generated. This quality was assessed by estimating the quantitative profiles of Oct4, Casp7, and Bcl2 mRNA transcripts. The nano-transfection efficiencies of zygotes that had been pre-treated with either 20 MPa or 40 MPa of HHP (13.5% and 13.7%, respectively) were insignificantly lower than those found in zygotes not exposed to HHP prior to their nano-transfection (20.1%; P≥0.05). Moreover, applying HHP treatment with the parameters of 20 MPa and 40 MPa followed by the nano-transfection of zygotes brought about an insignificant decrease in the rates of embryos at the blastocyst stage (30.4% and 23.0%, respectively) as compared to the control group of nano-transfected zygotes (40.4%; P≥0.05). Furthermore, analyzing the transcriptional activity of Oct4, Bcl2, and Casp7 genes revealed that HHP enhances the relative abundance (RA) of all mRNA transcripts in blastocysts derived from non-transfected rabbit zygotes. In turn, the augmented RAs found in the pro-apoptotic Casp7 and anti-apoptotic Bcl-2 transcripts confirmed the onset and progression of programmed cell death in blastocysts developed from nano-transfected zygotes that had undergone HHP pre-treatment. The conceptualization based not only on a novel nano-transfection approach used to genetically modify in vivo-fertilized rabbit zygotes but also on their HHP pre-treatment is elaborated here for the first time, with an emphasis on further investigations aimed at producing transgenic rabbit and other mammalian species embryos by somatic cell cloning.

Gene expression at different cell stages of in vitro-fertilized bovine embryos

The birth rate of embryos produced in vitro (IVF) is still lower than that of embryos produced in vivo. Three major steps for the success of the IVF technique are maturation of immature oocytes, fertilization of matured oocytes, and culture of the resulting embryos. Studying mRNA expression in early embryonic development stages is important and can help to assess embryo quality and optimize production protocols in vitro. The current study aimed to determine the expression levels of developmentally important genes in different stages of bovine embryos produced in vitro. Cumulus-oocyte complexes (COCs) were collected from bovine ovaries and cultured in synthetic oviduct fluid (SOF) medium for 7 - 9 days. Embryos were collected at the time-points listed above, and mRNA expression of genes involved in pluripotency (OCT4), DNA methylation (DNMT1), apoptosis (BAX), and metabolism (GLUT1) and a heat shock protein (HSP70) was estimated from the 2-cell stage to the blastocyst stage of embryos. The results showed statistically significant differences in the relative abundance (RA) of OCT4, DNMT1, BAX, and GLUT1 gene transcripts among the different stages, whereas there were non-significant differences in the RA of HSP70 between these stages. In conclusion, gene expression levels differ among the developmental stages of embryos produced in vitro, possibly because of the timing of embryonic genome activation (EGA).

Tricostatin A-treated round spermatid enhances preimplantation embryo developmental competency following round spermatid injection in mice.

It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.

Hormonal protocol used for cervical dilation in ewes does not affect morphological embryo quality but reduces recovery rate and temporarily alters gene expression.

Information on the impact of hormonal protocols for cervical dilation on the quality of ovine embryos is scarce. To compare the quality of embryos after cervical dilation protocol, ewes (n=64) were allocated into either a treated group (100μg estradiol benzoate intravenous and 0.12mg cloprostenol intramuscularly, 12hours before embryo collection plus 100 iu oxytocin intravenous 15 minutes before the collection procedure) or a control group (saline). Luteal function was analysed using ultrasonography and P4 measurement. Some collected embryos were frozen/thawed for gene expression, others were cultured in vitro, frozen/thawed for gene expression, and the remaining embryos were fixed for the apoptosis test (TUNEL test). The treatment reduced fluid (p=0.04) and structure (p=0.03) recovery rates, but the morphological quality, development stage, and apoptosis incidence of the embryos were not affected by treatment. The corpora lutea of the control group had greater blood perfusion (p=0.002) and greater P4 concentrations at 6, 9, and 12h after the treatment (p<0.0001). The expression of BAX, BCL2, PRDX1, and HSP90 genes were not affected by the treatment. However, the embryos in the treated group had fewer NANOG and OCT4 transcripts than control embryos (p=0.008; p=0.006, respectively). After culture, there was no difference between the groups in any gene. The hormonal protocol for cervical dilation reduced the efficiency of embryo collection. In addition, the treatment induced luteolysis and a transient alteration of embryo gene expression, however there were no detectable changes in embryo morphological quality, development stage, or incidence of apoptosis.

HN1L promotes stem cell-like properties by regulating TGF-β signaling pathway through targeting FOXP2 in prostate cancer.

Dysregulated hematological and neurological expressed 1-like (HN1L) has been implicated in carcinogenesis of difference cancers, including hepatocellular carcinoma and breast cancer. However, the role of HN1L in the progression of prostate cancer (PCA) remains unknown. Therefore, we aimed to investigate the role of HN1L in stemness and progression of PCA. The expression of HN1L in PCA tissues and cells was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blot analysis, and/or immunohistochemistry (IHC). CD133+ cells were sorted from PCA cells using magnetic fluorescence cell sorting technology and were considered as cancer stem cells (CSCs). Sphere formation assays, transwell assays, and animal experiments were conducted to assess cell stemness, migration, invasion, and in vivo tumorigenesis, respectively. The results showed that HN1L expression was higher in PCA tissues and cells as compared with normal tissues and cells, as well as in CD133+ cells as compared with CD133- cells. HN1L knockdown significantly decreased the expression levels of CSC markers including OCT4 (POU class 5 homeobox 1), CD44, and SRY-box transcription factor 2, inhibited cell migration, invasion, and tumorigenesis and decreased the number of tumor spheroids and CD133+ cell population. Furthermore, we found that HN1L could bind to forkhead box P2 (FOXP2) and positively regulated transforming growth factor-β (TGF-β) expression via upregulation of FOXP2. In addition, the overexpression of TGF-β in HN1L-knockdown PCA cells increased the number of tumor spheroids and CD133+ cell population, as well as enhanced cell migration and invasion. Collectively, this study demonstrates that HN1L promotes stem cell-like properties and cancer progression by targeting FOXP2 through TGF-β signaling pathway in PCA.

Bone marrow-derived mesenchymal stem cells combined with gonadotropin therapy restore postnatal oogenesis of chemo-ablated ovaries in rats via enhancing very small embryonic-like stem cells

BackgroundVery small embryonic-like stem cells (VSELs) are a rare population within the ovarian epithelial surface. They contribute to postnatal oogenesis as they have the ability to generate immature oocytes and resist the chemotherapy. These cells express markers of pluripotent embryonic and primordial germ cells.ObjectiveWe aimed to explore the capability of VSELs in restoring the postnatal oogenesis of chemo-ablated rat ovaries treated with bone marrow-derived mesenchymal stem cells (BM-MSCs) combined with pregnant mare serum gonadotropin (PMSG).MethodsFemale albino rats were randomly assigned across five groups: I (control), II (chemo-ablation), III (chemo-ablation + PMSG), IV (chemo-ablation + MSCs), and V (chemo-ablation + PMSG + MSCs). Postnatal oogenesis was assessed through measurement of OCT4, OCT4A, Scp3, Mvh, Nobox, Dazl4, Nanog, Sca-1, FSHr, STRA8, Bax, miR143, and miR376a transcript levels using qRT-PCR. Expression of selected key proteins were established as further confirmation of transcript expression changes. Histopathological examination and ovarian hormonal assessment were determined.ResultsGroup V displayed significant upregulation of all measured genes when compared with group II, III or IV. Protein expression confirmed the changes in transcript levels as group V displayed the highest average density in all targeted proteins. These results were confirmed histologically by the presence of cuboidal germinal epithelium, numerous primordial, unilaminar, and mature Graafian follicles in group V.ConclusionVSELs can restore the postnatal oogenesis in chemo-ablated ovaries treated by BM-MSCs combined with PMSG.

Cell-Permeable Oct4 Gene Delivery Enhances Stem Cell-like Properties of Mouse Embryonic Fibroblasts.

Direct conversion of one cell type into another is a trans-differentiation process. Recent advances in fibroblast research revealed that epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition. Conversely, fibroblasts can also give rise to epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell-like properties in fibroblasts, the Oct4 transcription factor acts as a master transcriptional regulator for reprogramming somatic cells. Notably, the production of gene complexes with cell-permeable peptides, such as low-molecular-weight protamine (LMWP), was proposed to induce reprogramming without cytotoxicity and genomic mutation. We designed a complex with non-cytotoxic LMWP to prevent the degradation of Oct4 and revealed that the positively charged cell-permeable LMWP helped condense the size of the Oct4-LMWP complexes (1:5 N:P ratio). When the Oct4-LMWP complex was delivered into mouse embryonic fibroblasts (MEFs), stemness-related gene expression increased while fibroblast intrinsic properties decreased. We believe that the Oct4-LMWP complex developed in this study can be used to reprogram terminally differentiated somatic cells or convert them into stem cell-like cells without risk of cell death, improving the stemness level and stability of existing direct conversion techniques.

Abstract 2419: Two-tiered inhibition of TGFBR2 signaling via ITD-1 and miR-149-3p targets CD44High glioma stem cells and non-stem-like GBM cells

Abstract The remarkable heterogeneity of glioblastoma (GBM), its therapy resistance and high recurrence rate present formidable challenges for effective treatment. A critical component of GBM malignancy derives from the heterogeneous population of glioma stem-like cells (GSCs) that are especially efficient in promoting tumorigenicity and chemoradiation resistance. Previous findings by our lab and others have established Oct4 and Sox2 transcription factors as key drivers of the GSC phenotype. Development of effective GBM therapeutics requires a deeper knowledge of the genetic and epigenetic drivers of GSCs, particularly the Oct4/Sox2-mediated mechanisms that promote and maintain more tumorigenic and resistant GSC subsets. In GBM, TGF-β has well-established oncogenic functions and is tied to multiple phenotypic aspects of GSCs. However, little has been done to elucidate the relevance of TGF-β type II receptor (TGFBR2) in GSCs and GBM as a whole. Here, we show that TGFBR2 is a direct transcriptional target of Oct4 and Sox2 and is upregulated in both newly diagnosed and recurrent GBM. Multidimensional analysis identified overactivation of canonical TGF-β signaling in Oct4/Sox2-expressing GSCs. Furthermore, transcriptional targets of Smad2/3 induced by Oct4 and Sox2 are upregulated in TGFBR2High clinical GBM samples, a subpopulation also shown to associate with poor patient outcome. Notably, scRNA-Seq analysis revealed enrichment of TGFBR2 in CD44High, but not CD133/SSEA-1High, GSCs, a subset known to be highly proliferative and resistant to standard therapy. Likewise, a strong correlation between TGFBR2 and CD44 expression was found in clinical GBM samples. Despite promising results in preclinical studies, inhibition of TGF-β signaling through ligand or receptor targeting has been ineffective against clinical GBM, due to multiple potential reasons including activation of compensatory signaling pathways. Therefore, we apply a multidimensional approach, incorporating direct inhibition of both TGFBR2 and its oncogenic downstream effectors, in an effort to produce a more robust response and minimize the potential for compensatory resistance. Bioinformatic analysis predicted miR-149-3p to target a collection of Oct4/Sox2-induced Smad2/3-regulated transcripts, most of which, including CD44, have well-established oncogenic function and/or overexpression in GBM. Transient expression of miR-149-3p reduced self-renewal capacity of GSC spheres, a necessary ability for in vivo tumor propagation. Furthermore, miR-149-3p enhanced the growth-inhibitory effects of ITD-1, a small molecule TGFBR2 inhibitor, in both GSCs and non-stem-like GBM cells. These findings demonstrate that Oct4/Sox2-induced TGFBR2 signaling is clinically relevant and has the potential to be effectively combated using a carefully structured two-tiered therapeutic approach in GBM. Citation Format: Amanda L. Johnson, Jack Korleski, Hernando Lopez-Bertoni, John Laterra. Two-tiered inhibition of TGFBR2 signaling via ITD-1 and miR-149-3p targets CD44High glioma stem cells and non-stem-like GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2419.