Expression Of Oct4 Research Articles

Optogenetic control of YAP reveals a dynamic communication code for stem cell fate and proliferation

YAP is a transcriptional regulator that controls pluripotency, cell fate, and proliferation. How cells ensure the selective activation of YAP effector genes is unknown. This knowledge is essential to rationally control cellular decision-making. Here we leverage optogenetics, live-imaging of transcription, and cell fate analysis to understand and control gene activation and cell behavior. We reveal that cells decode the steady-state concentrations and timing of YAP activation to control proliferation, cell fate, and expression of the pluripotency regulators Oct4 and Nanog. While oscillatory YAP inputs induce Oct4 expression and proliferation optimally at frequencies that mimic native dynamics, cellular differentiation requires persistently low YAP levels. We identify the molecular logic of the Oct4 dynamic decoder, which acts through an adaptive change sensor. Our work reveals how YAP levels and dynamics enable multiplexing of information transmission for the regulation of developmental decision-making and establishes a platform for the rational control of these behaviors.

Resistance to 2-Hydroxy-Flutamide in Prostate Cancer Cells Is Associated with the Downregulation of Phosphatidylcholine Biosynthesis and Epigenetic Modifications.

In this study, we examined the metabolic adaptations of a chemoresistant prostate cancer cell line in comparison to a sensitive cell line. We utilized prostate cancer LNCaP cells and subjected them to a stepwise increase in the antiandrogen 2-hydroxy-flutamide (FLU) concentration to generate a FLU-resistant cell line (LN-FLU). These LN-FLU cells displayed characteristics of cancer stem cells, exhibited drug resistance, and showed a significantly reduced expression of Cyclin D1, along with the overexpression of p16, pointing to a proliferation arrest. In comparing the cancer stem-like LN-FLU cells to the LNCaP cells, we observed a decrease in the expression of CTP-choline cytidylyl transferase α (CCTα), as well as a decline in choline kinase, suggesting altogether a downregulation of the phosphatidylcholine biosynthetic pathway. In addition, we found decreased levels of the protein methyl transferase PRMT2 and the upregulation of the histone deacetylase Sirtuin1 (Sirt1). Analysis of the human prostate cancer samples revealed similar results in a population with high expressions of the stem cell markers Oct4 and ABCB1A1. Our findings suggest that the adaptation of prostate cancer cells to antiandrogens could induce reprogramming into stem cells that survive in a low phosphocholine metabolism and cell cycle arrest and display drug resistance.

Maternal exposure to hyperbaric oxygen at the preimplantation stages increases apoptosis and ectopic Cdx2 expression and decreases Oct4 expression in mouse blastocysts via Nrf2-Notch1 upregulation and Nf2 downregulation.

The environmental oxygen tension has been reported to impact the blastocyst quality and cell numbers in the inner cell mass (ICM) during human and murine embryogenesis. While the molecular mechanisms leading to increased ICM cell numbers and pluripotency gene expression under hypoxia have been deciphered, it remains unknown which regulatory pathways caused the underweight fetal body and overweight placenta after maternal exposure to hyperbaric oxygen (HBO). The blastocysts from the HBO-exposed pregnant mice revealed significantly increased signals of reactive oxygen species (ROS) and nuclear Nrf2 staining, decreased Nf2 and Oct4 expression, increased nuclear Tp53bp1 and active caspase-3 staining, and ectopic nuclear signals of Cdx2, Yap, and the Notch1 intracellular domain (N1ICD) in the ICM. In the ICM of the HBO-exposed blastocysts, both Nf2 cDNA microinjection and Nrf2 shRNA microinjection significantly decreased the ectopic nuclear expression of Cdx2, Tp53bp1, and Yap whereas increased Oct4 expression, while Nrf2 shRNA microinjection also significantly decreased Notch1 mRNA levels and nuclear expression of N1ICD and active caspase-3. We show for the first time that maternal exposure to HBO at the preimplantation stage induces apoptosis and impairs ICM cell specification via upregulating Nrf2-Notch1-Cdx2 expression and downregulating Nf2-Oct4 expression.

MTOR Plays an Important Role in the Stemness of Human Fetal Cartilage Progenitor Cells (hFCPCs).

Mammalian target of rapamycin (mTOR) is known to regulate self-renewal ability and potency of embryonic stem cells (ESCs) and adult stem cells in opposite manners. However, its effects vary even among adult stem cells and are not reported in fetal stem/progenitor cells. This study investigated the role of mTOR in the function of human fetal cartilage-derived progenitor cells (hFCPCs). mTOR activity in hFCPCs was first examined via the level of phosphor-mTOR until passage 19, together with doubling time of cells and senescence-associated b-galactosidase (SA-bGal). Then, the effect of 100nM rapamycin, the inhibitor of mTOR, was investigated on self-renewal ability, proliferation rate and osteogenic/adipogenic potential of hFCPCs in vitro. Expression of stemness genes (Oct-4, Sox2 and Nanog) and cell cycle regulators (CDK4 and Cyclin D1) was measured at mRNA or protein levels. mTOR activity was maintained constantly at high levels in hFCPCs until passage 19, while their proliferation rate was decreasing from 48h at passage 13 to 70h at passage 9 and senescent cells were observed at passage 18 (8.3 ± 1.2%) and 19 (15.6 ± 1.9%). Inhibition of mTOR in hFCPCs impaired their colony forming frequency (CFU-F) by 4 folds, while showing no change in their doubling time and expression of CDK4 and Cyclin D1. Upon mTOR inhibition, Oct4 expression decreased by 2 folds and 4 folds at the mRNA and protein levels, respectively, while that of Sox2 and Nanog did not change significantly. Finally, mTOR inhibition reduced osteogenic and adipogenic differentiation of hFCPCs in vitro. This study has shown that mTOR plays an important role in the self-renewal ability of hFCPCS but not in their proliferation, The effect of mTOR appears to be associated with Oct-4 expression and important in the osteogenic and adipogenic differentiation ability of hFCPCs.

The Survey of the Fruit of Capparis spinosa Extracts Effects on the Expressional Profile of Essential Self-renewal Genes in MCF7 Cell Line

Background: Several drugs have been proposed for the treatment of breast cancer, but none has fully treated the disease so far. Methods: MCF7 cells were cultured in RPMI-1640 medium, consisting of different concentrations of aqueous-alcoholic extract of the fruit of Capparis spinosa (0.125, 0.25, 0.5, 1, 1.5, 2, 2.5, and 5 mg/mL) for 48 and 72 hours. MTT assay was used to determine the cell proliferation inhibition (IC50). The percentage of apoptotic cells was determined by flow cytometry analysis using Annexin V/PI apoptosis detection kit. RT-PCR method was carried out to assess the fold changes of OCT4, NANOG, and SOX2 genes. Two-way ANOVA (tukeys) and t-test (repeated measure) were used for the statistical analysis of obtained data MTT assay and RT-PCR method, respectively. p < 0.05 was considered significant. Results: Our results have shown that cell death was induced by increasing fruit of Capparis spinosa extract concentration. IC50 was observed at 48 h culture period with 4.81 mg/mL, and 72 h with 2.72 mg/mL fruit extract, respectively. Flow cytometry results exhibited an obviously significant augmentation in apoptotic MF7 cells. According to RT-PCR findings on the fruit of Capparis spinosa extract-treated cells, the mean expression of OCT4, NANOG, and SOX2 genes decreased after 48 and 72 h of incubation with IC50 concentration compared to controls. Conclusion: The fruit of the Capparis spinosa species was able to decrease the expression of self-renewal genes in the MCF7 cell line. Therefore, the fruit of Capparis spinosa extract can be considered a promising candidate for the management of human breast cancer after clinical trials.

Generation of two human iPSC lines (HIMRi002-A and HIMRi003-A) derived from Caveolinopathy patients with rippling muscle disease

Here we introduce the human induced pluripotent stem cell lines (hiPSCs), HIMRi002-A and HIMRi003-A, generated from cultured dermal fibroblasts of 61-year-old (HIMRi002-A) and 38-year-old (HIMRi003-A) female patients, carrying a known heterozygous pathogenic variant (p.A46T) in the Caveolin 3 (CAV3) gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi002-A and HIMRi003-A display typical embryonic stem cell-like morphology, carry the p.A46T CAV3 gene mutation, express several pluripotent stem cell markers, retain normal karyotype (46, XX) and can differentiate in all three germ layers. We postulate that the HIMRi002-A and HIMRi003-A iPSC lines can be used for the characterization of CAV3-associated pathomechanisms and for developing new therapeutic options.

Effect of Human Bone Marrow Mesenchymal Stem Cells with Ectopic High OCT4 Expression on T Lymphocyte Function

To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro. Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation. Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C. Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.

Multidrug resistance phenotype and its relation to stem cell characteristics in chronic myeloid leukemia

The present work aimed to evaluate the expression profile of genes related to stem cells (SC) characteristics during the acquisition of the multidrug resistance (MDR) phenotype in the chronic myeloid leukemia (CML). For this, the K562 (non MDR) and FEPS (MDR) cell lines were used. K562 cells had resistance induced by exposure to daunorubicin (DNR), and induction was confirmed by flow cytometry with an increase in ABCB1 expression in K562 cells treated at the highest concentration. Real-time PCR gene expression analysis showed a direct relationship in the expression of OCT4 and ABCB1 genes, with an increase in ABCB1 expression after exposure to DNR, followed by an increase in OCT4 gene expression. This direct relationship was confirmed in the MDR FEPS cells that had the ABCB1 gene silenced. For the ALOX5 gene, we observed an inverse relationship with ABCB1, with a decrease in the expression of ALOX5 in the DNR-transformed K562 cells, and an increase in the expression of this gene when ABCB1 was silenced in the FEPS cells. Thus, during the acquisition of the MDR phenotype by the K562 cells, it was possible to observe that there is an increase in the expression of ABCB1, accompanied by the expression of OCT4, while the expression of ALOX5 is decreased.

Metabolic and cell cycle shift induced by the deletion of Dnm1l attenuates the dissolution of pluripotency in mouse embryonic stem cells.

Mitochondria are versatile organelles that continuously change their morphology via fission and fusion. However, the detailed functions of mitochondrial dynamics-related genes in pluripotent stem cells remain largely unclear. Here, we aimed to determine the effects on energy metabolism and differentiation ability of mouse embryonic stem cells (ESCs) following deletion of the mitochondrial fission-related gene Dnml1. Resultant Dnm1l-/- ESCs maintained major pluripotency characteristics. However, Dnm1l-/- ESCs showed several phenotypic changes, including the inhibition of differentiation ability (dissolution of pluripotency). Notably, Dnm1l-/- ESCs maintained the expression of the pluripotency marker Oct4 and undifferentiated colony types upon differentiation induction. RNA sequencing analysis revealed that the most frequently differentially expressed genes were enriched in the glutathione metabolic pathway. Our data suggested that differentiation inhibition of Dnm1l-/- ESCs was primarily due to metabolic shift from glycolysis to OXPHOS, G2/M phase retardation, and high level of Nanog and 2-cell-specific gene expression.

Generation of a human iPSC line (HIMRi001-A) from a patient with filaminopathy

Here we introduce the human induced pluripotent stem cell (hiPSC) line HIMRi001-A generated from cultured dermal fibroblasts of a 60-year-old male patient with a myofibrillar myopathy, carrying a heterozygous c.4984C > T [p.Q1662X] mutation in the filamin C (FLNC)-gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi001-A displays typical embryonic stem cell-like morphology, carries the c.4984C > T FLNC gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and holds the potential to differentiate in all three germ layers. We postulate that HIMRi001-A can be used for the elucidation of FLNC-associated pathomechanisms and for developing new therapeutic options.

Proteomic Analysis and Reprogramming Potential of the Porcine Intra-Ooplasmic Nanovesicles.

Oocytes contain reprogramming machinery that can transform somatic cells into totipotent cells. In this study, we aimed to isolate and characterize nanovesicles from mature porcine oocytes and described them for the first time as "intra-ooplasmic vesicles (IOVs)". Isolated IOVs had an average diameter of 186.3 ± 10.8 nm. Proteomic analysis revealed 467 peptide reads, with the top 20 proteins related to reprogramming, antioxidative defense, cytoskeleton, heat shock proteins, and metabolism. Protein-protein interaction and gene ontology analysis indicated that these proteins were involved in various biological pathways, including protein folding, metabolism, and cellular responses to stress. Supplementing cultured fibroblasts with IOVs resulted in the expression of the pluripotency marker OCT4 and the early trophoblastic marker CDX2 and increased expression of the corresponding mRNAs together with increasing KLF4 and SALL4 expression. IOV treatment of fibroblasts for 14 consecutive days resulted in changes in cell morphology, with increased expression of ZEB2 and YBX3 as markers for epithelial-to-mesenchymal transition (EMT). These results provide a rationale for further characterization of IOVs, investigation of potential reprogramming capabilities for EMT, and the generation of induced pluripotent or oligopotent stem cells.

Cbx7 promotes the generation of induced pluripotent stem cells

The iPS cells were discovered in 2006. With their ability to differentiate into cells of all three germ layers, iPS cells have great potential for clinical applications. Oct4, Sox2, c-Myc, and Klf4 were identified as the most effective factors for generating iPS cells. Despite this, iPS cells manufactured with these factors would still be inefficient. As a member of the chromobox family, chromobox protein homolog 7 (Cbx7) binds to PRC1 and PRC2 to inhibit genes involved in differentiation. A decrease in the expression of Cbx7 is observed during embryonic stem cell differentiation. Currently, no report discusses the role of Cbx7 in the production of iPS cells. In this study, we hypothesized that Cbx7 could increase iPS cell generation. We confirmed that Cbx7 is highly expressed in pluripotent stem cells (including ES and iPS cells). In addition, transfecting Cbx7 into fibroblasts increased Oct4, Sox2, c-Myc, and Klf4 expression. Moreover, we describe a novel approach to producing iPS cells using Cbx7 in combination with Oct4, Sox2, c-Myc, and Klf4. In summary, we have demonstrated that Cbx7 enhances the reprogramming of iPS cells and characterized the stemness and pluripotency of iPS cells.

Pluripotency and embryonic lineage genes expression in the presence of small molecule inhibitors of FGF, TGFβ and GSK3 during pre-implantation development of goat embryos

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment markers (REX1, CDX2, GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFβ inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2–7 post-insemination in goat.Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG, OCT4 and GATA4, while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4, while the combination of the three molecules significantly decreases the expression of NANOG, SOX2 and CDX2.According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4, NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat.

Effects of adriamycin on cell differentiation and proliferation in rat testis

ABSTRACT Although adriamycin (ADR) is used to treat many cancers, it can be toxic to healthy organs including the testis. We investigated the effects of ADR on pluripotency in rat testis. Testicular damage was induced by either cumulative or single dose single dose administration of ADR in Wistar albino rats. Rats were divided randomly into three groups: untreated control, cumulative dose ADR group (2 mg/kg ADR every three days for 30 days) and single dose ADR group (15 mg/kg, single dose ADR). Testicular damage was evaluated and seminiferous tubule diameters were measured using light microscopy. Expression levels of Oct4, Sox2, Klf4, c-Myc, Utf1 and Dazl were assessed by immunohistochemistry and real time PCR. Serum testosterone levels were measured using ELISA assay. Histopathologic scores were lower and mean seminiferous tubule diameters were less compared to the ADR groups. Oct4, Sox2, Klf4 and Utf1 expressions were decreased significantly in spermatogenic cells of both cumulative and single dose ADR groups compared to the control group. We found that c-Myc expression in spermatogenic and Leydig cells were increased significantly in both ADR groups compared to the control group. Dazl expression was decreased in the cumulative adriamycin group compared to the control group, but increased in the single dose ADR group compared to both the control and cumulative ADR groups. Serum testosterone levels were decreased in both ADR groups compared to the control group. Our findings suggest that ADR is detrimental to regulation and maintenance of pluripotency in rat testis.

A functional crosstalk between the H3K9 methylation writers and their reader HP1 in safeguarding embryonic stem cell identity.

Histone H3 lysine 9 (H3K9) methylation, as a hallmark of heterochromatin, has a central role in cell lineage and fate determination. Although evidence of a cooperation between H3K9 methylation writers and their readers has started to emerge, their actual interplay remains elusive. Here, we show that loss of H3K9 methylation readers, the Hp1 family, causes reduced expression of H3K9 methyltransferases, and that this subsequently leads to the exit of embryonic stem cells (ESCs) from pluripotency and a reciprocal gain of lineage-specific characteristics. Importantly, the phenotypes of Hp1-null ESCs can be rescued by ectopic expression of Setdb1, Nanog, and Oct4. Furthermore, Setdb1 ablation results in loss of ESC identity, which is accompanied by a reduction in the expression of Hp1 genes. Together, our data support a model in which the safeguarding of ESC identity involves the cooperation between the H3K9 methylation writers and their readers.

The Stem Cell Expression Profile of Odontogenic Tumors and Cysts: A Systematic Review and Meta-Analysis.

Stem cells have been associated with self-renewing and plasticity and have been investigated in various odontogenic lesions in association with their pathogenesis and biological behavior. We aim to provide a systematic review of stem cell markers' expression in odontogenic tumors and cysts. The literature was searched through the MEDLINE/PubMed, EMBASE via OVID, Web of Science, and CINHAL via EBSCO databases for original studies evaluating stem cell markers' expression in different odontogenic tumors/cysts, or an odontogenic disease group and a control group. The studies' risk of bias (RoB) was assessed via a Joanna Briggs Institute Critical Appraisal Tool. Meta-analysis was conducted for markers evaluated in the same pair of odontogenic tumors/cysts in at least two studies. 29 studies reported the expression of stem cell markers, e.g., SOX2, OCT4, NANOG, CD44, ALDH1, BMI1, and CD105, in various odontogenic lesions, through immunohistochemistry/immunofluorescence, polymerase chain reaction, flow cytometry, microarrays, and RNA-sequencing. Low, moderate, and high RoBs were observed in seven, nine, and thirteen studies, respectively. Meta-analysis revealed a remarkable discriminative ability of SOX2 for ameloblastic carcinomas or odontogenic keratocysts over ameloblastomas. Stem cells might be linked to the pathogenesis and clinical behavior of odontogenic pathologies and represent a potential target for future individualized therapies.

The Effects of Pifithrin-µ on Spermatogonial Stem Cell Viability and Pluripotency

Introduction: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ), a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. Methods: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM/F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry technique by high PLZF and low c-Kit expressions. SSC colony formation assay was carried out and their viability was estimated by methylthiazolyldiphenyl-tetrazolium bromide (MTT, or 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5 µm). The apoptosis percentages were also measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. Results: The 0.6 and 1.2 µm PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5 µm PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentrations enhanced the colony size of SSCs and diminished their apoptosis. As well, exposure to PFT-µ upregulated Oct4 expression while downregulating the meiotic entry marker, Stra8. Conclusion: Based on findings, optimized concentrations of PFT-µ can decrease SSC apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

Bazi Bushen alleviates skin senescence by orchestrating skin homeostasis in SAMP6 mice.

Bazi Bushen, a Chinese-patented drug with the function of relieving fatigue and delaying ageing, has been proven effective for extenuating skin senescence. To investigate the potential mechanism, senescence-accelerated mouse prone 6 (SAMP6) was intragastrically administered with Bazi Bushen for 9 weeks to induce skin homeostasis. Skin homeostasis is important in mitigating skin senescence, and it is related to many factors such as oxidative stress, SASP, apoptosis, autophagy and stem cell. In our study, skin damage in SAMP6 mice was observed using HE, Masson and SA-β-gal staining. The content of hydroxyproline and the activities of SOD, MDA, GSH-PX and T-AOC in the skin were measured using commercial assay kits. The level of SASP factors (IL-6, IL-1β, TNF-α, MMP2 and MMP9) in skin were measured using ELISA kits. The protein expressions of p16, p21, p53, Bax, Bcl-2, Cleaved caspase-3, LC3, p62, Beclin1, OCT4, SOX2 and NANOG were measured by western blotting. The expression of ITGA6 and COL17A1 was measured by immunofluorescence staining and western blotting. Our findings demonstrated that Bazi Bushen alleviated skin senescence by orchestrating skin homeostasis, reducing the level of oxidative stress and the expression of SASP, regulating the balance of apoptosis and autophagy and enhancing the protein expressions of ITGA6 and COL17A1 to improve skin structure in SAMP6 mice. This study indicated that Bazi Bushen could serve as a potential therapy for alleviating skin senescence.

Nanog, Stat-3, and Sox-5 involvement in human fetal temporomandibular joint late development

Background and aimThe temporomandibular joint (TMJ) is a synovial joint that allows the complex movements essential for life. It connects the jawbone to the skull, working as a sliding hinge. Moreover, pluripotent stem cells are a source of precursors and tissue-specific cells in developing organisms, however, their biodistribution in developing fetal tissues is weakly studied. The aim of our study was analyse immunohistochemical expression of Nanog, Oct-4, Sox-2 and Stat-3 and Sox-5, in TMJ tissue samples from human fetuses aged between the 12th and 20th weeks of intrauterine life. Materials and methodsWe fixed and processed TMJ tissue samples from human fetuses, histological sections and immunohistochemical procedures were carried out. ResultsTMJ histological studies examination did not reveal any difference in the tissue organization between the samples in the studied periods. Immunohistochemical analysis demonstrated that Oct-4 and Sox-2 lack their expression in TMJ. In contrast, Nanog was expressed in nucleous of proliferative layer of mandibular condyle, Stat-3 was expressed in nuclear cells of articular disc, Stat-3 and Sox-5 showed positive nuclear and cytoplasmic immunostaining in codrocyte layers and in ossification areas. ConclusionsNanog acts in maintanence of pluripotency, Stat-3 in articular disc acts as a transcriptional factor. Stat-3 and Sox-2 act in chondrocyte and osteoblast diferentiation. Distribution of the cells, which express Nanog, Stat-3, and Sox-5 in TMJ tissue during fetal development, can help further understand its physiology, pathology, and repairing capacities.

Modeling human brain rhabdoid tumor by inactivating tumor suppressor genes in induced pluripotent stem cells

Atypical teratoid/rhabdoid tumor (ATRT) is a rare childhood malignancy that originates in the central nervous system. Over ninety-five percent of ATRT patients have biallelic inactivation of the tumor suppressor gene SMARCB1. ATRT has no standard treatment, and a major limiting factor in therapeutic development is the lack of reliable ATRT models. We employed CRISPR/Cas9 gene-editing technology to knock out SMARCB1 and TP53 genes in human episomal induced pluripotent stem cells (Epi-iPSCs), followed by brief neural induction, to generate an ATRT-like model. The dual knockout Epi-iPSCs retained their stemness with the capacity to differentiate into three germ layers. High expression of OCT4 and NANOG in neurally induced knockout spheroids was comparable to that in two ATRT cell lines. Beta-catenin protein expression was higher in SMARCB1-deficient cells and spheroids than in normal Epi-iPSC-derived spheroids. Nucleophosmin, Osteopontin, and Ki-67 proteins were also expressed by the SMARCB1-deficient spheroids. In summary, the tumor model resembled embryonal features of ATRT and expressed ATRT biomarkers at mRNA and protein levels. Ribociclib, PTC-209, and the combination of clofilium tosylate and pazopanib decreased the viability of the ATRT-like cells. This disease modeling scheme may enable the establishment of individualized tumor models with patient-specific mutations and facilitate high-throughput drug testing.