Pluripotent Cells Research Articles

Cohesin and condensin regulate chromosome topology and play an essential role in maintaining pluripotency in embryonic stem cells

Cohesin and condensin, two related protein complexes, play essential roles in ensuring the accurate segregation of the genome into daughter cells during cell division. However, the interaction between cohesin and condensin in embryonic stem cells remains unclear, as does the specific function of the meiosis-specific cohesin complex. Cohesin maintains the cohesion of replicated sister chromatids until their separation at anaphase, whereas condensin facilitates the reorganization of chromosomes into a highly compact structure characteristic of mitosis. First, we found via ChIP-seq analysis that cohesins (SMC3, RAD21, and REC8) and condensin (SMC4) share DNA binding sites in close proximity and directly interact with the insulator protein CTCF. Second, siRNA-regulated SMC3 depletion led to nuclear accumulation of SMC4. Third, embryonic stem (ES) cells uniquely harbor cohesin complexes containing the meiotic kleisin subunit REC8. RAD21 knockdown increased the proportion of SMC3–REC8 complexes. Our findings indicate that cohesin and condensin make important contributions to the functions of the chromosomal organization, and that meiotic cohesin may be specifically required for the mitotic program in ES cells.

New considerations in selecting donors for dental pulp stem cells: a pilot study

Background/purposeTissue engineering based on stem cell therapy necessitates a substantial quantity of high-quality stem cells. However, current sources face limitations, including narrow donor pools, compromised biological properties due to cryopreservation, and cellular senescence resulting from in vitro passaging and expansion. This study examines the impact of mild periodontitis on the biological performance of dental pulp stem cells (DPSCs) to explore the potential of broadening the donor pool for these cells.Materials and methodsThe experiment included two variables: age and the presence of periodontitis. DPSCs were isolated from six healthy subjects and six patients with mild periodontitis. Healthy subjects were categorized into Groups A (28–32 years) and B (52–54 years), and patients with mild periodontitis were categorized into Groups C (31–33 years) and D (50–53 years). The analyses included cell morphology, proliferation rate, multilineage differentiation capacity, apoptosis, and surface marker expression.ResultNo significant differences in cell morphology, pluripotency, or senescence were observed between healthy controls and periodontitis patients across age groups. Additionally, data on proliferation, pluripotency, and senescence were not significantly different. In healthy subjects, increased age was correlated with more elongated, flattened, and broader cells, alongside greater heterogeneity and intercellular granules. The proliferation and differentiation capacities decreased, whereas the degree of apoptosis increased. Similar trends were noted in patients with periodontitis.ConclusionThe biological properties of DPSCs remain unchanged in teeth with mild periodontitis, providing valuable insights for addressing the shortage of DPSCs in tissue engineering. Teeth with mild periodontitis have the potential to be pulp stem cell donors.

The Sotos syndrome gene Nsd1 safeguards developmental gene enhancers poised for transcription by maintaining the precise deposition of histone methylation.

Germline haploinsufficiency of NSD1 is implicated as the etiology of Sotos syndrome; however, the underlying mechanism remains far from being clear. Here, we use mouse embryonic stem cell (mESC) differentiation as a model system to address this question. We found Nsd1 to be indispensable for the faithful differentiation of mESCs into three primary germ layers, particularly, various meso-endodermal cell lineages related to development of the heart and the skeletal system. Time-course transcriptomic profiling following the mESC differentiation revealed that Nsd1 not only facilitates the basal expression but also permits the differentiation-accompanied rapid induction of a suite of meso-endoderm lineage-specifying transcription factor (TF) genes such as T and Gata4. Mechanistically, Nsd1 directly occupies putative distal enhancers of the lineage TF genes under the pluripotent cell state, where it deposits H3K36me2 to antagonizes the excessive H3K27me3 and maintain the basal H3K27ac level, thereby safeguarding these gene enhancers at a primed state that responds readily to differentiation cues. In agreement, gene rescue assays using the Nsd1 knockout mESCs showed that the H3K36me2 catalysis by Nsd1 requires several functional modules within Nsd1 (namely, PHD1-4, PWWP2 and SET) to a similar degree. Disruption of either one of these Nsd1 modules severely abrogated H3K36me2 in mESCs and significantly impaired appropriate induction of developmental genes upon mESC differentiation. Altogether, our study provides novel molecular insight into how the NSD1 perturbation derails normal development and causes the disease.

Cell therapy for liver disorders: past, present and future.

The liver fulfils a plethora of vital functions and, due to their importance, liver dysfunction has life-threatening consequences. Liver disorders currently account for more than two million deaths annually worldwide and can be classified broadly into three groups, considering their onset and aetiology, as acute liver diseases, inherited metabolic disorders and chronic liver diseases. In the most advanced and severe forms leading to liver failure, liver transplantation is the only treatment available, which has many associated drawbacks, including a shortage of organ donors. Cell therapy via fully mature cell transplantation is an advantageous alternative that may be able to restore a damaged organ's functionality or serve as a bridge until regeneration can occur. Pioneering work has shown that transplanting adult hepatocytes can support liver recovery. However, primary hepatocytes cannot be grown extensively in vitro as they rapidly lose their metabolic activity. Therefore, different cell sources are currently being tested as alternatives to primary cells. Human pluripotent stem cell-derived cells, chemically induced liver progenitors, or 'liver' organoids, hold great promise for developing new cell therapies for acute and chronic liver diseases. This Review focuses on the advantages and drawbacks of distinct cell sources and the relative strategies to address different therapeutic needs in distinct liver diseases.

A case of tracheal chondrosarcoma

Background. Chondrosarcoma of the trachea is an extremely rare malignant tumor, accounting for only 0.1 % of all tracheal tumors. It is believed that chondrosarcoma arises from malignant degeneration in mesenchymal pluripotent cells of cartilage tissue, ossified cartilage, and enchondromas. Chondrosarcoma of the trachea is the least-studied tumor, with only 35 cases described in the literature from 1959 to 2020. Surgery with R0 resection remains the gold standard treatment of tracheal chondrosarcoma. The median age of patients at the time of diagnosis was 68 years, and 91 % were male. The most common symptoms at the initial visit were dyspnea (up to 80 % of cases). Tumors located in the proximal third of the trachea were observed in 54 % of cases, while tumors in the distal third of the trachea were found in 29 % of cases. Low-grade tumors (g1) were the most common (54 %). Computed tomography of the chest is considered the gold standard for the detection of tracheal tumors. Purpose: To demonstrate a successful case of treatment of tracheal chondrosarcoma. Case presentation. A 67-year-old male patient was diagnosed with a tracheal tumor. On 04.07.2022, surgical treatment was performed: circular resection of the 2nd, 3rd, and 4th tracheal rings with lymph node dissection of the regional paratracheal lymph nodes R0. The postoperative period was uneventful. Adjuvant therapy was not given. At the 2-year follow-up, no anastomotic complications were reported. Conclusion. To date, circular resection of the trachea (R0) with end-to-end tracheal anastomosis is the gold standard for treating tracheal chondrosarcoma. this approach was performed in 77 % of cases with the median follow-up time of more than 30 months and relapse rate of only 6 %. It should be noted that adjuvant chemotherapy was not used in these cases due to the extremely low sensitivity of the tumor to chemotherapy drugs.

APC/C-mediated ubiquitylation of extranucleosomal histone complexes lacking canonical degrons

Non-degradative histone ubiquitylation plays a myriad of well-defined roles in the regulation of gene expression and choreographing DNA damage repair pathways. In contrast, the contributions of degradative histone ubiquitylation on genomic processes has remained elusive. Recently, the APC/C has been shown to ubiquitylate histones to regulate gene expression in pluripotent cells, but the molecular mechanism is unclear. Here we show that despite directly binding to the nucleosome through subunit APC3, the APC/C is unable to ubiquitylate nucleosomal histones. In contrast, extranucleosomal H2A/H2B and H3/H4 complexes are broadly ubiquitylated by the APC/C in an unexpected manner. Using a combination of cryo-electron microscopy (cryo-EM) and biophysical and enzymatic assays, we demonstrate that APC8 and histone tails direct APC/C-mediated polyubiquitylation of core histones in the absence of traditional APC/C substrate degron sequences. Taken together, our work implicates APC/C-nucleosome tethering in the degradation of diverse chromatin-associated proteins and extranucleosomal histones for the regulation of transcription and the cell cycle and for preventing toxicity due to excess histone levels.

Telomere length in offspring is determined by mitochondrial-nuclear communication at fertilization

The initial setting of telomere length during early life in each individual has a major influence on lifetime risk of aging-associated diseases; however there is limited knowledge of biological signals that regulate inheritance of telomere length, and whether it is modifiable is not known. We now show that when mitochondrial activity is disrupted in mouse zygotes, via exposure to 20% O2 or rotenone, telomere elongation between the 8-cell and blastocyst stage is impaired, with shorter telomeres apparent in the pluripotent Inner Cell Mass (ICM) and persisting after organogenesis. Identical defects of elevated mtROS in zygotes followed by impaired telomere elongation, occurred with maternal obesity or advanced age. We further demonstrate that telomere elongation during ICM formation is controlled by mitochondrial-nuclear communication at fertilization. Using mitochondrially-targeted therapeutics (BGP-15, MitoQ, SS-31, metformin) we demonstrate that it is possible to modulate the preimplantation telomere resetting process and restore deficiencies in neonatal telomere length.

Knockdown of Mageb16 disrupts cell proliferation and lineage specification during mouse preimplantation development.

Melanoma antigen family member B16 (Mageb16) is crucial for maintaining the pluripotency and differentiation of embryonic stem cells. However, the expression pattern and biological role of Mageb16 during preimplantation development remain unclear. In this study, we showed that Mageb16 mRNA expression was dynamic throughout preimplantation development, with the highest level occurring at the morula stage. The abundance of Mageb16 mRNA was effectively reduced via small interfering RNA (siRNA) microinjection. Mageb16 knockdown significantly reduced the blastocyst formation rate, outgrowth formation rate, and total number of cells per embryo. Importantly, the reduction in MAGEB16 blocked cell cycle progression at the G2/M phase and disrupted lineage segregation but did not induce DNA damage in preimplantation mouse embryos. Intriguingly, Mageb16 knockdown increased the level of histone H3 lysine 27 acetylation (H3K27ac) but attenuated transcriptional activity. Together, our results reveal a crucial role for Mageb16 in mouse preimplantation development, likely by controlling cell proliferation and lineage specification.

Regulatory dynamics of Nanog in chondrocyte dedifferentiation: role of KLF4/p53 and p38/AKT signaling.

Homeobox protein Nanog, a member of the transcription factor family, plays a crucial role in maintaining the pluripotency and self-renewal of embryonic stem cells. Due to its diverse activities, Nanog has been identified in multiple cell types, including embryonic stem cells (ESCs) and cancer stem cells (CSCs). However, its molecular mechanism in chondrocytes remains unclear. In this study, we explored the effects of Nanog on chondrocytes and its interaction with chondrocyte-specific proteins. Chondrocytes were transfected with a Nanog cDNA vector, resulting in reduced expression of the chondrogenic markers Type II collagen and SOX9, as confirmed by western blot, RT-PCR, and immunofluorescence. Following siRNA transfection, the dedifferentiation effect of Nanog was reversed, restoring Type II collagen and SOX9 expression. We also discovered that the mechanism by which Nanog affects chondrocytes is closely linked to p53 and KLF4. Overexpression of KLF4 induced the phosphorylation of p53, and phospho-p53 directly inhibited Nanog expression. Moreover, the p53 activator Nutlin-3A accelerated Nanog degradation, while the p53 inhibitor Pifithrin-α stabilized Nanog. Stabilized Nanog continued to promote chondrocyte dedifferentiation. Additional experiments were performed to identify the signaling pathways involved in Nanog-induced chondrocyte dedifferentiation. Our results showed that Nanog overexpression in chondrocytes significantly impacted the p38 kinase and AKT signaling pathways. Inhibition of p38 and AKT with SB203580 and LY294002 reduced Nanog expression and partially restored Type II collagen levels. Conversely, activation with anisomycin(ANS) and 740 Y-P enhanced Nanog expression, further reducing Type II collagen levels. To investigate Nanog's role in early development in vivo, we injected Nanog expression vectors into zebrafish embryos. The injected zebrafish exhibited structural defects in craniofacial cartilage, confirming Nanog's involvement in chondrocyte differentiation. These findings suggest that Nanog induces chondrocyte dedifferentiation, and this process can be modulated via the p53/KLF4 and p38/AKT pathways.

Parenteral Nanoemulsion for Optimized Delivery of GL-II-73 to the Brain—Comparative In Vitro Blood–Brain Barrier and In Vivo Neuropharmacokinetic Evaluation

Background/Objectives: GL-II-73 is a positive allosteric modulator that is selective for α5GABAA receptors and has physicochemical properties that favor nanocarrier formulations when parenteral delivery to the central nervous system is desired. Our aim was to develop an optimized nanoemulsion containing GL-II-73 and subsequently test whether this would improve permeation across the blood–brain barrier (BBB) and availability in the brain. Methods: The nanoemulsions were formulated and subjected to detailed physiochemical characterization. The optimized formulation was tested in comparison to a solution of GL-II-73 in the appropriate solvent in an in vitro model of the blood–brain barrier based on human induced pluripotent stem cell-derived microvascular endothelial cells, astrocytes, and pericytes. Plasma and brain exposure to GL-II-73 and its metabolite MP-III-022 was investigated in an in vivo neuropharmacokinetic study in rats exposed to the selected nanoemulsion and the conventional solution formulation. Results: The selected biocompatible nanoemulsion exhibited satisfactory physicochemical properties for parenteral administration, with a Z-ave of 122.0 ± 1.5, PDI of 0.123 ± 0.009 and zeta potential of −40.7 ± 1.5, pH of 5.16 ± 0.04, and adequate stability after one year of storage, and allowed the localization of GL-II-73 in the stabilization layer. The permeability of GL-II-73 through the BBB was twice as high with the selected nanoemulsion as with the solution. The availability of GL-II-73 and MP-III-022 (also a positive allosteric modulator selective for α5GABAA receptors) in the brain was 24% and 61% higher, respectively, after intraperitoneal administration of the nanoemulsion compared to the solution; the former increase was statistically significant. Conclusions: The increased permeability in vitro proved to be a good predictor for the improved availability of GL-II-73 in brain tissue in vivo from the formulation obtained by encapsulation in a nanoemulsion. The putative additive effect of the parent molecule and its metabolite MP-III-022 could lead to enhanced and/or prolonged modulation of α5GABAA receptors in the brain.

NK-cell cytotoxicity toward pluripotent stem cells and their neural progeny: impacts of activating and inhibitory receptors and KIR/HLA mismatch.

Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK-cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2, and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2, or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS-cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS-cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2-stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS-cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK-cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.

Genetic variation modulates susceptibility to aberrant DNA hypomethylation and imprint deregulation in naive pluripotent stem cells.

Genetic variation modulates susceptibility to aberrant DNA hypomethylation and imprint deregulation in naive pluripotent stem cells.

Generation and characterisation of seven induced pluripotent stem cell lines from two patients with Parkinson’s disease carrying the pathological variant c.1087G>T of the LGR4 gene

Parkinson’s disease is a neurodegenerative disorder affecting dopaminergic neurons of the substantia nigra pars compacta. The known pathological genetic variants may explain the cause of only 5 % of cases of the disease. In our study, we found two patients with a clinical diagnosis of Parkinson’s disease with the genetic va riant c.1087G>T (p.Gly363Cys) of the LGR4 gene. The LGR4 gene encodes the membrane receptor LGR4 (leucine rich repeat containing G protein-coupled receptor 4) associated with the G protein. We hypothesize that the LGR4 gene may be either a direct cause or a risk factor for this disease, since it is one of the main participants of the WNT/β-catenin signalling pathway. This signalling pathway is necessary for the proliferation of neurons during their differentiation, which may lead to Parkinson’s disease. To study the relationship between this genetic variant and Parkinson’s disease, an ideal tool is a cellular model based on induced pluripotent stem cells (iPSCs) and their differentiated derivatives, dopaminergic neurons. We reprogrammed the peripheral blood mononuclear cells of the two patients with the c.1087G>T variant of the LGR4 gene with non-integrating episomal vectors expressing OCT4, SOX2, KLF4, LIN28, L-MYC and mp53DD proteins. The obtained seven lines of induced pluripotent stem cells were characterised in detail. The iPSCs lines obtained meet all the requirements of pluripotent cells, namely, they stably proliferate, form colonies with a morphology characteristic of human pluripotent cells, have a normal diploid karyotype, express endogenous alkaline phosphatase and pluripotency markers (OCT4, NANOG, SSEA-4 and SOX2) and are capable to differentiate into derivatives of the three germ layers. The iPSC lines obtained in this work can be used as a tool to generate a relevant model to study the effect of the pathological variant c.1087G>T of the LGR4 gene on dopaminergic neuron differentiation.

Engineered microtissues to model the effects of dynamic heterotypic cell signaling on iPSC-derived human hepatocyte maturation.

Engineered microtissues to model the effects of dynamic heterotypic cell signaling on iPSC-derived human hepatocyte maturation.

PGC7 maintains the pluripotency of F9 embryonic carcinoma cells by promoting Nanog translation.

Primordial germ cell 7 (PGC7) is prominently expressed in primordial germ cells (PGCs) and embryonic stem cells (ESCs), serving as a pivotal marker for discerning stem cell pluripotency. However, the role of PGC7 in regulating core pluripotency factors remains unclear. In this study, the expression dynamics of PGC7 and pluripotency- associated proteins are systematically evaluated by quantitative reverse transcription PCR (RT-qPCR) and western blot analysis. Complementary experimental approaches including confocal immunofluorescence and Co- immunoprecipitation (Co-IP) assays are subsequently employed to establish subcellular colocalization patterns and elucidate the molecular mechanisms associated with PGC7 function. The results show that PGC7 is closely associated with the pluripotency status of F9 embryonal carcinoma (EC) cells. Notably, PGC7 can counteract the decrease in pluripotency induced by retinoic acid (RA). Ectopic expression of PGC7 in F9 EC cells enhances the translation of Nanog. Mechanistic analysis reveal that PGC7 activates Y-box binding protein 1 (YBX1) phosphorylation by enhancing the interaction between YBX1 and AKT1. The subsequent phosphorylation of YBX1 reduces its binding to Nanog mRNA and promotes the translation of Nanog. These results shed light on a previously unknown role of PGC7 in supporting the translation of Nanog, offering valuable insights into the functions of PGC7 in F9 EC cells.

The lncRNA DSCR9 is modulated in pulmonary arterial hypertension endothelial cell models and is associated with alterations in the nitric oxide pathway.

The lncRNA DSCR9 is modulated in pulmonary arterial hypertension endothelial cell models and is associated with alterations in the nitric oxide pathway.

Protocol for generating a pancreatic cancer organoid associated with heterogeneous tumor microenvironment.

Protocol for generating a pancreatic cancer organoid associated with heterogeneous tumor microenvironment.

Protocol for the differentiation of human alveolar epithelial type I cells from pluripotent stem cell-derived type II-like cells.

Protocol for the differentiation of human alveolar epithelial type I cells from pluripotent stem cell-derived type II-like cells.

Metabolism-epigenetic interaction-based bone and dental regeneration: From impacts and mechanisms to treatment potential.

Metabolism-epigenetic interaction-based bone and dental regeneration: From impacts and mechanisms to treatment potential.

Development of Methods for the Early Detection of Chemical Hazard and the Prevention of Pre-disease, Focusing on Environment, Food, and Health

Based on the perspectives of the environment, food, and health, this review reflects on previous research examining stem cells for the early detection of chemical hazards and the development of preventive health tools. The risks posed by endocrine-disrupting chemicals in the environment are investigated, including studies on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phthalate esters, and bisphenol A. Building on the findings of these studies, this review identifies emerging challenges in the field of endocrine-disrupting chemical research. Moreover, this paper explores innovative testing methods aimed at accurately evaluating the impact of chemicals on human health. The key topics covered include the implementation of developmental neurotoxicity testing methods, the species-specific effects of methylmercury, nanomaterials and the application of human pluripotent cells to assess the effects of low-dose radiation. Additionally, this review highlights transformative approaches in chemical health impact assessment that integrate cell science and artificial intelligence, and addresses challenges related to the application of multi-omics technologies in environmental health and toxicology.