Embryonic Stem Cells Research Articles

Cooperative role of LSD1 and CHD7 in regulating differentiation of mouse embryonic stem cells

AbstractLysine-specific histone demethylase 1 (LSD1) is a histone demethylase that plays a critical role in epigenetic regulation by removing the methyl group from mono- and di-methylated lysine 4 on histone H3 (H3K4me1/2), acting as a repressor of gene expression. Recently, catalytically independent functions of LSD1, serving as a scaffold for assembling chromatin-regulator and transcription factor complexes, have been identified. Herein, we show for the first time that LSD1 interacts with chromodomain-helicase-DNA-binding protein 7 (CHD7) in mouse embryonic stem cells (ESCs). To further investigate the CHD7–LSD1 crosstalk, we engineered Chd7 and Chd7/Lsd1 knockout (KO) mouse ESCs. We show that CHD7 is dispensable for ESC self-renewal and survival, while Chd7 KO ESCs can differentiate towards embryoid bodies (EBs) with defective expression of ectodermal markers. Intriguingly, Chd7/Lsd1 double KO mouse ESCs exhibit proliferation defects similar to Lsd1 KO ESCs and have lost the capacity to differentiate properly. Furthermore, the increased co-occupancy of H3K4me1 and CHD7 on chromatin following Lsd1 deletion suggests that LSD1 is required for facilitating the proper binding of CHD7 to chromatin and regulating differentiation. Collectively, our results suggest that LSD1 and CHD7 work in concert to modulate gene expression and influence proper cell fate determination.

Corrigendum: Advances in understanding the regulation of pluripotency fate transition in embryonic stem cells

Corrigendum: Advances in understanding the regulation of pluripotency fate transition in embryonic stem cells

Early autonomous patterning of the anteroposterior axis in gastruloids

ABSTRACT Minimal in vitro systems composed of embryonic stem cells (ESCs) have been shown to recapitulate the establishment of the anteroposterior (AP) axis. In contrast to the native embryo, ESC aggregates – such as gastruloids – can break symmetry, which is demarcated by polarization of the mesodermal marker T, autonomously without any localized external cues. However, associated earliest patterning events, such as the spatial restriction of cell fates and concomitant transcriptional changes, remain poorly understood. Here, we dissect the dynamics of AP axis establishment in mouse gastruloids, particularly before external Wnt stimulation. Through single-cell RNA sequencing, we identify key cell state transitions and the molecular signatures of T+ and T− populations underpinning AP polarization. We also show that this process is robust to modifications of aggregate size. Finally, transcriptomic comparison with the mouse embryo indicates that gastruloids develop similar mesendodermal cell types, despite initial differences in their primed pluripotent populations, which adopt a more mesenchymal state in lieu of an epiblast-like transcriptome. Hence, our findings suggest the possibility of alternate ESC states in vivo and in vitro that can converge onto similar cell fates.

Subcellular mRNA kinetic modeling reveals nuclear retention as rate-limiting.

Eukaryotic mRNAs are transcribed, processed, translated, and degraded in different subcellular compartments. Here, we measured mRNA flow rates between subcellular compartments in mouse embryonic stem cells. By combining metabolic RNA labeling, biochemical fractionation, mRNA sequencing, and mathematical modeling, we determined the half-lives of nuclear pre-, nuclear mature, cytosolic, and membrane-associated mRNAs from over 9000 genes. In addition, we estimated transcript elongation rates. Many matured mRNAs have long nuclear half-lives, indicating nuclear retention as the rate-limiting step in the flow of mRNAs. In contrast, mRNA transcripts coding for transcription factors show fast kinetic rates, and in particular short nuclear half-lives. Differentially localized mRNAs have distinct rate constant combinations, implying modular regulation. Membrane stability is high for membrane-localized mRNA and cytosolic stability is high for cytosol-localized mRNA. mRNAs encoding target signals for membranes have low cytosolic and high membrane half-lives with minor differences between signals. Transcripts of nuclear-encoded mitochondrial proteins have long nuclear retention and cytoplasmic kinetics that do not reflect co-translational targeting. Our data and analyses provide a useful resource to study spatiotemporal gene expression regulation.

Pseudo-trajectory inference for identifying essential regulations and molecules in cell fate decisions.

Cell fate decision is crucial in biological development and plays fundamental roles in normal development and functional maintenance of organisms. By identifying key regulatory interactions and molecules involved in these fate decisions, we can shed light on the intricate mechanisms underlying the cell fates. This understanding ultimately reveals the fundamental principles driving biological development and the origins of various diseases. In this study, we present an overarching framework which integrates pseudo-trajectory inference and differential analysis to determine critical regulatory interactions and molecules during cell fate transitions. To demonstrate feasibility and reliability of the approach, we employ the differentiation networks of hepatobiliary system and embryonic stem cells as representative model systems. By applying pseudo-trajectory inference to biological data, we aim to identify critical regulatory interactions and molecules during the cell fate transition processes. Consistent with experimental observations, the approach can allow us to infer dynamical cell fate decision processes and gain insights into the underlying mechanisms which govern cell state decisions.

Proteomic and functional comparison between human induced and embryonic stem cells.

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

Abstract A004: RNA splicing alterations in the development of acquired MAPKi resistance in melanoma

Abstract The standard treatment for melanoma with a BRAF mutation involves Mitogen-Activated Protein Kinase inhibitors (MAPKi), which target the aberrant MAPK signaling pathway. Although patients initially respond well to this therapy, many develop acquired resistance over time. This resistance can be attributed to various altered signaling pathways, including the reactivation of the MAPK pathway, activation of alternative survival pathways like PI3K/PTEN/AKT, engagement of receptor tyrosine kinases (RTKs) such as PDGFRβ and EGFR, and developmental pathways. Despite extensive omics studies aimed at deciphering the mechanisms behind acquired resistance, the specific alterations within these pathways remain poorly understood. To gain further insights into acquired MAPKi resistance in melanoma, we explored the role of RNA splicing events by analyzing publicly available datasets of pre- and post-MAPKi treated melanoma from patient-derived cell lines and in vitro studies using PDX models. We investigated differential transcript usage (DTU) to detect specific splice variants altered during the development of resistance. Our analysis revealed significant transcript alterations during on-treatment that reverted to baseline states once resistance was established, underscoring the dynamic and adaptive nature of these changes. Genes with DTU were enriched in pathways related to MAPKi resistance, such as the MAPKi signaling pathway, PI3K/AKT pathway, and signaling by RTKs. Furthermore, the DTU-centered analysis provided better insights into MAPKi resistance mechanisms compared to standard differential gene expression (DEG) analysis, with DTUs showing higher enrichment scores in MAPKi resistance-related pathways. Further, identifying developmental splicing signatures highlights the complexity of MAPKi resistance, as it reveals significant upregulation of transcripts associated with embryonic melanoblast stem cells (MSCs) among the altered transcripts in on-treatment cell lines. This reprogramming through RNA splicing may confer an adaptive advantage, enabling melanoma cells to revert to a more plastic, stem-like state, with developmental splicing events playing a crucial role in the adaptive response to MAPKi therapy. Next, using a regulatory model based on the expression of splicing factors (SFs), we accurately predicted transcript alterations in on-treatment samples and identified key SFs. Our study highlights the crucial role of RNA splicing in the adaptive response of melanoma cells to MAPKi treatment. These insights pave the way for future research and therapeutic strategies focusing on RNA splicing to combat drug resistance in cancer. Citation Format: Sumit Mukherjee, Arashdeep Singh, Hyunjeong Joo, Sumeet Patiyal, Hyungsoo Kim, Lipika R. Pal, Kun Wang, Chi-Ping Day, Ze’ev A Ronai, Eytan Ruppin, Sridhar Hannenhalli. RNA splicing alterations in the development of acquired MAPKi resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A004.

Spinal cord neural stem cells derived from human embryonic stem cells promote synapse regeneration and remyelination in spinal cord injury model rats.

Spinal cord injury (SCI) is a devastating injury that significantly impairs patients' quality of life. To date, there is no effective treatment to mitigate nerve tissue damage and restore neurological function. Neural stem cells (NSCs) derived from human embryonic stem cells (hESCs) are considered an important cell source for reconstructing damaged neural circuits and enabling axonal regeneration. Recent preclinical studies have shown that NSCs are potential therapeutic cell sources for neuroprotection and neuroregeneration in SCI animal models. NSCs can be derived from different sources and the spinal cord-specific NSCs have a higher potential for the regeneration of SCI. However, the long-term therapeutic efficacy of spinal cord-specific NSCs remains unproven. Here, we generated human spinal cord NSCs (hSCNSCs) and investigated the effects of transplanted hSCNSCs on the repair of the SCI model rats for 60 days. The transplanted hSCNSCs improved BBB scores, reduced the lesion area and promoted an increase in the number of Nestin-positive cells in the spinal cord compared to the model rats. Meanwhile, hSCNSC transplantation promoted the expression of synaptophysin, a synaptic signature protein and MBP, a protein associated with remyelination. Interestingly, BAF45D, a chromatin remodelling factor that contributes to the induction of hSCNSCs with region-specific spinal cord identity, were increased by the hSCNSC transplantation. In addition, conditioned medium derived from the hSCNSCs also promoted regenerative repair of the injured spinal cord. These results demonstrate that hSCNSCs may play a critical role in the regenerative repair of SCI.

Abstract PR002: Hypoxia-induced epigenetic changes coordinate transcription start site selection and translational control through remodeling of 5’UTRs

Abstract Adaptation to hypoxia enhances cancer cell malignancy and negatively impacts patient outcomes. Reprogramming of mRNA translation is an essential component of the hypoxia-adaptive response. During hypoxia, cells undergo a global decrease in protein synthesis. However, translation of subsets of transcripts encoding stress-response, survival and stemness factors is increased. This selective regulation is thought to be governed largely via the interactions between specific mRNA features and remodeling of the translational apparatus. However, our understanding of the mechanisms that govern translational perturbations under hypoxia remains incomplete. Here, we interrogated the effects of hypoxia on global changes in histone methylation and transcription start site (TSS) selection, coupled with monitoring corresponding alterations in total mRNA levels and translation efficiencies on a genome-wide scale in T47D breast cancer cells and H9 human embryonic stem cells. This revealed widespread epigenetic alterations leading to TSS switching and extensive remodeling of 5’UTRs, and allowed us to map the impact of specific 5’UTR features on translation efficiency under hypoxia. Pyruvate Dehydrogenase Kinase 1 (PDK1), a key enzyme in orchestrating metabolic adaption to hypoxia, undergoes TSS switching leading to increased availability of efficiently translated 5’UTR isoforms, and we show that this effect is coordinated at an epigenetic level. Therefore, our findings uncover a mechanism driving translational reprogramming under hypoxia, whereby alterations in translational apparatus are orchestrated with epigenetic perturbations that result in 5’UTR remodeling of the transcriptome. Citation Format: Kathleen Watt, Bianca Dauber, Krzysztof J Szkop, Laura Lee, Predrag Jovanovic, Shan Chen, Laia Masvidal, Kristofersson Tandoc, Ranveer Palia, Ivan Topisirovic, Ola Larsson, Lynne-Marie Postovit. Hypoxia-induced epigenetic changes coordinate transcription start site selection and translational control through remodeling of 5’UTRs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR002.

Sambucus williamsii induced embryonic stem cells differentiated into neurons

Sambucus williamsii induced embryonic stem cells differentiated into neurons

Lactylation of Hdac1 regulated by Ldh prevents the pluripotent-to-2C state conversion.

Cellular metabolism regulates the pluripotency of embryonic stem cells (ESCs). Yet, how metabolism regulates the transition among different pluripotent states remains elusive. It has been shown that protein lactylation, which uses lactate, a metabolic product of glycolysis, as a substrate, plays a critical role in various biological events. Here we focused on that glycolysis regulates the conversion between ESCs and 2-cell-like cells (2CLCs) through protein lactylation. RNA-seq revealed the activation of 2-cell (2C) genes by suppression of Ldh. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with lactylated peptide enrichment and quantitative mass spectrometric analysis was carried out to investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition. And we focused on Hdac1. Lactylation of Hdac1 required for silencing 2C genes was proved by quantitative reverse-transcription PCR (qRT-PCR), immunofluorescence (IF), Western blot and chimeric embryos. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and in vitro deacetylation assay confirmed lactylation of Hdac1 promoting its binding at 2C genes and enhancing its deacetylase activity, thereby facilitating the removal of H3K27ac and the silencing of 2C genes. We found that inhibition or depletion of Ldha, the enzyme converting pyruvate to lactate, leads to the activation of 2C genes, as well as reduced global lactylation in ESCs. To investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition, quantitative lactylome analysis was performed, and 1716 lactylated proteins were identified. We then focused on Hdac1, a histone deacetylase involved in the silencing of 2C genes. Lactylation of Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes. In summary, our study reveals a mechanistic link between cellular metabolism and pluripotency regulation through protein lactylation. Our research is the first time to reveal that quantitative lactylome analysis in mouse ESCs. We found that lactylated Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes.

KAT6B is required for histone 3 lysine 9 acetylation and SOX gene expression in the developing brain.

Heterozygous mutations in the histone lysine acetyltransferase gene KAT6B (MYST4/MORF/QKF) underlie neurodevelopmental disorders, but the mechanistic roles of KAT6B remain poorly understood. Here, we show that loss of KAT6B in embryonic neural stem and progenitor cells (NSPCs) impaired cell proliferation, neuronal differentiation, and neurite outgrowth. Mechanistically, loss of KAT6B resulted in reduced acetylation at histone H3 lysine 9 and reduced expression of key nervous system development genes in NSPCs and the developing cortex, including the SOX gene family, in particular Sox2, which is a key driver of neural progenitor proliferation, multipotency and brain development. In the fetal cortex, KAT6B occupied the Sox2 locus. Loss of KAT6B caused a reduction in Sox2 promoter activity in NSPCs. Sox2 overexpression partially rescued the proliferative defect of Kat6b -/- NSPCs. Collectively, these results elucidate molecular requirements for KAT6B in brain development and identify key KAT6B targets in neural precursor cells and the developing brain.

Transplanted deep-layer cortical neuroblasts integrate into host neural circuits and alleviate motor defects in hypoxic-ischemic encephalopathy injured mice.

Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although intensive studies and therapeutic approaches, there are limited restorative treatments till now. Human embryonic stem cell (hESCs)-derived cortical neural progenitors have shown great potentials in ischemic stroke in adult brain. However, it is unclear whether they are feasible for cortical reconstruction in immature brain with hypoxic-ischemic encephalopathy. By using embryonic body (EB) neural differentiation method combined with DAPT pre-treatment and quantitative cell transplantation, human cortical neuroblasts were obtained and transplanted into the cortex of hypoxic-ischemic injured brain with different dosages 2 weeks after surgery. Then, immunostaining, whole-cell patch clamp recordings and behavioral testing were applied to explore the graft survival and proliferation, fate commitment of cortical neuroblasts in vitro, neural circuit reconstruction and the therapeutic effects of cortical neuroblasts in HIE brain. Transplantation of human cortical neural progenitor cells (hCNPs) in HIE-injured cortex exhibited long-term graft overgrowth. DAPT pre-treatment successfully synchronized hCNPs from different developmental stages (day 17, day 21, day 28) to deep layer cortical neuroblasts which survived well in HIE injured brain and greatly prevented graft overgrowth after transplantation. Importantly, the cortical neuroblasts primarily differentiated into deep-layer cortical neurons and extended long axons to their projection targets, such as the cortex, striatum, thalamus, and internal capsule in both ipsilateral and contralateral HIE-injured brain. The transplanted cortical neurons established synapses with host cortical neurons and exhibited spontaneous excitatory or inhibitory post-synaptic currents (sEPSCs or sIPSCs) five months post-transplantation. Rotarod and open field tests showed greatly improved animal behavior by intra-cortex transplantation of deep layer cortical neuroblasts in HIE injured brain. Transplanted hESCs derived cortical neuroblasts survive, project to endogenous targets, and integrate into host cortical neural circuits to rescue animal behavior in the HIE-injured brain without graft overgrowth, providing a novel and safe cell replacement strategy for the future treatment of HIE.

Mammalian ubiquitous promoter isolated from proximal regulatory region of bovine MSTN gene.

Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin (MSTN) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy.

Abstract 4120919: Cell physiology and multi-omics analysis revealed disordered embryogenesis as early as mesoderm specification in models of Holt-Oram Syndrome

Introduction: Congenital heart diseases (CHDs) are the leading cause of childhood morbidity and mortality. The dysregulation of several cardiac transcription factors (TFs) leads to CHD. The coordination of several cardiac TFs is required and essential for cardiogenesis. However, the mechanisms to acquire its cardiac cell identity remain unclear. Hypothesis: Mutant Tbx5 dysregulates embryogenesis during mesoderm specification, prior to its expression in the tissues in Holt-Oram syndrome. Methods: Using cellular physiology and multiomics analysis in both human ES cells and a zebrafish model of TBX5 germline mutation. we evaluated embryonic structure and function, prior to the onset of gastrulation in the context of both heterozygous and homozygous TBX5 mutation. Results: Zebrafish time course transcriptome profiles over gastrulation revealed that loss of Tbx5 impacted transcriptional profiles at the blastula stage. Single cell RNA sequencing (scRNA-seq) on 16243 zebrafish blastula stage cells showed loss of Tbx5 impacted transcription noise at the blastula stage prior to the expression of zygotic Tbx5 with associated effects on chromatin accessibility using omni-assay for transposase-accessible chromatin (ATAC) and evidence of aberrant Wnt signaling even at the blastula stage. Embryo-wide cell structure and function were abnormal in both Tbx5 mutant zebrafish heterozygotes or homozygotes. To validate Undifferentiated human ES cell H3K4me3 Cleavage Under Targets&Release Using Nuclease (CUT&RUN) also showed abnormal Wnt signaling in TBX5 homozygous mutation and TBX5 CUT&RUN showed aberrant mesoderm pathway. Calcium imaging analysis demonstrated the lowest excitation frequency in TBX5 homozygous mutation prior to mesoderm specification. TBX5 homozygous human ES derived mesoderm cells exhibited low expression levels of mesoderm marker genes compared to TBX5 wild type mesoderm. Conclusion: Integrating single cell physiology and multi-omics technologies, we idneifified fundamental dysregulation of embryogenesis in mutant cardiac-restricted gene disorder prior to mesoderm specification or the zygotic expression of the mutant gene. These findings suggest that Tbx5 started to determine cell fate prior to mesoderm specification by altering chromatin accessibilities and histone modification. Tbx5 affected mesoderm differentiation by influencing Wnt signaling and cell physiology.

Progress of induced pluripotent stem cell (iPSC)-derived renal organoids in clinical application

Background: Kidney disease has become a growing public health problem worldwide, and there is an urgent need to develop reliable models for investigating novel and effective treatment strategies. In recent years, kidney organoids, as novel models different from traditional two-dimensional cells and model animals, have attracted more and more attention. Current advances have allowed the generation of kidney organoids from the directed differentiation of induced pluripotent stem cells (iPSCs), which possess similar characteristics to embryonic stem cells (ESCs), but bypass ethical constraints and have a wide range of sources. Summary: Herein, the methods of generating renal organoids from iPSCs, the applications of iPSC-derived renal organoids in disease modeling, drug effectiveness detection, and regenerative medicine as well as the challenges were reviewed. Key messages: iPSC-derived renal organoids can be used to model kidney diseases, and are great models for studying kidney injury and toxicity. Many efforts are needed to finally apply organoids into clinical application.

TMIC-51. NEUROIMMUNE-COMPETENT ORGANOID MODELING OF MICROGLIA/TUMOR INTERACTIONS AND DRUG RESPONSIVENESS

Abstract INTRODUCTION Due to its highly aggressive and infiltrative nature, lack of effective treatment options and localization in the skull base, diffuse intrinsic pontine glioma (DIPG) is universally fatal with less than a year median survival time. Studying how DIPG cells interact with brain cells in its microenvironment, including microglia which is the primary brain immune cell type, may lead to new therapeutic strategies to halt the invasion of DIPG into brain tissue. We developed a clinically relevant human immunocompetent brain organoid-DIPG fusion model for investigating the tumor invasion process at the cellular and molecular levels. METHODS Neuronal progenitor and GFP-labeled myeloid precursor cells were derived from human embryonic stem cells and combined to self-assemble and co-develop over two months into microglia-containing brain organoids (MiCBOs). The development of the MiCBOs as well as the distribution and motility of the microglia within the brain organoid was characterized by confocal and multiphoton microscopy, immunofluorescence staining and Western Blots. Confocal live-cell imaging was used to investigate the invasion of TdTomato-labeled tumor cells into MiCBO. We further performed single-cell RNA-Sequencing (scRNA-Seq) analysis to gain insight into the cell-specific gene expression changes after the confrontation of the MiCBOs with tumor spheroids. RESULTS MiCBOs contain viable, widely distributed and highly motile microglia as well as functionally mature neurons, highly resembling those in the human brain. scRNA-Seq showed distinct gene expression patterns in both microglia and tumor cells after confronting MiCBOs with DIPG spheroids, suggesting candidate signaling pathways between those two cell types. CONCLUSION Our novel MiCBO-DIPG fusion model provides a pathophysiologically-relevant system to elucidate molecular mechanisms underlying cell-cell interactions during tumor invasion in a human brain tissue context. This scalable model can be adapted to drug screening applications to identify therapeutics that modulate neuroimmune/tumor interactions and to establish the therapeutic index for anti-cancer medications.

Single-cell landscape identified SERPINB9 as a key player contributing to stemness and metastasis in non-seminomas.

Embryonal carcinoma (EC), characterized by a high degree of stemness similar to that of embryonic stem cells, is the most malignant subtype within non-seminomatous testicular germ cell tumors (TGCTs). However, the mechanisms underlying its malignancy remain unknown. In this study, we employed single-cell RNA sequencing to analyze four non-seminoma samples. Our differential expression analysis revealed high expression of SERPINB9 in metastatic EC cells. We conducted in vitro experiments to further investigate SERPINB9's role in the progression of EC. Functionally, the knockdown of SERPINB9 in NCCIT and NTERA-2 leads to a diminished migratory capability and decreased cis-platin resistance, as demonstrated by Transwell migration assay and drug sensitivity assay. Moreover, embryoid bodies showed reduced size and lower OCT4 expression, alongside heightened expression of differentiation markers AFP, ACTA2, and CD57 in shSERPINB9 cells. In vivo, the role of SERPINB9 in maintaining cancer stemness was validated by the limiting dilution assay. Mechanistically, Bulk RNA-seq further showed downregulation of ERK1/2 signaling and WNT signaling pathways with concomitant upregulation of differentiation pathways subsequent to SERPINB9 knockdown. Additionally, the analysis indicated increased levels of cytokines linked to tertiary lymphoid structures (TLS), such as IL6, IL11, IL15, CCL2, CCL5, and CXCL13 in shSERPINB9 cells, which were further validated by ELISA. Our research indicates that SERPINB9 plays a key role in driving tumor progression by enhancing tumor stemness and suppressing TLS. This study stands as the first to elucidate the molecular signature of non-seminomas at a single-cell level, presenting a wealth of promising targets with substantial potential for informing the development of future therapeutic interventions.

Predicting tumour resistance to paclitaxel and carboplatin utilising genome-wide screening in haploid human embryonic stem cells.

Taxanes and platinum molecules, specifically paclitaxel and carboplatin, are widely used anticancer drugs that induce cell death and serve as first-line chemotherapy for various cancer types. Despite the efficient effect of both drugs on cancer cell proliferation, many tumours have innate resistance against paclitaxel and carboplatin, which leads to inefficient treatment and poor survival rates. Haploid human embryonic stem cells (hESCs) are a novel and robust platform for genetic screening. To gain a comprehensive view of genes that affect or regulate paclitaxel and carboplatin resistance, genome-wide loss-of-function screens in haploid hESCs were performed. Both paclitaxel and carboplatin screens have yielded selected plausible gene lists and pathways relevant to resistance prediction. The effects of mutations in selected genes on the resistance to the drugs were demonstrated. Based on the results, an algorithm that can predict resistance to paclitaxel or carboplatin was developed. Applying the algorithm to the DNA mutation profile of patients' tumours enabled the separation of sensitive versus resistant patients, thus, providing a prediction tool. As the anticancer drugs arsenal can offer alternatives in case of resistance to either paclitaxel or carboplatin, an early prediction can provide a significant advantage and should improve treatment. The algorithm assists this unmet need and helps predict whether a patient will respond to the treatment and may have an immediate clinically actionable application.

Zygotic activin a is dispensable for the mouse preimplantation embryo development and for the derivation and pluripotency of embryonic stem cells†.

In this work, we aimed to determine the role of activin A during crucial events of mouse embryogenesis and distinguish the function of the protein of zygotic origin and the one secreted by the maternal reproductive tract. To this end, we recorded the progression of development and phenotype of Inhba knockout embryos and compared them with the heterozygotes and wild-type embryos using time-lapse imaging and detection of lineage-specific markers. We revealed that the zygotic activin A deficiency does not impair the course and rate of development of embryos to the blastocyst stage. Inhba knockout embryos form functional epiblast, as evidenced by their ability to give rise to embryonic stem cells. Our study is the first to show that derivation, maintenance in culture, and pluripotency of embryo-derived embryonic stem cells are exogenous and endogenous activin A-independent. However, the implantation competence of activin A-deficient embryos may be compromised as indicated in the outgrowth assay.