Lineage Differentiation Research Articles

Abstract 6407: Identification of novel chemotherapy-related CHIP variations in patients with high-grade serous ovarian cancer

Abstract Background: Hematopoietic lineage differentiation is subjected to genetic mutations that, due to fitness advantages, might give rise to clonally expanded populations. Clonal hematopoiesis of indeterminate potential (CHIP) is defined as the outgrowth of a single clone driven by acquired somatic mutations in hematopoietic stem cells (HSCs), in the absence of hematological abnormalities. Previous studies have shown the association of CH with aging and a higher risk of developing secondary hematologic malignancies in cancer patients treated with chemotherapy agents. It is therefore of great interest to study CH incidence prior to and post chemotherapy exposure and its association with the evolution of hematologic malignancies. We aimed to characterize CHIP variants of a highly selected group of patients with high-grade serous ovarian cancer (HGSC) who underwent neoadjuvant chemotherapy (NACT). Methods: Comprehensive ultra-high-depth whole exome sequencing, employing unique molecular barcode technologies, was performed using plasma-derived cell-free DNA (cfDNA) and matched white blood cells (WBC) DNA and tumor DNA from pre-NACT (n=10) and post-NACT (n=10) samples of patients with HGSC. CHIP variants were defined as heterozygous variants found in both the WBC and cfDNA samples from the same patients with variant allele frequency less than 5% in the WBC and higher than 0.5% in cfDNA samples. Results: Our analysis identified 93,088 variants, impacting 13,780 genes. Among them, 463 variants were already known and published, and 5,548 variants were common to the COSMIC genes signature. The number of CHIP variants significantly decreased after chemotherapy (p = 0.004). We then focused on CHIP variants enriched after chemotherapy and identified 77 that showed higher VAF in post-NACT cfDNA samples, which may be associated with the development of therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS). Among them, interestingly, we found a missense mutation with protein change in CDK12 (chr17_39492765_G_T). We noticed that 38% of the CHIP variants found in cfDNA samples, were also found in matched tumor samples. Among these, another CDK12 missense mutation (chr17_39492768_A_T) showed higher VAF in post-NACT tumors. Conclusions: Our innovative approach detected a higher number of CHIP variants than currently characterized. Analysis of cfDNA from HGSC patients consistently demonstrated a reduction in CHIP mutations post-NACT. The subset of variants with increased VAF post-NACT may be associated with therapy-related t-AML/MDS. The detection of cfDNA CHIP variants in matched tumors highlights the potential predictive role of CHIP in t-AML/MDS development, emphasizing its consideration in therapeutic protocols. Citation Format: Sara Corvigno, Amma Asare, Jun Yao, Li Zhao, Nicole Fleming, Joseph Celestino, Richard A. Hajek, Ency A. Arboleda Goette, Ridge T. Rogers, Tri V. Nguyen, Raymond N. Montoya, Karen H. Lu, Amir A. Jazaeri, Shannon N. Westin, Anil K. Sood, Sanghoon Lee. Identification of novel chemotherapy-related CHIP variations in patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6407.

Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4−CD8− double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.

Abstract 1242: Foxa1/2-dependent epigenomic reprogramming drives lineage switching in lung adenocarcinoma

Abstract The ability of cancer cells to alter their identity is essential for tumor survival and progression. Loss of the pulmonary lineage specifier NKX2-1 within KRAS-driven lung adenocarcinoma (LUAD) enhances tumor progression and results in a pulmonary-to-gastric lineage switch that is dependent upon the activity of pioneer factors FoxA1 and FoxA2; however, the underlying mechanism remains largely unknown. Here, we perform transcriptional and epigenetic assays to show that FoxA1/2 reprogram the chromatin landscape of NKX2-1-negative LUAD to facilitate a gastric differentiation program. Using sequential recombination models, we find that FoxA1/2 are required for demethylation of gastric-defining genes after Nkx2-1 deletion. FoxA1 colocalizes with TET3, an enzyme that mediates DNA demethylation, in NKX2-1-negative tumors. Deletion of Foxa1/2 results in loss of TET3 occupancy at key gastric marker genes, indicating that FoxA1/2 recruit TET3 to lineage-specific sites. H3K27ac ChIP-seq and HiChIP show that FoxA1/2 also control the activity of enhancers and promoters as well as their 3D interactions at gastric target genes following NKX2-1 loss. Furthermore, oncogenic KRAS is required for the FoxA1/2-dependent epigenetic reprogramming in NKX2-1-negative LUAD. This work demonstrates the role of FoxA1/2 in rewiring the methylation and histone landscape and cis-regulatory dynamics of NKX2-1-negative LUAD to drive cancer cell lineage switching. Citation Format: Katherine Gillis, Walter Orellana, Emily Wilson, Gabriela Fort, Headtlove Essel Dadzie, Timothy Parnell, Xiaoyang Zhang, Eric Snyder. Foxa1/2-dependent epigenomic reprogramming drives lineage switching in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1242.

Abstract 3055: Characterizing the role of HNF4α isoforms in human PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly and recalcitrant malignancy with a 5-year survival rate of 12%. Transcriptomic studies have classified PDAC into a classical or basal-like subtype. The classical subtype has a better prognosis, response to chemotherapy, and express higher levels of endodermal lineage specifiers compared to the basal-like subtype. However, the field lacks a comprehensive understanding of the key subtype-specific regulators. Functional analysis of transcription factors associated with each subtype are needed to elucidate their roles in regulation of PDAC subtypes and to identify therapeutically targetable vulnerabilities. Previous work in our lab demonstrated that HNF4α is a key regulator of the classical subtype. Using genetically engineered mouse models, our lab showed that loss of HNF4α leads to an increase in tumor burden, a shift from glandular to poorly differentiated histology, and a decrease in the classical subtype score. However, while murine PDAC only express one set of HNF4α isoforms, human PDAC express both sets of HNF4α isoforms, suggesting murine PDAC may not fully recapitulate human disease. There are 2 subsets of HNF4α isoforms driven by expression from 2 distinct promoters, P1 and P2. P1 and P2 isoforms are differentially expressed within the gastrointestinal (GI) tract and play dichotomous roles in GI malignancies. In HNF4α-positive human PDAC, P2 isoforms are always expressed but there is variable expression of P1 isoforms. Based on the differential role of HNF4α isoforms in both normal tissue and cancer, we hypothesize that P1 isoforms will restrain tumor growth and promote greater classical/GI differentiation compared to P2 isoforms. We utilized CRISPRi to selectively inhibit transcription at the P1 or P2 promoter in HNF4α-positive, human cell lines expressing both isoforms. To test for sufficiency, we exogenously expressed P1 or P2 isoforms in HNF4α-negative, human cell lines. We then performed Incucyte, CRISPRi competition, and anchorage-independent growth assays. We performed RNA-seq and ChIP-seq on both exogenous and endogenous HNF4α isoforms. Functional assays revealed that HNF4α isoforms regulated growth in a context dependent manner. Exogenous P1 isoforms significantly inhibited growth while loss of endogenous P1 isoforms was dispensable for growth. Exogenous P2 isoforms inhibited growth to a lesser degree while loss of endogenous P2 isoforms led to a significant reduction in growth. Despite ChIP-seq analysis revealing very similar binding profiles, RNAseq analysis revealed that P1 isoforms are significantly more transcriptionally active than P2 isoforms and are stronger inducers of GI differentiation. Taken together, our data demonstrates that P2 isoforms are more compatible with growth in PDAC compared to P1 isoforms, which promote a more differentiated cell state. Mechanistically, this may be due to the increased transcriptional activity of P1 isoforms. Citation Format: Pengshu Fang, Acramul Kabir, Gabriela Fort, Eric L. Snyder. Characterizing the role of HNF4α isoforms in human PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3055.

Abstract 3052: Investigating the role of FOXA1/2 & HNF4⍺ in pancreatic ductal adenocarcinoma

Abstract PDAC represents a formidable challenge with an alarmingly low 5-year survival rate of less than 12%. Integrated genomic, transcriptomic, and proteomic analyses have identified two distinct PDAC subtypes: classical and basal. The critical difference between these subtypes lies in the expression of endodermal lineage specifiers, with the classical subtype expressing FOXA1, FOXA2 (FOXA1/2), and HNF4⍺, critical for pancreatic cell-fate determination, while the basal subtype downregulates these genes, leading to the loss of endodermal identity. We reason that the transcription factors (TF), FOXA1/2 and HNF4α, that play critical roles in pancreatic development, are not only biomarkers of the classical subtype but are essential regulators of PDAC subtype differentiation state. Despite their relevance in other cancers, their specific functions in PDAC remain largely unexplored. We aim to define the functional roles of FOXA1/2 and HNF4⍺ in the classical PDAC subtype, exploring their regulation of growth and cellular identity. Previous published work in our lab has shown that exogenous HNF4⍺ restrains growth in a panel of human and murine PDAC cell lines. Furthermore, we have shown that HNF4⍺ regulates the classical subtype program of PDAC. While FOXA1/2 and HNF4⍺ cooperatively enact gene expression changes in normal tissue, the extent to which cooperative interactions between these TFs are required to activate the classical gene expression program of PDAC remains unknown. Overall, we will test the central hypothesis that FOXA1/2 and HNF4⍺ are critical regulators of the transcriptional network governing the cellular identity of classical PDAC. To test this hypothesis, we will be reconstituting and modulating the expression of these factors in human and murine PDAC cell and organoid lines. We will assess gene expression changes through RNA-seq and genomic localization of these TFs through ChIP-seq. We have conducted bulk RNA sequencing on Foxa1/2-positive and negative organoids to transcriptionally identify differences brought on by Foxa1/2 deletion and found over 2,200 differentially expressed genes. Foxa1/2 deletion led to a significant decline in the classical PDAC signature based on gene ontology. Furthermore, we show that HNF4α can inhibit organoid growth in the absence of FOXA1/2. We discovered that HNF4⍺ targets, such as Lgals4, expression levels are unaltered at the RNA level independent of FOXA1/2. By assessing the roles of FOXA1/2 and HNF4α in PDAC, we aim to determine the transcriptional network governed by these factors in the classical subtype of PDAC Citation Format: Walter A. Orellana, Katherine Gillis, Pengshu Fang, Gabriela Fort, Eric L. Snyder. Investigating the role of FOXA1/2 & HNF4⍺ in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3052.

Abstract 3950: Dedifferentiation unveils origin of Ewing sarcoma

Abstract Ewing sarcoma (ES) is an aggressive pediatric bone tumor in adolescents primarily driven by the expression of the fusion oncogene EWS::FLI1. Although the etiology of ES remains controversial, human mesenchymal stem cells (hMSCs) are considered to be the likely cell of origin. This is not only due to their ability to tolerate ectopic EWS::FLI1 expression, but also because the transcriptome of EWS::FLI1 expressing hMSCs is highly similar to that of primary Ewing tumors. However, to date a cellular differentiation-based model of ES does not exist. With the intent to understand ES histogenesis, this study aims to establish the first comprehensive single-cell differentiation atlas (both transcriptome and epigenome) of juvenile bone marrow-derived hMSCs with and without EWS::FLI1 expression. We sought to study the consequences of inducing the EWS::FLI1 oncogene expression at various time points of hMSC differentiation into adipo-, chondro-, osteo- and neuro-genic lineages. Our workflow utilizes physiology-mimicking conditions including serum-free differentiation, hypoxia, and growth/differentiation as spheroids. We pooled bone marrow-derived hMSCs isolated from eleven healthy juvenile donors, engineered them to conditionally express EWS::FLI1, and carried out multi-lineage differentiation for 10 days where EWS::FLI1 was induced daily for 24h or 72h. This temporally resolved atlas of 105 samples representing normal and perturbed differentiation allowed us to map ES patient tumor data to multiple lineages and time-points across stages of lineage commitment. Single-cell RNA-seq analysis of ~271,000 transcriptomes revealed an apparent and dynamic dedifferentiation in the presence of EWS::FLI1 across all four lineages. Preliminary analysis of genes detected in the EWS::FLI1 high cluster uncovers a specific lineage preference and time window that shares the transcriptomic signature with tumor cells, indicating the potential cell/stage of tumor-origin. Our results provide new insight into the enigmatic cell of origin of Ewing sarcoma, laying the foundation for the development of much-needed pre-clinical models for tailoring therapies. This study is supported by a generous grant (#20) of the Alex’s Lemonade Stand Foundation (ALSF) Crazy8 Program. Citation Format: Utkarsh Kapoor, Christoph Hafemeister, Lisa Shaw, Karin Muelbacher, Julia Moldaschl, Dominik Egger, Stefan Toegel, Cornelia Kasper, Matthias Farlik, Florian Halbritter, Heinrich Kovar. Dedifferentiation unveils origin of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3950.

Cimetidine Attenuates Therapeutic Effect of Anti-PD-1 and Anti-PD-L1 and Modulates Tumor Microenvironment in Colon Cancer.

Histamine modulates immunity by binding to histamine receptor 2 (H2R). Cimetidine, an H2R antagonist that inhibits gastric acid secretion and treats gastrointestinal ulcers, interferes with histamine-mediated immunomodulation and may have anticancer activity. This study examined cimetidine's effect on the anticancer effect of anti-PD-L1 in colon cancer. The MTT assay, colony formation assay, and DNA histograms assessed cell viability, clonogenicity, and cell cycle distribution, respectively. Flow cytometry measured H2R and PD-L1 expression and estimated specific immune cell lineages. For the in vivo study, tumor cells were subcutaneously implanted into the right flank of BALB/c mice. Cimetidine had no significant effect on CT26 cell viability, clonogenicity, or cell cycle distribution. It also did not affect H2R and PD-L1 expression levels in CT26 cells. In vivo, anti-PD-1 and anti-PD-L1 suppressed CT26 tumor growth, whereas cimetidine showed mild antitumor activity. In the combined experiment, cimetidine significantly attenuated anti-PD-1 and anti-PD-L1' antitumor effects without major toxicity. In the tumor microenvironment, anti-PD-L1 increased CD3+ T, CD4+ T, and CD8+ T cells and M1 macrophages. Combined treatment with cimetidine reversed this. Cimetidine also reversed anti-PD-1 and anti-PD-L1's decrease in circulating and tumor-associated neutrophils. Cimetidine attenuated anti-PD-L1's antitumor effect and modulated the tumor microenvironment in colon cancer.

Endothelial gene regulatory elements associated with cardiopharyngeal lineage differentiation

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. The enhancers activated during endothelial differentiation within the cardiopharyngeal mesoderm are not completely known. Here, we use a cardiogenic mesoderm differentiation model that activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identify 101 putative regulatory elements of EC genes. We then apply a machine-learning strategy, trained on validated enhancers, to predict enhancers. Using this computational assay, we determine that 50% of these sequences are likely enhancers, some of which are already reported. We also identify a smaller set of regulatory elements of well-known EC genes and validate them using genetic and epigenetic perturbation. Finally, we integrate multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we show EC regulatory sequences with a high likelihood to be enhancers, and we validate a subset of them using computational and cell culture models. Motif analyses show that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC regulation in cardiopharyngeal mesoderm.

Clinical and prognostic significance of baseline microRNA 223 in acute ischemic stroke

BackgroundAcute ischemic stroke (AIS) is the second leading cause of disability and death worldwide. Micro-RNA (miRNA)-223 was first identified as a regulator of hematopoietic lineage differentiation. Later, its diverse roles were discovered in a wide spectrum of pathological conditions. The present study aimed to assess the clinical and prognostic significance of miR-223 in patients with acute ischemic stroke (AIS). The study included 93 patients with AIS diagnosed on the basis of clinical and radiological findings. In addition, there were 50 healthy subjects who served as controls. Patients were classified into two categories: Those with favorable functional outcome (modified Rankin Scale (mRS): 0–2) and others with unfavorable functional outcome (mRS: 3–6) at 6 months post-stroke.ResultsThe present prospective longitudinal study included 93 patients with AIS. They included 60 males (64.5%) and 33 females (35.5%) with an age of 64.5 ± 12.4 years. At the end of 6-month follow up, 44 patients (47.3%) had favorable outcome while the remainder 49 patients (52.7%) had unfavorable outcome. Patients with favorable outcome had significantly lower baseline miR-223 levels [median (IQR): 4.4 (2.0–6.3) versus 8.4 (4.5–14.9), p < 0.001], lower HbA1c levels (5.6 ± 1.0 versus 6.2 ± 1.2, p = 0.006) and lower C-reactive protein (CRP) levels [median (IQR): 8.9 (5.1–26.7) versus 15.2 (6.2–39.3) mg/dL, p = 0.02]. Multivariate binary logistic regression analysis recognized high baseline miR-223 [OR (95% CI) 1.13 (1.06–1.24), p = 0.011], infarct size [OR (95% CI) 2.58 (1.66–4.77), p = 0.001] and National Institutes of Health Stroke Scale (NIHSS) [OR (95% CI) 2.11 (1.74–3.09), p = 0.004] as significant predictors of unfavorable outcome in the studied patients.ConclusionsElevated baseline miR-223 levels are associated with high NIHSS and larger infarct size at baseline and can effectively predict patients’ outcome at 6-months post-stroke.

Long COVID across SARS-CoV-2 variants, lineages, and sublineages

This prospective study aimed to determine the prevalence of long COVID in patients hospitalized for SARS-CoV-2 infection from March 2020 to July 2022 and assess the impact of different viral lineages. A total of 2,524 patients were followed up for 12months, with persistent symptoms reported in 35.2% at one month, decreasing thereafter. Omicron variant patients initially showed higher symptom intensity, but this trend diminished over time. Certain viral lineages, notably Delta lineages AY.126 and AY.43, and Omicron sublineages BA.1.17, BA.2.56, and BA.5.1, consistently correlated with more severe symptoms. Overall, long COVID prevalence and severity were similar across SARS-CoV-2 variants. Specific lineages may influence post-COVID sequelae persistence and severity.

Evolutionary dynamics of the accessory genomes of Staphylococcus aureus.

Staphylococcus aureus is a ubiquitous commensal and opportunistic bacterial pathogen that can cause a wide gamut of infections, which are exacerbated by the presence of multidrug-resistant and methicillin-resistant S. aureus. S. aureus is genetically heterogeneous and consists of numerous distinct lineages. Using 558 complete genomes of S. aureus, we aim to determine how the accessory genome content among phylogenetic lineages of S. aureus is structured and has evolved. Bayesian hierarchical clustering identified 10 sequence clusters, of which seven contained major sequence types (ST 1, 5, 8, 30, 59, 239, and 398). The seven sequence clusters differed in their accessory gene content, including genes associated with antimicrobial resistance and virulence. Focusing on the two largest clusters, BAPS8 and BAPS10, and each consisting mostly of ST5 and ST8, respectively, we found that the structure and connected components in the co-occurrence networks of accessory genomes varied between them. These differences are explained, in part, by the variation in the rates at which the two sequence clusters gained and lost accessory genes, with the highest rate of gene accumulation occurring recently in their evolutionary histories. We also identified a divergent group within BAPS10 that has experienced high gene gain and loss early in its history. Together, our results show highly variable and dynamic accessory genomes in S. aureus that are structured by the history of the specific lineages that carry them.IMPORTANCEStaphylococcus aureus is an opportunistic, multi-host pathogen that can cause a variety of benign and life-threatening infections. Our results revealed considerable differences in the structure and evolution of the accessory genomes of major lineages within S. aureus. Such genomic variation within a species can have important implications on disease epidemiology, pathogenesis of infection, and interactions with the vertebrate host. Our findings provide important insights into the underlying genetic basis for the success of S. aureus as a highly adaptable and resistant pathogen, which will inform current efforts to control and treat staphylococcal diseases.

Deep Learning Powered Identification of Differentiated Early Mesoderm Cells from Pluripotent Stem Cells.

Pluripotent stem cells can be differentiated into all three germ-layers including ecto-, endo-, and mesoderm in vitro. However, the early identification and rapid characterization of each germ-layer in response to chemical and physical induction of differentiation is limited. This is a long-standing issue for rapid and high-throughput screening to determine lineage specification efficiency. Here, we present deep learning (DL) methodologies for predicting and classifying early mesoderm cells differentiated from embryoid bodies (EBs) based on cellular and nuclear morphologies. Using a transgenic murine embryonic stem cell (mESC) line, namely OGTR1, we validated the upregulation of mesodermal genes (Brachyury (T): DsRed) in cells derived from EBs for the deep learning model training. Cells were classified into mesodermal and non-mesodermal (representing endo- and ectoderm) classes using a convolutional neural network (CNN) model called InceptionV3 which achieved a very high classification accuracy of 97% for phase images and 90% for nuclei images. In addition, we also performed image segmentation using an Attention U-Net CNN and obtained a mean intersection over union of 61% and 69% for phase-contrast and nuclear images, respectively. This work highlights the potential of integrating cell culture, imaging technologies, and deep learning methodologies in identifying lineage specification, thus contributing to the advancements in regenerative medicine. Collectively, our trained deep learning models can predict the mesoderm cells with high accuracy based on cellular and nuclear morphologies.

The use of a SOX10 reporter toward ameliorating oligodendrocyte lineage differentiation from human induced pluripotent stem cells.

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC-derived OLs.

Coactivator condensation drives cardiovascular cell lineage specification.

During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here, we show that the cardiovascular transcriptional coactivator myocardin (MYOCD) activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The carboxyl-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region's ability to form condensates disrupts gene activation and smooth muscle cell reprogramming. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation and smooth muscle cell reprogramming. Our findings demonstrate that MYOCD condensate formation is required for gene activation during cardiovascular differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during development.

Improved Neural Inductivity of Size-Controlled 3D Human Embryonic Stem Cells Using Magnetic Nanoparticles.

Background: To improve the efficiency of neural development from human embryonic stem cells, human embryoid body (hEB) generation is vital through 3-dimensional formation. However, conventional approaches still have limitations: long-term cultivation and laborious steps for lineage determination. Methods: In this study, we controlled the size of hEBs for ectodermal lineage specification using cell-penetrating magnetic nanoparticles (MNPs), which resulted in reduced time required for initial neural induction. The magnetized cells were applied to concentrated magnetic force for magnet-derived multicellular organization. The uniformly sized hEBs were differentiated in neural induction medium (NIM) and suspended condition. This neurally induced MNP-hEBs were compared with other groups. Results: As a result, the uniformly sized MNP-hEBs in NIM showed significantly improved neural inductivity through morphological analysis and expression of neural markers. Signaling pathways of the accelerated neural induction were detected via expression of representative proteins; Wnt signaling, dopaminergic neuronal pathway, intercellular communications, and mechanotransduction. Consequently, we could shorten the time necessary for early neurogenesis, thereby enhancing the neural induction efficiency. Conclusion: Overall, this study suggests not only the importance of size regulation of hEBs at initial differentiation stage but also the efficacy of MNP-based neural induction method and stimulations for enhanced neural tissue regeneration.

Pituitary tumours without distinct lineage differentiation express stem cell marker SOX2

ContextThe recent WHO 2022 Classification of pituitary tumours identified a novel group of ‘plurihormonal tumours without distinct lineage differentiation (WDLD)’. By definition, these express multiple combinations of lineage commitment transcription factors, in a monomorphous population of cells.ObjectivesTo determine the expression of stem cell markers (SOX2, Nestin, CD133) within tumours WDLD, immature PIT-1 lineage and acidophil stem cell tumours, compared with committed cell lineage tumours.MethodsRetrospective evaluation of surgically resected pituitary tumours from St Vincent’s Hospital, Sydney. Patients were selected to cover a range of tumour types, based on transcription factor and hormone immunohistochemistry. Clinical data was collected from patient files. Radiology reports were reviewed for size and invasion. Samples were analysed by immunohistochemistry and RT-qPCR for SF-1, PIT-1, T-PIT, SOX2, Nestin and CD133. Stem cell markers were compared between tumours WDLD and those with classically “mature” types.ResultsOn immunohistochemistry, SOX2 was positive in a higher proportion of tumours WDLD compared with those meeting WHO lineage criteria, 7/10 v 10/42 (70 v 23.4%, p = 0.005). CD133 was positive in 2/10 tumours WDLD but 0/41 meeting lineage criteria, P = 0.003. On RT-qPCR, there was no significant difference in relative expression of stem cell markers (SOX2, CD133, Nestin) between tumours with and WDLD.ConclusionsOur study is the first to biologically characterise pituitary tumours WDLD. We demonstrate that these tumours exhibit a higher expression of the stem cell marker SOX2 compared with other lineage-differentiated tumours, suggesting possible involvement of stem cells in their development.

The Musashi RNA binding proteins direct the translational activation of key pituitary mRNAs

The pituitary functions as a master endocrine gland that secretes hormones critical for regulation of a wide variety of physiological processes including reproduction, growth, metabolism and stress responses. The distinct hormone-producing cell lineages within the pituitary display remarkable levels of cell plasticity that allow remodeling of the relative proportions of each hormone-producing cell population to meet organismal demands. The molecular mechanisms governing pituitary cell plasticity have not been fully elucidated. Our recent studies have implicated a role for the Musashi family of sequence-specific mRNA binding proteins in the control of pituitary hormone production, pituitary responses to hypothalamic stimulation and modulation of pituitary transcription factor expression in response to leptin signaling. To date, these actions of Musashi in the pituitary appear to be mediated through translational repression of the target mRNAs. Here, we report Musashi1 directs the translational activation, rather than repression, of the Prop1, Gata2 and Nr5a1 mRNAs which encode key pituitary lineage specification factors. We observe that Musashi1 further directs the translational activation of the mRNA encoding the glycolipid Neuronatin (Nnat) as determined both in mRNA reporter assays as well as in vivo. Our findings suggest a complex bifunctional role for Musashi1 in the control of pituitary cell function.

Contrasting response of comammox Nitrospira, ammonia oxidising bacteria, and archaea to soil pH and nitrogen inputs

This study aimed to investigate the effect of soil pH change, and nitrogen amendment on ammonia oxidiser abundance and comammox Nitrospira community composition. The experimental design used soil mesocosms placed in a temperature-controlled incubator for 90 days. A Templeton silt loam was used as its physiochemical properties are typical of the region's dairy farms. The results showed that comammox Nitrospira clade B preferred the natural (pH 6.1–6.2) soil pH with no applied nitrogen. Furthermore, synthetic urine (N700) decreased the abundance of comammox Nitrospira clade B. This may have been because the large amounts of available ammonia in the N700 treatments inhibited the growth of comammox Nitrospira.These results suggest that while comammox Nitrospira clade B are present in New Zealand dairy farm soils, but their role in nitrification in the very high nitrogen environment under a urine patch in grazed pastures may be limited. Further research is needed to confirm this.In contrast to comammox, the AOB community (dominated by Nitrosospira) responded positively to the application of synthetic urine. The response was greatest in the high pH soil (7.1), followed by the natural and then the low pH (4.9) soils. This may be due to the difference in ammonia availability. At high pH, the ammonia/ammonium equilibrium favours ammonia production. Calculated ammonia availability in the N700 treatments accurately predicted the AOB amoA gene abundance.Interestingly, the AOA community abundance (which was predominantly made up of Thaumarchaeota group I.1b clade E) seemed to prefer the natural and high pH soils over the low pH. This may be due to the specific lineage of AOA present. AOA did not respond to the application of nitrogen.

Specific lineage transition of tumor-associated macrophages elicits immune evasion of ascitic tumor cells in gastric cancer with peritoneal metastasis.

Gastric cancer with peritoneal metastasis (PM-GC), recognized as one of the deadliest cancers. However, whether and how the tumor cell-extrinsic tumor microenvironment (TME) is involved in the therapeutic failure remains unknown. Thus, this study systematically assessed the immunosuppressive tumor microenvironment in ascites from patients with PM-GC, and its contribution to dissemination and immune evasion of ascites-disseminated tumor cells (aDTCs). Sixty-three ascites and 43 peripheral blood (PB) samples from 51 patients with PM-GC were included in this study. aDTCs in ascites and circulating tumor cells (CTCs) in paired PB were immunophenotypically profiled. Using single-cell RNA transcriptional sequencing (scRNA-seq), crosstalk between aDTCs and the TME features of ascites was inspected. Further studies on the mechanism underlying aDTCs-immune cells crosstalk were performed on in vitro cultured aDTCs. Immune cells in ascites interact with aDTCs, prompting their immune evasion. Specifically, we found that the tumor-associated macrophages (TAMs) in ascites underwent a continuum lineage transition from cathepsinhigh (CTShigh) to complement 1qhigh (C1Qhigh) TAM. CTShigh TAM initially attracted the metastatic tumor cells to ascites, thereafter, transitioning terminally to C1Qhigh TAM to trigger overproliferation and immune escape of aDTCs. Mechanistically, we demonstrated that C1Qhigh TAMs significantly enhanced the expression of PD-L1 and NECTIN2 on aDTCs, which was driven by the activation of the C1q-mediated complement pathway. For the first time, we identified an immunosuppressive macrophage transition from CTShigh to C1Qhigh TAM in ascites from patients with PM-GC. This may contribute to developing potential TAM-targeted immunotherapies for PM-GC.

Strain background of Candida albicans interacts with SIR2 to alter phenotypic switching.

The genetic background between strains of a single species and within a single strain lineage can significantly impact the expression of biological traits. This genetic variation may also reshape epigenetic mechanisms of cell identity and environmental responses that are controlled by interconnected transcriptional networks and chromatin-modifying enzymes. Histone deacetylases, including sirtuins, are critical regulators of chromatin state and have been directly implicated in governing the phenotypic transition between the 'sterile' white state and the mating-competent opaque state in Candida albicans, a common fungal commensal and pathogen of humans. Here, we found that a previously ambiguous role for the sirtuin SIR2 in C. albicans phenotypic switching is likely linked to the genetic background of mutant strains produced in the RM lineage of SC5314. SIR2 mutants in a specific lineage of BWP17 displayed increased frequencies of switching to the opaque state compared to the wild-type. Loss of SIR2 in other SC5314-derived backgrounds, including newly constructed BWP17 sir2Δ/Δ mutants, failed to recapitulate the increased white-opaque switching frequencies observed in the original BWP17 sir2Δ/Δ mutant background. Whole-genome sequencing revealed the presence of multiple imbalanced chromosomes and large loss of heterozygosity tracts that likely interact with SIR2 to increase phenotypic switching in this BWP17 sir2Δ/Δ mutant lineage. These genomic changes are not found in other SC5314-derived sir2Δ/Δ mutants that do not display increased opaque cell formation. Thus, complex karyotypes can emerge during strain construction that modify mutant phenotypes and highlight the importance of validating strain background when interpreting phenotypes.