Differentiated Cell Types Research Articles

Abstract 4335: High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors

Abstract This study is aimed at testing the hypothesis that increased patient age fosters conditions that are favorable for head and neck squamous cell carcinomas (HNSCC) of the oral cavity development. There is a well-documented increase in cancer incidence with age observed across diverse cancer types, where elderly patients exhibit diminished survival outcomes compared to their younger counterparts. While the convergence of aging and cancer hallmarks offers valuable insights, further work is needed to elucidate the age-specific mechanisms influencing HNSCC, beyond the shared characteristics of these biological processes. To this end, we have assembled a high-quality human single-cell RNA-sequencing HNSCC atlas profiling more than 230,000 cells across more than 50 patients with ages ranging between 18 and 89, which provides a resource to investigate age-associated changes in the disease heterogeneity. For this library, we integrated six publicly available single-cell RNAseq datasets from 54 HPV-negative patients to create the atlas. Cells were clustered, classified, characterized by gene set enrichment analysis, both in the epithelial cell compartment and in the tumor microenvironment (TME). Differential cell type proportion analysis was performed to identify cell types enriched in young (<49 years) or old (>70 years) patients. Cell-cell communication analysis was performed to identify signaling events occurring between different populations, and how these and specific ligand-receptor pairs differed in patients of different ages. CNV analyses were performed to identify cancer subclones and assess level of variation across tumors. Interestingly, we identified distinct cell populations and signaling events that associate with age, and we were able to compare, validate and recapitulate these observations in the 4MOSC1 OSCC isograft tumor model in old and young mice. Additional notable findings include a differential enrichment for cytotoxic CD8 T cells over dysfunctional CD8 T cells in young patients when compared to old, suggesting younger patients have a more effective immune TME. We also found an enrichment for cancer-associated fibroblast populations in old patients (myCAFs) that send collagen signals to malignant epithelial cells, a result we have recapitulated using the 4MOSC1 model, and hypothesize may play a role in ECM stiffening, EMT, and metastasis. Further analyses are ongoing, and we plan to validate the hypotheses generated, specifically the presence of differential abundance of specific cell populations, and age-specific ligand-receptor signaling events that lead to tumor growth. Citation Format: Lina Kroehling, Anthony Spinella, Maria Kukuruzinska, Xaralabos Varelas, Stefano Monti. High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors [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 4335.

Abstract PR007: Patient derived organoids as a model to study estrogen mediated endometrial cancer

Abstract The purpose of our study was to develop a more optimal in vitro model to study the mechanisms of hormone action on endometrial cancer. Endometrial cancer (EC) represents a major health disparity with a 21% increase in mortality in African American women. The cause for this excess mortality is unknown. Endometrial cancer is commonly characterized by histological subtype into Endometrioid or Non-Endometrioid with estrogen being a driver for many endometroid type tumors. Most EC cell lines do not have high expression of estrogen receptor or do not respond to physiological levels of hormones in vitro; this lack of functional experimental models has hampered progress in the study of this disease. We have developed Patient Derived Organoids (PDOs) from 9 patient-derived xenografts (PDX) that are from both African American and Non-Hispanic Caucasian women with various stages and subtypes of EC, as a model to study EC. This model system captures the characteristics of different histological and molecular subtypes in vitro, as they maintain not only EC tumor cells but also cells from the adjacent tumor stroma. Our developed PDOs were derived from multiple molecular subtypes of EC that have different characteristics and genomic features, allowing us to observe differences in the basal properties of each tumor in vitro. The use of this organoid model system is advantageous for studying EC because growth characteristics are similar to an in vivo model, with the organoids preserving the genotype and phenotype of the native EC, allowing us to observe cellular interactions. We have observed inter-organoid variation of protein expression of β-catenin, E-cadherin, and vimentin, proteins playing a pivotal role in cellular contact, signaling, and epithelial-mesenchymal transition (EMT). Using qPCR and Western blot analysis we have observed high expression levels of estrogen receptor and progesterone receptor in endometrioid type PDOs to begin the exploration of hormonal signaling in this model. Treatment of these same PDOs with β-estradiol resulted in an increase in proliferation of the stromal cells. Single cell RNA sequencing was performed on two PDO’s that had the highest expression of progesterone and estrogen receptors. This analysis demonstrated cell population heterogeneity with multiple cell clusters forming with differential gene expression. From this analysis we can see that the cells recapitulate the gene expression from the tumor of origin, with cells containing the same genetic mutation as the original patient. These results make this model potentially good for studying hormone signaling cascades seen in EC patients that have not been previously observed in established EC cell lines. These studies will also aid in the opportunity to explore the function of differential cell types in the pathogenesis of this disease. Citation Format: Breanna M.W. Jeffcoat, Andrew B. Galdden, Russell R. Broaddus. Patient derived organoids as a model to study estrogen mediated endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR007.

Comparing the osteogenesis outcomes of different lumbar interbody fusions (A/O/X/T/PLIF) by evaluating their mechano-driven fusion processes

BackgroundIn lumbar interbody fusion (LIF), achieving proper fusion status requires osteogenesis to occur in the disc space. Current LIF techniques, including anterior, oblique, lateral, transforaminal, and posterior LIF (A/O/X/T/PLIF), may result in varying osteogenesis outcomes due to differences in biomechanical characteristics. MethodsA mechano-regulation algorithm was developed to predict the fusion processes of A/O/X/T/PLIF based on finite element modeling and iterative evaluations of the mechanobiological activities of mesenchymal stem cells (MSCs) and their differentiated cells (osteoblasts, chondrocytes, and fibroblasts). Fusion occurred in the grafting region, and each differentiated cell type generated the corresponding tissue proportional to its concentration. The corresponding osteogenesis volume was calculated by multiplying the osteoblast concentration by the grafting volume. ResultsTLIF and ALIF achieved markedly greater osteogenesis volumes than did PLIF and O/XLIF (5.46, 5.12, 4.26, and 3.15 cm3, respectively). Grafting volume and cage size were the main factors influencing the osteogenesis outcome in patients treated with LIF. A large grafting volume allowed more osteoblasts (bone tissues) to be accommodated in the disc space. A small cage size reduced the cage/endplate ratio and therefore decreased the stiffness of the LIF. This led to a larger osteogenesis region to promote osteoblastic differentiation of MSCs and osteoblast proliferation (bone regeneration), which subsequently increased the bone fraction in the grafting space. ConclusionTLIF and ALIF produced more favorable biomechanical environments for osteogenesis than did PLIF and O/XLIF. A small cage and a large grafting volume improve osteogenesis by facilitating osteogenesis-related cell activities driven by mechanical forces.

Source cell-type epigenetic memory persists in induced pluripotent cells but is lost in subsequently derived germline cells.

Introduction: Retention of source cell-type epigenetic memory may mitigate the potential for induced pluripotent stem cells (iPSCs) to fully achieve transitions in cell fate in vitro. While this may not preclude the use of iPSC-derived somatic cell types for therapeutic applications, it becomes a major concern impacting the potential use of iPSC-derived germline cell types for reproductive applications. The transition from a source somatic cell type to iPSCs and then on to germ-cell like cells (GCLCs) recapitulates two major epigenetic reprogramming events that normally occur during development in vivo-embryonic reprogramming in the epiblast and germline reprogramming in primordial germ cells (PGCs). We examined the extent of epigenetic and transcriptomic memory persisting first during the transition from differentiated source cell types to iPSCs, and then during the transition from iPSCs to PGC-like cells (PGCLCs). Methods: We derived iPSCs from four differentiated mouse cell types including two somatic and two germ cell types and tested the extent to which each resulting iPSC line resembled a) a validated ES cell reference line, and b) their respective source cell types, on the basis of genome-wide gene expression and DNA methylation patterns. We then induced each iPSC line to form PGCLCs, and assessed epigenomic and transcriptomic memory in each compared to endogenous PGCs/M-prospermatogonia. Results: In each iPSC line, we found residual gene expression and epigenetic programming patterns characteristic of the corresponding source differentiated cell type from which each was derived. However, upon deriving PGCLCs, we found very little evidence of lingering epigenetic or transcriptomic memory of the original source cell type. Discussion: This result indicates that derivation of iPSCs and then GCLCs from differentiated source cell types in vitro recapitulates the two-phase epigenetic reprogramming that normally occurs in vivo, and that, to a significant extent, germline cell types derived in vitro from pluripotent cells accurately recapitulate epigenetic programming and gene expression patterns corresponding to equivalent endogenous germ cell types, suggesting that they have the potential to form the basis of in vitro gametogenesis as a useful therapeutic strategy for treatment of infertility.

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

Genome-wide transcriptome analysis of Echinococcus multilocularis larvae and germinative cell cultures reveals genes involved in parasite stem cell function.

The lethal zoonosis alveolar echinococcosis is caused by tumour-like growth of the metacestode stage of the tapeworm Echinococcus multilocularis within host organs. We previously demonstrated that metacestode proliferation is exclusively driven by somatic stem cells (germinative cells), which are the only mitotically active parasite cells that give rise to all differentiated cell types. The Echinococcus gene repertoire required for germinative cell maintenance and differentiation has not been characterised so far. We herein carried out Illumina sequencing on cDNA from Echinococcus metacestode vesicles, from metacestode tissue depleted of germinative cells, and from Echinococcus primary cell cultures. We identified a set of ~1,180 genes associated with germinative cells, which contained numerous known stem cell markers alongside genes involved in replication, cell cycle regulation, mitosis, meiosis, epigenetic modification, and nucleotide metabolism. Interestingly, we also identified 44 stem cell associated transcription factors that are likely involved in regulating germinative cell differentiation and/or pluripotency. By in situ hybridization and pulse-chase experiments, we also found a new general Echinococcus stem cell marker, EmCIP2Ah, and we provide evidence implying the presence of a slow cycling stem cell sub-population expressing the extracellular matrix factor Emkal1. RNA-Seq analyses on primary cell cultures revealed that metacestode-derived Echinococcus stem cells display an expanded differentiation capability and do not only form differentiated cell types of the metacestode, but also cells expressing genes specific for protoscoleces, adult worms, and oncospheres, including an ortholog of the schistosome praziquantel target, EmTRPMPZQ. Finally, we show that primary cell cultures contain a cell population expressing an ortholog of the tumour necrosis factor α receptor family and that mammalian TNFα accelerates the development of metacestode vesicles from germinative cells. Taken together, our analyses provide a robust and comprehensive characterization of the Echinococcus germinative cell transcriptome, demonstrate expanded differentiation capability of metacestode derived stem cells, and underscore the potential of primary germinative cell cultures to investigate developmental processes of the parasite. These data are relevant for studies into the role of Echinococcus stem cells in parasite development and will facilitate the design of anti-parasitic drugs that specifically act on the parasite germinative cell compartment.

THE DEVELOPMENT OF HUMAN PRIMARY CULTURE MODELS TO MODEL INFLAMMATORY SIGNALING IN CROHN’S DISEASE AND ULCERATIVE COLITIS

Abstract Intestinal inflammation is a major pathological feature associated with gastrointestinal disorders such as Crohn’s disease and ulcerative colitis (UC). To unravel the complex mechanisms underlying these conditions and to develop effective therapeutic strategies, it is crucial to recapitulate intestinal inflammatory signaling using accurate and biologically relevant experimental systems. To establish primary cell-based models that reprise the inflammatory signaling in Crohn’s and UC, we developed the RepliGut platform. Briefly, we established a biobank of primary intestinal stem cells, derived from healthy full-length intestines from a range of donors of different sexes, ages, and ethnicities. From each donor, intestinal epithelial stem cells were isolated from each region of the small intestine and colon (duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon). The intestinal stem cells were passed to generate large and quality-controlled cell lots of “ready-to-use” vials which are cryopreserved and can be thawed directly onto 12- or 96-well Transwell inserts, generating the RepliGut platform. The RepliGut culture conditions allow for investigation into the physiology of intestinal epithelial cells as either proliferative stem or differentiated cell types and enable studies on focused regional differences in the intestinal tract, while the Transwell platform allows apical and basal media access that facilitates barrier function measures and convenient bi-lateral media sampling. Using this system, Crohn’s and UC-like inflammation can be produced via treatment with the most clinically and therapeutically validated cytokines associated with disease pathophysiology, TNF-α and IFN-γ. Circulating concentrations of both TNF-α and IFN-γ are observed to be elevated in patients with inflammatory bowel diseases and therapeutic antagonists including adalimumab (TNF-α) and JAK inhibitors such as tofacitinib (IFN-γ) represent the current standard of care. On the RepliGut platform, both TNF-α and IFN-γ produced a dose-dependent loss in epithelial barrier function, increased cytokine secretion, and caused cellular death, all of which were mitigated by adalimumab and tofacitinib, respectively. Moreover, at clinically relevant concentrations, combinations of TNF-α and IFN-γ produced synergistic inflammatory response on the intestinal epithelial cultures. In summary, the RepliGut platform provides a convenient and high-throughput platform that reproduces cytokine-induced intestinal inflammation for mechanistic studies and drug screening.

Identification of a Chondrocyte-Specific Enhancer in the Hoxc8 Gene.

Hox genes encode transcription factors whose roles in patterning animal body plans during embryonic development are well-documented. Multiple studies demonstrate that Hox genes continue to act in adult cells, in normal differentiation, in regenerative processes, and, with abnormal expression, in diverse types of cancers. However, surprisingly little is known about the regulatory mechanisms that govern Hox gene expression in specific cell types, as they differentiate during late embryonic development, and in the adult organism. The murine Hoxc8 gene determines the identity of multiple skeletal elements in the lower thoracic and lumbar region and continues to play a role in the proliferation and differentiation of cells in cartilage as the skeleton matures. This study was undertaken to identify regulatory elements in the Hoxc8 gene that control transcriptional activity, specifically in cartilage-producing chondrocytes. We report that an enhancer comprising two 416 and 224 bps long interacting DNA elements produces reporter gene activity when assayed on a heterologous transcriptional promoter in transgenic mice. This enhancer is distinct in spatial, temporal, and molecular regulation from previously identified regulatory sequences in the Hoxc8 gene that control its expression in early development. The identification of a tissue-specific Hox gene regulatory element now allows mechanistic investigations into Hox transcription factor expression and function in differentiating cell types and adult tissues and to specifically target these cells during repair processes and regeneration.

Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC& Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

Cellular Reprogramming, Transdifferentiation and Alleviation of the Aging Pathology

Cellular reprogramming and transdifferentiation have emerged as transformative technologies in regenerative medicine, holding significant promise for addressing age-related pathologies and rare genetic disorders. Dr. Shinya Yamanaka's pioneering work in cellular reprogramming paved the way for the restoration of pluripotency in terminally differentiated cells. The understanding that exogenous introduction of the key transcription factors (Oct4, Sox2, KLF4 and c-Myc) enables cells for reprogramming, regaining pluripotency has provided tremendous opportunities and better platforms for therapeutic implications. Transdifferentiation, the conversion of one differentiated cell type to another, offers a complementary approach to cellular reprogramming and can be procured by modulating the expression of specific transcription factors, miRNAs, or other small molecules. Therapeutic approaches that utilize transdifferentiated cells are promising in addressing the age-related pathologies of vital organs such as the pancreas, liver, cardiovascular system as well as neurological disorders. Additionally, the emerging field of epigenetic rejuvenation offers extended possibilities for targeted interventions as it focuses on targeting age-associated CpG signatures of the epigenome. Induced pluripotent stem cells (iPSCs) obtained by cellular reprogramming have become invaluable tools for studying rare monogenic syndromes and premature aging diseases that include Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner syndrome. iPSCs enable researchers to model diseases and gain insights into their underlying mechanisms. Translation of these technologies into clinical practice just needs bypassing hurdles related to safe, efficient, specific therapeutic outcomes. Thus, recent advancements in stem cell technologies highlight that cellular reprogramming and transdifferentiation are poised to revolutionize healthcare, providing novel approaches to address age-related pathologies and other genetic disorders.

Modelling the development of biological structures displaying longitudinal geometries in vitro: culturing pluripotent stem cells on plasma-treated, growth factor-coupled polycaprolactone fibres

Many biological structures such as nerves, blood and lymphatic vessels, and muscle fibres exhibit longitudinal geometries with distinct cell types extending along both the length and width of internal linear axes. Modelling these three-dimensional structures in vitro is challenging: the best-defined stem-cell differentiation systems are monolayer cultures or organoids using pluripotent stem cells. Pluripotent stem cells can differentiate into functionally mature cells depending on the signals received, holding great promise for regenerative medicine. However, the integration of in vitro differentiated cell types into diseased tissue remains a challenge. Engineered scaffolds can bridge this gap if the appropriate signalling systems are incorporated into the scaffold. Here, we have taken a biomimicry approach to generate longitudinal structures in vitro. In this approach, mouse embryonic stem cells are directed to differentiate to specific cell types on the surface of polycaprolactone (PCL) fibres treated by plasma-immersion ion implantation and to which with lineage-specifying molecules have been covalently immobilised. We demonstrate the simplicity and utility of our method for efficiently generating high yields of the following cell types from these pluripotent stem cells: neurons, vascular endothelial cells, osteoclasts, adipocytes, and cells of the erythroid, myeloid, and lymphoid lineages. Strategically arranged plasma-treated scaffolds with differentiated cell types could ultimately serve as a means for the repair or treatment of diseased or damaged tissue.

Evolution of hierarchy and irreversibility in theoretical cell differentiation model.

The process of cell differentiation in multicellular organisms is characterized by hierarchy and irreversibility in many cases. However, the conditions and selection pressures that give rise to these characteristics remain poorly understood. By using a mathematical model, here we show that the network of differentiation potency (differentiation diagram) becomes necessarily hierarchical and irreversible by increasing the number of terminally differentiated states under certain conditions. The mechanisms generating these characteristics are clarified using geometry in the cell state space. The results demonstrate that the hierarchical organization and irreversibility can manifest independently of direct selection pressures associated with these characteristics, instead they appear to evolve as byproducts of selective forces favoring a diversity of differentiated cell types. The study also provides a new perspective on the structure of gene regulatory networks that produce hierarchical and irreversible differentiation diagrams. These results indicate some constraints on cell differentiation, which are expected to provide a starting point for theoretical discussion of the implicit limits and directions of evolution in multicellular organisms.

Reprogramming and multi-lineage transdifferentiation attenuate the tumorigenicity of colorectal cancer cells

Significant advances have been made in reprogramming various somatic cells into induced pluripotent stem cells (iPSCs) and in multi-lineage differentiation (transdifferentiation) into different tissues. These manipulable transdifferentiating techniques may be applied in cancer therapy. Limited works have been reported that cancer cell malignancy can be switched to the benign phenotypes through reprogramming techniques. Here, we reported that two colorectal cancer (CRC) cell lines (DLD1, HT29) could be reprogrammed into iPSCs (D-iPSCs, H-iPSCs). D- and H-iPSCs showed reduced tumorigenesis. Furthermore, we successfully induced D- and H-iPSCs differentiation into terminally differentiated cell types such as cardiomyocyte, neuron, and adipocyte-like cells. Impressively, the differentiated cells exhibited further attenuated tumorigenesis in vitro and in vivo. RNA-Seq further indicated that epigenetic changes occurred after reprogramming and transdifferentiation that caused reduce tumorigenicity. Overall, our study indicated that CRC cells can be reprogrammed and further differentiate to terminally differentiated lineages with attenuation of their malignancy in vitro and in vivo. The current work sheds light on a potential multi-lineage differentiation therapeutic strategy for colorectal cancer.

Stromal localization of inactive CD8+ T cells in metastatic mismatch repair deficient colorectal cancer.

The determinants of metastasis in mismatch repair deficiency with high levels of microsatellite instability (MSI-H) in colorectal cancer (CRC) are poorly understood. Here, we hypothesized that distinct immune and stromal microenvironments in primary tumors may discriminate between non-metastatic MSI-H CRC and metastatic MSI-H CRC. We profiled 46,727 single cells using high-plex imaging mass cytometry and analyzed both differential cell type abundance, and spatial distribution of fibroblasts and immune cells in primary CRC tumors with or without metastatic capacity. We validated our findings in a second independent cohort using immunohistochemistry. High-plex imaging mass cytometry and hierarchical clustering based on microenvironmental markers separated primary MSI-H CRC tumors with and without metastatic capacity. Primary tumors with metastatic capacity displayed a high stromal content and low influx of CD8+ T cells, which expressed significantly lower levels of markers reflecting proliferation (Ki67) and antigen-experience (CD45RO) compared to CD8+ T cells in non-metastatic tumors. CD8+ T cells showed intra-epithelial localization in non-metastatic tumors, but stromal localization in metastatic tumors, which was validated in a second cohort. We conclude that localization of phenotypically distinct CD8+ T cells within stroma may predict metastasis formation in MSI-H CRC.

Human placental vascular and perivascular cell heterogeneity differs between first trimester and term, and in pregnancies affected by foetal growth restriction.

Growth-restricted placentae have a reduced vascular network, impairing exchange of nutrients and oxygen. However, little is known about the differentiation events and cell types that underpin normal/abnormal placental vascular formation and function. Here, we used 23-colour flow cytometry to characterize placental vascular/perivascular populations between first trimester and term, and in foetal growth restriction (FGR). First-trimester endothelial cells had an immature phenotype (CD144+/lowCD36-CD146low), while term endothelial cells expressed mature endothelial markers (CD36+CD146+). At term, a distinct population of CD31low endothelial cells co-expressed mesenchymal markers (CD90, CD26), indicating a capacity for endothelial to mesenchymal transition (EndMT). In FGR, compared with normal pregnancies, endothelial cells constituted 3-fold fewer villous core cells (P < 0.05), contributing to an increased perivascular: endothelial cell ratio (2.6-fold, P < 0.05). This suggests that abnormal EndMT may play a role in FGR. First-trimester endothelial cells underwent EndMT in culture, losing endothelial (CD31, CD34, CD144) and gaining mesenchymal (CD90, CD26) marker expression. Together this highlights how differences in villous core cell heterogeneity and phenotype may contribute to FGR pathophysiology across gestation.

Endothelial to mesenchymal transition in kidney fibrosis.

Fibrotic diseases are characterized by the uncontrolled accumulation of extracellular matrix (ECM) components leading to disruption of tissue homeostasis. Myofibroblasts as the main ECM-producing cells can originate from various differentiated cell types after injury. Particularly, the process of endothelial-to-mesenchymal transition (endMT), describing phenotypic shifts of endothelial cells to adopt a fully mesenchymal identity, may contribute to the pool of myofibroblasts in fibrosis, while leading to capillary rarefaction and exacerbation of tissue hypoxia. In renal disease, incomplete recovery from acute kidney injury (AKI) and the ensuing fibrotic reaction stand out as major contributors to chronic kidney disease (CKD) development. While the focus has largely been on impaired tubular epithelial repair as a potential fibrosis-driving mechanism, alterations in the renal microcirculation post-AKI, and in particular endMT as a maladaptive response, could hold equal significance. Dysfunctional interplays among various cell types in the kidney microenvironment can instigate endMT. Transforming growth factor beta (TGF-β) signaling, with its downstream activation of canonical/Smad-mediated and non-canonical pathways, has been identified as primary driver of this process. However, non-TGF-β-mediated pathways involving inflammatory agents and metabolic shifts in intercellular communication within the tissue microenvironment can also trigger endMT. These harmful, maladaptive cell-cell interactions and signaling pathways offer potential targets for therapeutic intervention to impede endMT and decelerate fibrogenesis such as in AKI-CKD progression. Presently, partial reduction of TGF-β signaling using anti-diabetic drugs or statins may hold therapeutic potential in renal context. Nevertheless, further investigation is warranted to validate underlying mechanisms and assess positive effects within a clinical framework.

Pan-cancer scRNA-seq analysis reveals immunological and diagnostic significance of the peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMCs) reflect systemic immune response during cancer progression. However, a comprehensive understanding of the composition and function of PBMCs in cancer patients is lacking, and the potential of these features to assist cancer diagnosis is also unclear. Here, the compositional and status differences between cancer patients and healthy donors in PBMCs were investigated by single-cell RNA sequencing (scRNA-seq), involving 262,025 PBMCs from 68 cancer samples and 14 healthy samples. We observed an enhanced activation and differentiation of most immune subsets in cancer patients, along with reduction of naïve T cells, expansion of macrophages, impairment of NK cells and myeloid cells, as well as tumor promotion and immunosuppression. Based on characteristics including differential cell type abundances and/or hub genes identified from weight gene co-expression network analysis (WGCNA) modules of each major cell type, we applied logistic regression to construct cancer diagnosis models. Furthermore, we found that the above models can distinguish cancer patients and healthy donors with high sensitivity. Our study provided new insights into using the features of PBMCs in non-invasive cancer diagnosis.

Bone Marrow Microenvironment and Hematopoietic Cell Types Are Differentially Affected By Transcriptomic Quality Control Filtering Metrics in Single Cell Data

A crucial step prior to analysis of single cell transcriptomic data includes filtering out low-quality (e.g., dying) cells and artifacts (e.g., empty droplets). Choosing appropriate quality control parameters leads to correct cell assignment, so that the data for downstream analysis is high-quality and is representative of all processed cell types. These metrics include high and low cut-offs for mitochondrial content, total number of genes, number of different genes, and ribosomal content per cell. They have been found to vary based on cell and tissue type, as well biological variables such as age and disease, and technical variables such as sequencing technology. The aim of this study was to investigate the variation of these quality control metrics in a data set that included stem, progenitor, and differentiated cell types of the hematopoietic, mesenchymal, and endothelial lineages. Furthermore, we set out to determine whether applying standard cut-offs may differentially affect different cell types. We calculated individual filtering parameters for each cell type using the R package ddqc (Subramanian et al. Genome Biol. 2022). Thus, any biological variability based on mitochondrial, ribosomal, and gene counts was accounted for. Notably, distribution of measurements for each of the above quality control metrics varied by cluster, and certain cell-type means were adjacent to commonly used cut-off values. For instance, we observed higher content of mitochondrial genes in osteoblasts, sinusoidal endothelial cells, a subset of adipocyte-primed mesenchymal cells (MSPC), as well as hematopoietic progenitor cells, pre-B, and B cells. On the other hand, subsets of erythroblasts, neutrophils, as well as arteriolar endothelial cells had the lowest mitochondrial content. In addition, lower total gene counts and lower gene complexity were found in adipocyte-primed MSPCs, neutrophils, and megakaryocyte-erythrocyte progenitors, while higher counts were found in granulocyte-monocyte progenitor cells, megakaryocyte/erythroid-primed multipotent progenitor cells and subsets of erythroblasts. Applying universal cutoff values to this data set would greatly reduce, and some cases, eliminate cell types that have high mitochondrial content or lower gene counts and complexity, while leaving those cell types on the opposite spectrum unaffected. This would also skew proportions of cells in downstream analyses, thus affecting biological interpretation of the results. Overall, our results demonstrate biological variability of the quality control metric distributions in a single cell transcriptomic data set that includes diverse bone marrow niche and hematopoietic cell types. This reveals novel biological information regarding distinct cell types as well as cautions that applying standard filtering paraments on such a data set would eliminate some cell types and bias downstream analysis for others. Optimization of filtering criteria will be important when broadening single cell analysis to multiplexed approaches incorporating transcriptome, genome, epigenome, and proteome-level data.

OR2803 Corticotroph Fate Weighted Cell Differentiation Paradigm For Human Induced Pluripotent Stem Cells

Abstract Disclosure: M.E. Moore: None. K.G. Chen: None. K. Park: None. D. Asuzu: None. N. Ramavenkat: None. S. Walbridge: None. D. Mullaney: None. M. Arhin: None. D. Maric: None. D. Mandal: None. P. Chittiboina: None. Introduction: The vertebrate anterior pituitary gland is a highly conserved ‘master’ organ that controls hormonal milieu in real-time. Pituitary adenomas and their treatments (surgery, chemotherapy or radiation) can result in pituitary organ failure, specifically hypocortisolism, a challenging condition to manage. Here, we created an optimized paradigm (anterior pituitary corticotroph or APcor) to preferentially drive human induced pluripotent stem cells (hiPSCs) towards a pituitary corticotroph fate. Methods: We directed U112I hiPSCs towards terminally differentiated cell types using a combination of rho-associated kinase inhibitor (Y-27632), bone morphogenic protein 4 (BMP4), transforming growth factor β inhibitor (SB431542), sonic hedgehog (SHH), fibroblast growth factor 8b (FGF8b), fibroblast growth factor 10 (FGF10) and leukemia inhibitory factor (LIF). Markers for pluripotent stem cells, cranial placode (CP), Rathke’s Pouch (RP), anterior pituitary progenitors and mature cell types were assessed at 10-day intervals using quantitative qRT-PCR and multiplex immunocytochemistry (mICC) or immunohistochemistry (IHC). ACTH activity was measured using ELISA (MD Bio). Results: We tested initial differentiation conditions and found optimal CP initiation in presence of SB431542. We observed decrease in embryonic stem cell markers OCT4 and NANOG by day 10, with concomitant increase in CP/RP markers SIX1, PAX6 and SIX6 using mICC/IHC and qRT-PCR. Continuation of differentiation paradigm with LIF showed strong expression of anterior pituitary progenitor markers PITX1, TBX19 and POU1F1 at day 20. pRT-PCR analysis at this time point confirms differentiation towards pituitary cell types without needing enrichment for SIX1. At day 30, POMC expression was observed by IHC in 10-15% of cells as measured by quantitative analysis (Qupath 0.2.0), indicating mature corticotrophs. Corticotroph cell fate was confirmed with corticotropin releasing hormone (CRH) stimulation which led to up to 40x-fold increase in ACTH expression detected by ELISA (D30 w/CRH vs Pluripotent w/CRH). Conclusions: We found with the APcor paradigm, we can reliably drive towards corticotroph cell fate. We successfully differentiated hiPSCs into mature functioning corticotrophs with ability to recapitulate pituitary organ function. Our method is amenable to 3D organoid formation and pre-clinical implantation in animal models of hypophysectomy as a novel hormone replacement therapy. Presentation: Sunday, June 18, 2023

Vascular endothelial cell-specific disruption of the profilin1 gene leads to severe multiorgan pathology and inflammation causing mortality.

Actin-binding protein Profilin1 is an important regulator of actin cytoskeletal dynamics in cells and critical for embryonic development in higher eukaryotes. The objective of the present study was to examine the consequence of loss-of-function of Pfn1 in vascular endothelial cells (ECs) in vivo. We utilized a mouse model engineered for tamoxifen-inducible biallelic inactivation of the Pfn1 gene selectively in EC (Pfn1EC-KO). Widespread deletion of EC Pfn1 in adult mice leads to severe health complications presenting overt pathologies (endothelial cell death, infarct, and fibrosis) in major organ systems and evidence for inflammatory infiltrates, ultimately compromising the survival of animals within 3 weeks of gene ablation. Mice deficient in endothelial Pfn1 exhibit selective bias toward the proinflammatory myeloid-derived population of immune cells, a finding further supported by systemic elevation of proinflammatory cytokines. We further show that triggering Pfn1 depletion not only directly upregulates proinflammatory cytokine/chemokine gene expression in EC but also potentiates the paracrine effect of EC on proinflammatory gene expression in macrophages. Consistent with these findings, we provide further evidence for increased activation of Interferon Regulatory Factor 7 (IRF7) and STAT1 in EC when depleted of Pfn1. Collectively, these findings for the first time demonstrate a prominent immunological consequence of loss of endothelial Pfn1 and an indispensable role of endothelial Pfn1 in mammalian survival unlike tolerable phenotypes of Pfn1 loss in other differentiated cell types.