Lineage Tracing Studies Research Articles

Tracing the origin of alveolar stem cells in lung repair and regeneration

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

250 Harnessing the Endogenous Stem Cell Response After Spinal Cord Injury: A Novel and Translationally Relevant Therapeutic Strategy

INTRODUCTION: The adult spinal cord contains a population of endogenous ependymal-derived neural stem/progenitor cells (epNSPCs) that hold the potential to promote regeneration and repair after injury. epNSPCs are normally quiescent but are acutely activated after spinal cord injury (SCI). Despite this, the activation of these cells remains insufficient to promote robust neural regeneration and recovery. Understanding the mechanisms that govern the biology of epNSPCs is critical in developing a therapeutic strategy to harness their regenerative potential after SCI. METHODS: epNSPCs isolated from the adult spinal cord were used for in vitro mechanistic analysis. In vivo, cervical SCI was induced in adult rodents using a clinically relevant compression-contusion model of cervical SCI. FoxJ1-Cre-tdT reporter mice were used for lineage tracing studies. One-week post-SCI, animals (n = 15/group) were randomized to receive CX546 (a positive AMPA receptor modulator), or vehicle-control. Animals underwent weekly behavioural testing and spinal cords were extracted for histological/molecular analysis. RESULTS: Glutamate excitotoxicity, a hallmark in the pathogenesis of acute SCI, leads to calcium influx in spinal cord epNSPCs via AMPA receptors (AMPARs) and this change in calcium in concert with Notch signaling drives their proliferation and astrocytic differentiation. Blockade of AMPARs in vivo inhibits the activation of epNSPCs post-SCI. Positive modulation of AMPARs after SCI using the clinically relevant class of drugs known as ampakines, enhances epNSPC proliferation, increases pro-regenerative astrocytes and neurotrophic factor production, and promotes neuronal survival and motor recovery. CONCLUSIONS: We report on a novel mechanism by which AMPARs regulate the growth and phenotype of endogenous epNSPCs. Pharmacological targeting of this mechanism could be used to harness the regenerative potential of endogenous neural stem cells and improve motor recovery after SCI.

Abstract 6923: Deconvolution of breast cancer dissemination and therapy resistance via single-cell transcriptional lineage tracing

Abstract Chemotherapy remains the most used systemic treatment option for triple-negative breast cancer (TNBC). Lineage tracing studies demonstrated that treatment does not significantly alter clonal composition of primary tumors (PTs). The specific transcriptional phenotype(s) conferring chemoresistance in PTs and metastasis, however, are still largely undetermined, and have not been analyzed at single-clone resolution. We first modelled clinically relevant contexts of chemoresistance in our recently published single-cell lineage tracing approach (DOI: 10.1158/0008-5472.CAN-22-2717), using the standard-of-care combination of adriamycin and cyclophosphamide (A+C) in TNBC xenografts. Treatment was administered in neoadjuvant or adjuvant settings, or both together. In all cases, surgical PT resection was followed by metastatic disease. The neoadjuvant treatment resulted into a stable disease, thus resembling a significant fraction of the treated patients. To investigate clonal and transcriptional dynamics we performed scRNAseq analysis of 12 replicates (3 matched PT-lung replicates for each condition, for a total of 24 samples). Clustering analyses revealed a separation of treatments and tissue of origin. We retrieved a large number of longitudinal clones, including rare clones with high metastatic potential, in all the experimental conditions. A+C did not significantly alter clonal abundances, neither in PTs nor in metastases. Pseudobulk transcriptional analyses revealed key transcriptional traits associated with chemoresistance. Differential expression on longitudinal clones gave insights about transcriptional features of chemoresistant and pro-metastatic clones under chemotherapy. Integration at single-clone level of cluster analysis, pseudobulk and differential expression revealed cystatin (CST) family genes as highly-enriched in the chemoresistant and pro-metastatic phenotype. We are currently validating the role of CST genes in tumor growth, dissemination and chemoresistance in vivo using a panel of specific shRNAs. In parallel, using the same experimental model, we successfully deconvoluted the metastatic cascade through a single-cell lineage tracing approach on matched PT-CTC (circulating tumor cells)-lung replicates. scRNAseq analyses of pro-metastatic, circulating and metastatic clones is ongoing. Our preliminary data showed that all the dominant clones in the metastatic lungs are also found as CTCs in the bloodstream, thus suggesting that all pro-metastatic cells are also capable of surviving in the bloodstream and reinforcing our concept of pro-metastatic clone. This approach led us to gain a complete overview around clonal and transcriptional evolution of breast cancer dissemination. Citation Format: Alberto Dalmasso, Andrea Cossa, Giulia Bertolini, Niccolò Roda, Ilaria Servidio, Francesco Antonio Tucci, Salvatore Pece, Gabriella Sozzi, Pier Giuseppe Pelicci. Deconvolution of breast cancer dissemination and therapy resistance via single-cell transcriptional lineage tracing [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 6923.

Cardiac and perivascular myofibroblasts, matrifibrocytes, and immune fibrocytes in hypertension; commonalities and differences with other cardiovascular diseases.

Hypertension is a major cause of cardiovascular diseases such as myocardial infarction and stroke. Cardiovascular fibrosis occurs with hypertension and contributes to vascular resistance, aortic stiffness, and cardiac hypertrophy. However, the molecular mechanisms leading to fibroblast activation in hypertension remain largely unknown. There are two types of fibrosis: replacement fibrosis and reactive fibrosis. Replacement fibrosis occurs in response to the loss of viable tissue to form a scar. Reactive fibrosis occurs in response to an increase in mechanical and neurohormonal stress. Although both types of fibrosis are considered adaptive processes, they become maladaptive when the tissue loss is too large, or the stress persists. Myofibroblasts represent a subpopulation of activated fibroblasts that have gained contractile function to promote wound healing. Therefore, myofibroblasts are a critical cell type that promotes replacement fibrosis. Although myofibroblasts were recognized as the fibroblasts participating in reactive fibrosis, recent experimental evidence indicated there are distinct fibroblast populations in cardiovascular reactive fibrosis. Accordingly, we will discuss the updated definition of fibroblast subpopulations, the regulatory mechanisms, and their potential roles in cardiovascular pathophysiology utilizing new knowledge from various lineage tracing and single-cell RNA sequencing studies. Among the fibroblast subpopulations, we will highlight the novel roles of matrifibrocytes and immune fibrocytes in cardiovascular fibrosis including experimental models of hypertension, pressure overload, myocardial infarction, atherosclerosis, aortic aneurysm, and nephrosclerosis. Exploration into the molecular mechanisms involved in the differentiation and activation of those fibroblast subpopulations may lead to novel treatments for end-organ damage associated with hypertension and other cardiovascular diseases.

Pericytes as the Orchestrators of Vasculature and Adipogenesis.

Pericytes (PCs) are located surrounding the walls of small blood vessels, particularly capillaries and microvessels. In addition to their functions in maintaining vascular integrity, participating in angiogenesis, and regulating blood flow, PCs also serve as a reservoir for multi-potent stem/progenitor cells in white, brown, beige, and bone marrow adipose tissues. Due to the complex nature of this cell population, the identification and characterization of PCs has been challenging. A comprehensive understanding of the heterogeneity of PCs may enhance their potential as therapeutic targets for metabolic syndromes or bone-related diseases. This mini-review summarizes multiple PC markers commonly employed in lineage-tracing studies, with an emphasis on their contribution to adipogenesis and functions in different adipose depots under diverse metabolic conditions.

Abstract B093: Alcoholic chronic inflammation driven CREB mediates acinar-to-ductal reprogramming and promote neoplastic progression

Abstract Introduction: Alcoholic chronic pancreatitis (ACP) is a progressive fibroinflammatory disease of the pancreas often characterized by extensive acinar cell injury leading to acinar-to-ductal metaplasia (ADM). However, the impact of ADM associated with ACP on the acceleration of oncogenic mutant Kras (Kras*)-driven tumorigenesis remains poorly understood. In this study, we aimed to unravel the underlying mechanism by which ACP-induced dysfunction and with Kras*, leads to the development of irreversible ADM and premalignant lesions that establish a tumor-permissive microenvironment for the emergence of high-grade pancreatic intraepithelial neoplasia (PanINs). Methods: ACP mice model was developed in acinar cell-specific Ptf1aCreERTM/+, Ptf1aCreERTM/+; LSL-KrasG12D/+ (KC), and genetic ablation of cyclic AMP response element-binding protein 1 (CREB) in KC (KCC−/−) mice crossed with or without R26R-EYFP reporter mice, and these mice were fed with alcohol-enriched or regular diet along with repetitive caerulein administration. Lineage tracing studies and acinar explants were employed to investigate the fate of adult acinar cells. ACP-induced mice were subsequently euthanized either 3 days or 21 days after ACP induction, the pancreas was collected for immunohistochemical analysis and examined for ADM, PanINs, fibrosis, and inflammation. To gain insights into the molecular mechanisms; kinase array analysis, acinar cell explant culture, Western blot and qPCR-based studies were conducted. Additionally, single-cell RNA sequencing (scRNA-seq) was employed to investigate cell-specific transcriptomic changes focusing on distinct cellular compartments. Results: Lineage tracing studies showed that ACP-induced ADM stems from direct transdifferentiating adult acinar cells to a ductal phenotype in Kras* mice. ACP induction with Kras* cooperatively provided a sustained Kras - dependent oncogenic program in inflammation-induced metaplastic progenitor cells. The upregulation of CREB accompanied this process of malignant transformation. CREB activation persists even after ACP withdrawal in KC mice compared to Ptf1aCreERTM/+ mice without mutant Kras. Acinar-specific CREB ablation attenuated advanced PanIN lesion development, impeding tumor progression in Kras* mice. Additionally, it reduced inflammation-induced tissue injury and facilitated acinar cell recovery. ScRNA-seq analysis revealed gene enrichment favoring acinar cell homeostasis and downregulation of ductal gene signatures in KCC−/− mice, further emphasizing CREB's pivotal role in modulating these cellular processes. Conclusion: Our findings demonstrate that Kras* sustains an irreversible ADM and PanIN through continuous activation of CREB. Additionally, we have identified that CREB mediates an oncogenic transcriptional program that drives tumor progression in the context of inflammation-induced disease. Importantly, our study reveals that ACP synergistically cooperates with Kras*-driven activated CREB, promoting the progression of ADM toward high-grade PanINs. Citation Format: Siddharth Mehra, Supriya Srinivasan, Anna Bianchi, Austin Dosch, Samara Singh, Vanessa Garrido, Iago De Castro Silva, Varunkumar Krishnamoorthy, Sudhakar Jinka, Jashodeep Datta, Nipun B. Merchant, Nagaraj Nagathihalli. Alcoholic chronic inflammation driven CREB mediates acinar-to-ductal reprogramming and promote neoplastic progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B093.

Abstract C032: The involvement of the splanchnic program at the cellular and molecular levels in pancreatic cancer associated fibroblasts

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a dense stroma primarily composed of cancer associated fibroblasts (CAFs). Our group recently performed a lineage tracing study using genetically engineered mouse models. This study demonstrated that CAFs and pancreatic tissue resident fibroblasts (TRFs) are both derived from the fetal splanchnic mesenchyme, an area of mesoderm next to the foregut endoderm. To determine whether two major subtypes of CAFs are derived from the splanchnic mesenchyme, I analyzed co-expression of the lineage tracing marker Tomato and CAF subtype marker a smooth muscle actin (aSMA). This suggests that both myofibroblasts (aSMA high) and inflammatory fibroblasts (aSMA low) are derived from the splanchnic mesenchyme. Furthermore, we found that transcription factor Gata6 is expressed in the splanchnic mesenchyme and in some CAFs. We constructed mouse genetic models where Gata6 is specifically deleted in fibroblasts. These Gata6 conditional knock out mice had increased tumor burden, suggesting that stromally-expressed Gata6 has a tumor restraining role. We derived CAF and TRF cell lines from these genetic mouse models and their littermate normal mice. My preliminary data suggest that several important fibroblast factors, including type I collagen, aSMA, and Interleukin 6, are upregulated in the Gata6 KO CAFs and TRFs relative to normal CAFs and TRFs. In summary, our study showed that two major subtypes of pancreatic CAFs are derived from the splanchnic mesenchyme, and splanchnic factors may play a role in adult pancreatic fibroblasts to regulate the expression of critical fibroblast genes. Citation Format: Tom Walter, Lu Han, Michael Ostrowski. The involvement of the splanchnic program at the cellular and molecular levels in pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C032.

Abstract A045: Persistence of the splanchnic gene signature along the mesenchymal cell trajectory during pancreatic cancer progression

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. While fibroblasts are sparse in the normal adult pancreas, CAFs are a major component in the desmoplastic stroma of PDAC and may account for half of the entire tumor tissue. Here we aimed to investigate the origin, diversification, and function of CAFs. We constructed a dual-DNA-recombinase mouse genetic model, which allowed for genetic alternations in pancreatic epithelial cells and fibroblasts independently and simultaneously. The splanchnic mesenchyme is a layer of tissue adjacent to the pancreatic endoderm during fetal development. Our lineage tracing studies demonstrated that the splanchnic mesenchyme is the fetal origin of adult pancreatic fibroblasts during homeostasis and tumorigenesis. Notably, the other two postulated CAF sources, bone marrow and epithelial cells, have minimal contributions to fibroblasts. We further compared mice carrying no genetic mutations, only Kras mutation, and Kras/p53 dual mutations in pancreatic epithelial cells. We found that fibroblasts in all three conditions are derived from the splanchnic mesenchyme, suggesting a continuous mesenchymal trajectory in coordination with the pancreatic epithelium. Additionally, single cell transcriptomic analysis indicated persistent and dynamic gene expressions along the pancreatic mesenchymal trajectory during development, homeostasis, precancer lesion and cancer. Intriguingly, certain splanchnic factors are expressed in adult pancreatic fibroblasts in temporally and spatially distinct patterns. To determine the function of splanchnic factors in fibroblasts, we constructed mouse genetic models to delete GATA6 specifically in CAFs, which resulted in increased tumor burden in the pancreas. This suggests a non-cell autonomous function of GATA6 in CAFs to restrain pancreatic cancer progression. In summary, this study delineated a continuous cell trajectory of the mesenchymal lineage in the pancreas across different life stages. Furthermore, persistent gene expressions along the mesenchymal trajectory contributes to pancreatic CAF heterogeneity. Importantly, such persistence may constitute an inherent mechanism to restrain pancreatic cancer. Citation Format: Lu Han, Tom Walter, Joseph Beaudet, Caroline Everett, Julia Piermattei, Alex Adams, Kun Fang, Michael Zimmermann, Angela Mathison, Raul Urrutia, Victor Jin, Gustavo Leone, Michael Ostrowski. Persistence of the splanchnic gene signature along the mesenchymal cell trajectory during pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A045.

CRISPR Knock-Ins in Organoids to Track Tumor Cell Subpopulations.

The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.

Genetic targeting of lymphatic endothelial cells in mice: current strategies and future perspectives.

Lymphatic vessels within different organs have diverse developmental origins, depend on different growth factor signaling pathways for their development and maintenance, and display notable tissue-specific adaptations that contribute to their roles in normal physiology and in various diseases. Functional studies on the lymphatic vasculature rely extensively on the use of mouse models that allow selective gene targeting of lymphatic endothelial cells (LECs). Here, we discuss LEC diversity and provide an overview of some of the commonly used LEC-specific inducible Cre lines and induction protocols, outlining essential experimental parameters and their implications. We describe optimized treatment regimens for embryonic, postnatal and adult LECs, efficiently targeting organs that are commonly studied in lymphatic vascular research, such as the mesentery and skin. We further highlight the anticipated outcomes and limitations associated with each induction scheme and mouse line. The proposed protocols serve as recommendations for laboratories initiating studies involving targeting of the lymphatic vasculature, and aim to promote uniformity in lineage tracing and functional studies within the lymphatic vascular field.

Cardiac Development at a Single-Cell Resolution.

Mammalian cardiac development is a complex, multistage process. Though traditional lineage tracing studies have characterized the broad trajectories of cardiac progenitors, the advent and rapid optimization of single-cell RNA sequencing methods have yielded an ever-expanding toolkit for characterizing heterogeneous cell populations in the developing heart. Importantly, they have allowed for a robust profiling of the spatiotemporal transcriptomic landscape of the human and mouse heart, revealing the diversity of cardiac cells-myocyte and non-myocyte-over the course of development. These studies have yielded insights into novel cardiac progenitor populations, chamber-specific developmental signatures, the gene regulatory networks governing cardiac development, and, thus, the etiologies of congenital heart diseases. Furthermore, single-cell RNA sequencing has allowed for the exquisite characterization of distinct cardiac populations such as the hard-to-capture cardiac conduction system and the intracardiac immune population. Therefore, single-cell profiling has also resulted in new insights into the regulation of cardiac regeneration and injury repair. Single-cell multiomics approaches combining transcriptomics, genomics, and epigenomics may uncover an even more comprehensive atlas of human cardiac biology. Single-cell analyses of the developing and adult mammalian heart offer an unprecedented look into the fundamental mechanisms of cardiac development and the complex diseases that may arise from it.

Abstract IA008: Mechanisms and consequences of pancreatic cancer stromal evolution

Abstract The mechanisms underlying evolution of tumor-associated stroma remain poorly understood. In solid tumors featuring a prominent stromal reaction, an improved understanding of the functions and origins of abundant stromal cell types may facilitate the development of new and effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is the quintessence of a fibro-inflammatory malignancy, with 50-90% of tumor volume occupied by a dense, desmoplastic stroma. Cancer-associated fibroblasts (CAFs) are the key cell type which drives the stromal reaction in PDAC, and recent reports suggest that stromal CAFs represent a heterogeneous population of cells from diverse origins, potentially including cell types which support and others which suppress tumor growth. Pancreatic stellate cells (PSCs) are lipid-storing cells in healthy pancreas which can transdifferentiate to an activated CAF phenotype. PSCs have been suggested as the predominant source of fibroblasts in the PDAC tumor microenvironment. However, proper lineage tracing studies have never been performed, such that the relative contribution and specific functions of PSCs in the tumor microenvironment are unknown. We have developed a novel mouse model to track PSC differentiation and function during pancreatic tumor progression in vivo. This model revealed that PSC-derived CAFs in fact give rise to a numerically minor but functionally significant subset of PDAC CAFs, and may represent a viable therapeutic target. We adapted our mouse model to enable targeted ablation of PSCs and derivative CAFs within their host tissue for the first time. Functional studies in this model reveal non-redundant functions for PSC-derived CAFs in shaping the PDAC microenvironment, and highlight the significance of mesenchymal lineage heterogeneity for pancreatic tumorigenesis. Further, we find that tumor genotype with respect to p53 status dictates stromal evolutionary routes with respect to cell of origin, a finding that may extend to additional cancer cell-intrinsic genomic alterations. Together, these findings shed light on mechanisms that shape the tumor microenvironment during pancreatic cancer progression, and may hold relevance in additional solid tumor types. Citation Format: Mara Sherman. Mechanisms and consequences of pancreatic cancer stromal evolution [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA008.

Clonal Derivation and Dynamics in Rhesus Macaques Following Hematopoietic Stem Cell Transplantation and the Impact of Clonal Hematopoiesis

Microglia (MG) are the resident macrophages of the central nervous system (CNS). Under steady-state conditions, MG play essential roles in immune surveillance; phagocytosis and processing of pathogens, dying cells and protein aggregates; and synaptic pruning to maintain neuronal health. MG dysfunction has been linked to various CNS disorders, including neurodegenerative disorders such as Alzheimer's disease, with multiple pathways implicated, including increased and prolonged activation with excessive release of pro-inflammatory mediators. Lineage tracing studies in mice have demonstrated that yolk sac macrophages form the CNS MG compartment during development, with little ongoing replacement from circulating monocyte/macrophages at steady state or even following hematopoietic stem/progenitor cell (HSPC) transplantation. However, improvements in disease outcomes in human patients with CNS disorders such as adrenoleukodystrophy and metachromatic leukodystrophy following HSPC gene therapies have suggested that adult HSPC-derived cells can contribute to MG function. Recently, Bouzid et al reported that individuals with somatic HPSC mutations in hematopoietic expansion driver genes (clonal hematopoiesis/CH) had a significantly lower risk of Alzheimer's disease (AD). The same study utilized nuclear chromatin profiling of a small set of postmortem brain samples to suggest substantial contribution of CH-derived HSPC to the MG compartment. The authors suggested that enhanced function of CH-derived MG enables them to better clear AD plaques. To more directly investigate the replacement and clonal source of adult CNS resident MG/myeloid cells by HSPC-derived cells and to and assess the impact of CH mutations on MG replacement, we utilized clinically-relevant rhesus macaque (RM) models. Fresh MG and other tissue-resident myeloid cells were purified from the brains of RM that had undergone autologous transplantation 2- 9 years previously with HSPCs transduced with a barcoded lentiviral vector expressing GFP following ablative total body irradiation (TBI, n=6) (Koelle et al, Blood, 2017) or busulfan (n=1, vector without GFP)(Abraham et al, MTMCD, 2023); or HSPCs engineered to carry TET2 mutations using CRISPR/Cas9 (n=2)(Shin et al, Blood, 2022). In addition, we studied an aged RM with natural CH ( RUNX1 mutation) in order to investigate MG replacement in the absence of conditioning and transplantation. We optimized RM MG (CD45+CD11b+CX3CR1+) and CNS myeloid cell (CD45+CD11B+CX3CR1-) purification. To ensure separation of bona fide tissue-resident CNS cells away from perivascular cells or contaminating blood, we incorporated CD45-specific antibody serial intravascular staining (SIVS) (Mortlock et al., 2022) and sorted SIVS- parenchymal MG, SIVS- perivascular MG, and SIVS+ myeloid cells. In all 6 TBI-conditioned lentiviral-barcoded animals, at most 2% of tissue-resident SIVS- MG cells were GFP+ (Fig 1A), with a mean ratio of <0.01 compared to the level of GFP+ circulating blood monocytes in each animal (Fig 1B). Barcode analyses demonstrated similar clonal derivation of GFP+ MG cells to peripheral blood (PB) monocytes, representing output from stable long-term multipotent HSPC clones. A higher MG repopulation level was found in the busulfan-conditioned animal by vector copy number. MG replacement with CH-derived cells was no higher absolutely or relative to blood monocytes in the two engineered TET2-CH animals nor in the aged animal with natural CH (Fig 1B). These results demonstrate only low-level replacement of MG from HSPCs even many years following ablative conditioning and autologous transplantation, and no evidence for enhanced repopulation in the setting of engineered or natural clonal hematopoiesis. The clonal ontogeny of the HSPC-derived MG cells matched that of blood myeloid cells. Further characterization of MG derived from transplanted HSPCs versus control MG is ongoing, as is functional characterization of CH versus unmutated macrophages, asking whether mutated cells are hyper-functional. Our clinically-relevant macaque model provides insights into MG ontogeny, and information relevant to improving CNS-directed HSPC gene therapies, as well as information relevant to interpreting a potential protective impact of CH on human AD.

MODL-07. NF1 MUTATION IN OLIGODENDROCYTE PRECURSOR CELLS INDUCES PRENEOPLASTIC LESIONS IN THE BRAIN

Abstract Oligodendrocyte precursor cells (OPCs) are progenitor cells that give rise to oligodendrocytes in the central nervous system throughout the lifespan. Transcriptomic and lineage tracing studies suggest that OPCs may be the putative cell of origin for many gliomas in children and adults. While children with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome typically develop optic pathway and brainstem gliomas, as many as 50% of children develop gliomas in other brain regions using radiologic criteria. Relevant to these gliomas, discrete patches of hyperdense OPCs were found in the brains of Nf1+/- mice. The OPCs within the lesions display a transient increase in proliferation and hypercellularity. When the brains of mice carrying different heterozygous NF1 patient derived germline Nf1 gene mutations were examined, hyperdense OPC lesions were only seen in mice whose Nf1 mutations also predispose to optic gliomas. Mechanistically, monoallelic KRAS hyperactivation also results in similar OPC hyperdense lesions. Moreover, OPC-specific Nf1-heterozygous mice display similar lesions, while OPC-specific Nf1-knockout results in generalized OPC hyperdensity throughout the entire brain. Taken together, these data suggest the existence of a preneoplastic state conferred by Nf1 mutation in OPCs and establish a tractable model system to explore the transition to glioma in children with NF1.

Abstract 13284: New Elastic Fiber Formation by Non-Smooth Muscle Lineage Cells During the Progression of Aortic Dissections

Introduction: Aortic dissection (AD) is a life-threatening vascular disease displaying the disruption of the extracellular matrix (ECM). Elastic fibers are a major component of ECM and confer structural integrity to the aortic wall. Normally, elastic fibers are minimally synthesized in adulthood. However, it remains unknown whether and how elastic fibers are synthesized following ADs. Methods and Results: AD was induced by administration of β-aminopropionitrile (BAPN, 0.5% wt/vol) to male C57BL/6J mice at 4 weeks of age. Thoracic aortas were collected after 4 or 12 weeks of BAPN administration. False lumen formation with fresh hematomas was observed at 4 weeks of BAPN administration, indicating development of acute ADs. The false lumen developed striking remodeling at 12 weeks, suggesting progression to the chronic phase. RT-qPCR revealed that mRNA abundance of tropoelastin, a precursor of elastic fibers, was increased significantly at the chronic phase of ADs, but not at the acute phase. Tropoelastin mRNA was observed primarily in the vascular wall of the false lumen, as evidenced by in situ hybridization. Of interest, elastic fibers were formed in the wall of the false lumen in chronic ADs. These data suggest that elastic fibers were newly synthesized during the progression of ADs. Immunostaining revealed that the walls of true and false lumens of chronic ADs were mainly composed of cells expressing both αSMA (encoding by Acta2 ) and MYH11, common markers for smooth muscle cells (SMCs). However, in a lineage tracing study, the walls of the false lumen of chronic ADs were negative for the Acta2 lineage, suggesting that cells in the wall of the false lumen were derived from a non-SMC lineage but acquired SMC properties during AD progression. Bulk RNA sequencing identified the increased abundance of 2,799 genes in chronic ADs and 102 transcription factors were predicted to regulate these genes. Among them, JunB was the most abundant molecule that can be aligned in the promotor region of tropoelastin. Of note, JunB was expressed adjacent to both αSMA-positive cells and new elastic fibers in the dissected aortas. Conclusion: ADs exhibit vascular remodeling with newly elastic fiber formation by non-SMC lineage cells during the disease progression.

Abstract 17260: Endothelium-Targeted Nanoparticle Delivery of FOXM1 Reactivates Aging-Impaired Endothelial Regeneration for Vascular Repair and Inflammatory Lung Injury Resolution: Precision Gene Therapy of Acute Respiratory Distress Syndrome

Introduction: Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS). However, the underlying molecular mechanisms remain elusive and there is no effective therapy for ARDS. Hypothesis: Impaired endothelial regeneration and vascular repair in aged lungs causes persistent lung injury leading to high incidence of ARDS and high mortality in elderly patients following sepsis challenge. Methods: Genetic lineage tracing was employed to determine the cell origin of endothelial regeneration. Aged Foxm1Tg mice was used for assessing the role of FoxM1 in activating endothelial regeneration. Endothelium-targeted nanoparticles were employed to deliver FOXM1 gene selectively to endothelial cells (ECs) in aged mice. Methylation analysis was employed to determine how aging impairs FoxM1 expression. Results: Genetic lineage tracing study showed that endothelial regeneration was mediated by lung endothelial proliferation in adult mice whereas this intrinsic regenerative program was impaired in aged mice. Expression of FoxM1, an important mediator of endothelial regeneration in young adult mice, was not induced in lungs of aged mice due to aging-induced hypermethylation of the Foxm1 promoter. Transgenic FOXM1 expression or in vivo endothelium-targeted nanoparticle delivery of FOXM1 gene reactivated endothelial regeneration, normalized vascular repair and promoted survival of aged mice following sepsis. Furthermore, nanoparticle delivery of FoxM1 gene selectively in lung ECs also reactivated endothelial regeneration in aged mice, supporting the novel concept of precision gene therapy. In COVID-19 lung autopsy samples, FOXM1 expression was not induced in vascular ECs of elderly patients in contrast to mid-age patients, validating the clinical relevance of our findings. Conclusions: We have demonstrate that persistent lung injury and high mortality in aged mice following sepsis challenge is ascribed to impaired endothelial regeneration and vascular repair. Therapeutic reactivation of FoxM1-dependent endothelial regeneration and vascular repair represents a potential effective therapy for elderly ARDS patients by endothelium-targeted nanoparticle delivery of FOXM1 gene.

Control of murine brown adipocyte development by GATA6.

Brown adipose tissue (BAT) is a thermogenic organ that protects animals against hypothermia and obesity. BAT derives from the multipotent paraxial mesoderm; however, the identity of embryonic brown fat progenitor cells and regulators of adipogenic commitment are unclear. Here, we performed single-cell gene expression analyses of mesenchymal cells during mouse embryogenesis with a focus on BAT development. We identified cell populations associated with the development of BAT, including Dpp4+ cells that emerge at the onset of adipogenic commitment. Immunostaining and lineage-tracing studies show that Dpp4+ cells constitute the BAT fascia and contribute minorly as adipocyte progenitors. Additionally, we identified the transcription factor GATA6 as a marker of brown adipogenic progenitor cells. Deletion of Gata6 in the brown fat lineage resulted in a striking loss of BAT. Together, these results identify progenitor and transitional cells in the brown adipose lineage and define a crucial role for GATA6 in BAT development.

SAT268 Regulation Of Stem/Progenitor Cells Of The HPA Axis During Stress Adaptation

Abstract Disclosure: P. Siatra: None. D. Cozma: None. I. Berger: None. I. Oikonomakos: None. M. Theodoropoulou: None. S.R. Bornstein: None. C.L. Andoniadou: None. C. Steenblock: None. Chronic stress is a pervasive concern in the modern society. A long-term hyper activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to elevated amounts of stress hormones [e.g. ACTH and glucocorticoids (GCs)]. This can incur maladaptation that eventually contributes to mental illness, cardiovascular dysfunctions, diabetes, cancer and autoimmune diseases. Emerging key players for stress adaptation of the HPA axis are stem/progenitor cell populations. In this study, we aim to unravel the mechanisms involved in the regulation of stem/progenitor cells of the HPA axis during stress adaptation. To that aim, we incorporated an in vitro system of non-adherent spheroids from primary adrenocortical mouse cells that were stimulated with ACTH. The treatment resulted in the increased growth of the spheroids compared to unstimulated controls indicating a potential role of the hormone in cell proliferation and stem cell behaviour. This result is in line with the enlarged adrenals observed in patients with Cushing’s syndrome. To assess the contribution of stem cells during stress adaptation in the adrenal cortex in vivo, we performed immobilization stress and lineage tracing studies using the tamoxifen inducible Gli1:CreERT2/R26R:eYFP mouse line. By combining these results along with single-cell RNA-seq technology, we aim to shed light into molecular factors involved in the adult adrenal cortex regeneration and remodelling. To follow the effect of GC excesses on the negative feedback loop observed during a normal stress response on the pituitary gland, we stimulated adherent primary anterior pituitary colonies with dexamethasone and corticosterone. GC treatment led to downregulation of Pomc expression, a precursor polypeptide for ACTH and other hormones, in the treated colonies compared to the control. This downregulation caused by GCs could be mediated through different gene targets of the glucocorticoid receptor as well as via paracrine signalling from pituitary stem cells. Altogether, our results will provide us with more profound knowledge of mechanisms underlying interactions between stressors and stem/progenitor cells of the HPA axis along with novel therapeutic targets for stress-associated disorders. Presentation: Saturday, June 17, 2023

SAT272 Insulin Signaling In Hyperactivation Of The HPA Axis In Metabolic Diseases

Abstract Disclosure: D. Cozma: None. P. Siatra: None. I. Oikonomakos: None. D. Kalra: None. U.A. Friedrich: None. A. Dahl: None. S.R. Bornstein: None. S. Zeissig: None. C.L. Andoniadou: None. C. Steenblock: None. Stress can lead to an increase in body fat and obesity as main clinical conditions preceding the metabolic syndrome [1]. In metabolic stress, hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis has been observed, resulting in increased steroidogenesis and alteration of cortisol secretion [1]. This increased sensitivity of the HPA axis may be related to the development of comorbidities or severe illness in patients with metabolic diseases and vice versa. Disorders of the HPA axis, e.g., Cushing’s disease, resulting from overproduction of ACTH, share similar clinical symptoms with metabolic diseases [1]. The molecular signaling mechanisms that cause such changes in the HPA axis under metabolic stress remain elusive. Previous studies indicate a role of insulin in the regulation of adrenal steroidogenesis by upregulating the transcriptional activity of NR5A1 [2]. Here, we study the molecular mechanisms to insulin-induced hyperactivation of the HPA axis and characterize the molecular changes that lead to altered cortisol secretion. We generated non-adherent spheroids from primary adrenocortical cells in vitro and treated these with insulin. This resulted in an increase of spheroid diameter over time [Slope: 5.163 ± 0.994; P=0.0139; n=11], compared to control [Slope: -4.058 ± 0.987; P=0.0261; n=10], indicating greater cell expansion following insulin treatment. Similarly, repeating this experiment with adherent primary anterior pituitary stem cell colonies showed an increase in colony size when treated with insulin compared to control [Diameter: 1221.0 ± 290.9 µm vs. 914.5 ± 256.2 µm; P=0.0079; n=3]. This indicates that the stem cell populations of the adrenal and pituitary gland are responsive to insulin stimulation, likely leading to increased proliferation. This is complementary to our previous studies where we revealed increased secretion of aldosterone in insulin-stimulated adrenocortical cultures [3]. To identify signaling pathways related to insulin stimulation, we performed bulk RNA sequencing of in vitro primary cultures of adrenocortical and pituitary cells with and without insulin treatment. Analysis of these data sets reveals specific signaling pathways that are candidates in regulation of metabolic stress in vivo. Using mouse models of obesity and diabetes along with genetic lineage-tracing studies, we aim to decipher the role of these pathways on HPA axis stem/progenitor cells during metabolic stress. The outcomes will pave the way for a better understanding of HPA axis activity during metabolic stress adaption, and the role of the HPA axis in patients suffering from metabolic diseases.

Revealing the developmental origin and lineage predilection of neural progenitors within human bone marrow via single-cell analysis: implications for regenerative medicine

BackgroundHuman bone marrow stromal cells (BMSCs) are an easily accessible and expandable progenitor population with the capacity to generate neural cell types in addition to mesoderm. Lineage tracing studies in transgenic animals have indicated Nestin + BMSCs to be descended from the truncal neural crest. Single-cell analysis provides a means to identify the developmental origin and identity of human BMSC-derived neural progenitors when lineage tracing remains infeasible. This is a prerequisite towards translational application.MethodsWe attained transcriptomic profiles of embryonic long bone, adult human bone marrow, cultured BMSCs and BMSC-derived neurospheres. Integrated scRNAseq analysis was supplemented by characterization of cells during culture expansion and following provision of growth factors and signalling agonists to bias lineage.ResultsReconstructed pseudotime upon the integrated dataset indicated distinct neural and osteogenic differentiation trajectories. The starting state towards the neural differentiation trajectory consisted of Nestin + /MKI67 + BMSCs, which could also be diverted towards the osteogenic trajectory via a branch point. Nestin + /PDGFRA + BMSCs responded to neurosphere culture conditions to generate a subpopulation of cells with a neuronal phenotype according to marker expression and gene ontogeny analysis that occupied the end state along the neural differentiation trajectory. Reconstructed pseudotime also revealed an upregulation of BMP4 expression during culture of BMSC-neurospheres. This provided the rationale for culture supplementation with the BMP signalling agonist SB4, which directed progenitors to upregulate Pax6 and downregulate Nestin.ConclusionsThis study suggested BMSCs originating from truncal neural crest to be the source of cells within long bone marrow possessing neural differentiation potential. Unravelling the transcriptomic dynamics of BMSC-derived neural progenitors promises to enhance differentiation efficiency and safety towards clinical application in cell therapy and disease modelling.