Early Progenitor Cells Research Articles

The role of MCP-1 and SDF-1 in impaired mobilization of endothelial progenitor cells from the bone marrow in coronary heart disease

It is relevant to study of angiogenesis mediators and the mobilization of early endothelial progenitor cells (EPС) from bone marrow into the blood in patients with coronary heart disease (CHD), suffering and not suffering from ischemic cardiomyopathy (ICMP).СHD patients: 30 people with ICMP and 22 people without ICMP, 15 healthy donors. The content of early EPС (VEGFR2+CD34+CD14+) was determined in the blood and bone marrow by flow cytofluorometry, the concentration of MSP-1, SDF-1, VEGF-A – by multiplex analysis, of HIF-1α – by ELISA.Тhe content of SDF-1 and HIF-1α in peripheral blood in patients with CHD without cardiomyopathy was higher than in healthy individuals (respectively 60.00 (50.00-80.00) pg/mL and 6.00 (5.00-6.20) ng/mL versus 30.00 (5.00-45.00) pg/mL, p = 0.049 and 4.60 (3.28-5.11) ng/mL, p = 0.049), with ICMP corresponded to the norm. Тhe concentration of SDF-1 in the bone marrow was higher, and the level of HIF-1α was less than their content in the bloodstream, regardless of the presence of ICMP (respectively 130.0 (90.0-170.0) pg/mL, p = 0.005 and 0.97 (0.80-1.11) ng/mL, p < 0.001). The level of MCP-1 in the blood varied within the normal range in patients with CHD of both study groups (190.0 (168.0-215.0) pg/mL), and in the bone marrow was higher only in patients with ICMP (406.5 (265.0-583.0) pg/mL, p = 0.028). Regardless of ICMP presence, the content of VEGF-A in the blood of patients with CHD corresponded to the norm (3.80 (1.00-6.50) pg/mL) and in myeloid tissue. The number of EPC was increased in the blood of patients with CHD without cardiomyopathy (0.70 (0.46-1.23) and 0.19 (0.13-0.32) %, p < 0.001) and corresponded to their number in the bone marrow. And in patients with ICMP, normal values of the indicator were recorded in the blood with the accumulation of EPC in myeloid tissue (0.57 (0.45-0.98) %, p = 0.019).The development of ICMP is associated with the accumulation of early EPC in myeloid tissue due to their increased retention by an excess of MCP-1 in the bone marrow with weak involvement in the bloodstream due to the lack of a surplus of SDF-1 and HIF-1a in the blood.

Transcription factor-based transdifferentiation of human embryonic to trophoblast stem cells.

During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

Integrated transcriptomics of human blood vessels defines a spatially controlled niche for early mesenchymal progenitor cells

Human blood vessel walls show concentric layers, with the outermost tunica adventitia harboring mesenchymal progenitor cells. These progenitor cells maintain vessel homeostasis and provide a robust cell source for cell-based therapies. However, human adventitial stem cell niche has not been studied in detail. Here, using spatial and single-cell transcriptomics, we characterized the phenotype, potential, and microanatomic distribution of human perivascular progenitors. Initially, spatial transcriptomics identified heterogeneity between perivascular layers of arteries and veins and delineated the tunica adventitia into inner and outer layers. From this spatial atlas, we inferred a hierarchy of mesenchymal progenitors dictated by a more primitive cell with a high surface expression of CD201 (PROCR). When isolated from humans and mice, CD201Low expression typified a mesodermal committed subset with higher osteogenesis and less proliferation than CD201High cells, with a downstream effect on canonical Wnt signaling through DACT2. CD201Low cells also displayed high translational potential for bone tissue generation.

Abstract PO5-24-02: Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer

Abstract Background: A subpopulation of tumor cells known to have intrinsic resistance to therapies and contribute to metastasis function as “cancer stem”, or tumor-initiating, cells (TICs). TICs can be identified by various biomarkers, including fluorescent reporters for signaling pathways that regulate TIC function such as STAT signaling. However, standard TIC reporters have limitations. Their expression is unstable and there is no established method to follow TICs as they undergo cell state changes. Furthermore, we do not completely understand the transcriptional features of TICs. Improved methods of identifying TICs as well as the development of high-resolution transcriptional signatures are essential to understand TIC biology. An improved understanding of TIC biology can inform the development of novel therapeutics to selectively ablate the TIC compartment, thus improving response to standard cancer therapies. Methods: We screened a panel of PDX models to determine whether a STAT reporter identifies TICs in patient-derived xenograft (PDX) models. To augment existing STAT TIC reporters, we developed a two-component STAT signaling-specific lineage-tracing (LT) system. The first component labels active STAT signaling cells by expression of enhanced green fluorescent protein (EGFP+), followed by a self-cleaving P2A peptide and a TAM-inducible Cre-recombinase (4M67-EGFP-P2A-CreERT2). The second component is a constitutively expressed dual-color switching Cre-dependent reporter vector (EFS-loxPdsRedloxP-mNeptune2). We determined which LT cell populations (EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP−/mNeptune2+, and EGFP−/dsRed+) from SUM159 xenografts functioned as TICs. To probe TIC transcriptional features, we performed scRNA seq on the two tumor models with STAT TICs and a PDX model that did not have STAT TICs. We performed lineage trajectory analysis and differential gene expression analysis to characterize candidate TIC transcriptional signatures. To confirm whether these transcriptional features are associated with TICs, we identified a biomarker of this cell state (bone marrow stromal antigen 2 [BST2]) and explored the relationship between this biomarker and TICs. Results: We identified four PDX models with STAT reporter activity, one of which had STAT TICs. We validated our LT system in several models both in vitro and in vivo, then demonstrated EGFP+/mNeptune2+ from SUM159 xenografts are enriched for TICs and that lineage-tagged cells (mNeptune2+) function as proliferative early progenitor cells. scRNA seq of three xenograft models uncovered a distinct antiviral cell state in all three models that represent a more highly enriched TIC subpopulation. Critically, transcriptional features were shared between the antiviral cell states across xenograft models. To determine whether cells in this antiviral state represent TICs, we FACS-enriched BST2 cells (an IFN-stimulated cell surface protein that was a marker of the antiviral cell state) and demonstrated enrichment of the TIC population by mammosphere formation assays and limiting dilution transplantation. We demonstrated that BST2 expression is increased in residual PDX tumors following chemotherapy. Furthermore, we demonstrated similar antiviral cell states are present in human breast cancer scRNA seq datasets. Conclusions: These data demonstrate TICs adopt an antiviral cell state in some triple-negative breast cancers, and BST2 is a function marker of TICs in this cell state. Therefore, targeting genes associated with this cell state or associated antiviral pathways may represent a therapeutic vulnerability to eliminate TICs in some breast cancers. Citation Format: Eric Souto, Ping Gong, John Landua, Ram Srinivasan, Lacey Dobrolecki, Abhinaya Ganesan, Michael Lewis. Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-02.

A new atlas to study embryonic cell types in Xenopus

This paper introduces a single-cell atlas for pivotal developmental stages in Xenopus, encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessors, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropicalis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference.

Abstract 1772: A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified

Abstract Despite being the most common subtype of human peripheral T-cell lymphoma (PTCL), PTCL-not otherwise specified (PTCL-NOS) remains a poorly understood diagnosis of exclusion with poor survival times and treatment responses. Canine PTCL clinically and immunophenotypically resembles human PTCL-NOS, and PTCL is more common in dogs than humans, leading to their interest as a naturally occurring preclinical model. Canine models of cancer offer several unique advantages: in contrast to traditional rodent models, these are spontaneous tumors in genetically diverse and immunocompetent animals living in a shared environment with humans, but relative to their human counterparts, they offer accelerated lifespans with shorter clinical courses of disease. Here, we analyzed the gene expression profile of 96 canine CD4+ PTCLs to determine similarities to human PTCL-NOS and investigate a possible cell of origin. Bulk RNA-seq was performed on lymph node aspirates of 96 cases of canine CD4+ PTCL diagnosed by flow cytometry and sorted CD4+ nodal lymphocytes and CD4+ thymocytes from healthy control dogs. Raw reads were aligned to the Ensembl canine reference genome (ROS_Cfam_1.0) and tabulated. Data normalization and differential expression analysis was conducted with DESeq2, and differentially expressed genes were compared to human PTCL-NOS and various stages of T-cell development via gene set enrichment analyses (GSEA). Global gene expression in canine CD4+ PTCL was analogous to that of human PTCL-NOS (NES = 2.3, p < 0.001). Additionally, canine CD4+ PTCL had increased expression of GATA3 (log2fc = 1.9, padj = 6.5e-07) and was enriched for gene signatures associated with downregulation of PTEN (NES = 1.7, p = 0.0001) and upregulation of PI3K/AKT/mTOR signaling (NES = 1.5, padj = 0.0007), resembling the more aggressive human GATA3-PTCL subtype. Canine CD4+ PTCL was also enriched for human and murine gene signatures associated with early thymocyte progenitor cells and had increased expression of genes of immaturity, including CD34 (log2fc = 7.8, padj = 2.6e-14), KIT (log2fc = 3.3, padj = 7.7e-05), and CCR9 (log2fc = 4.8, padj = 3.9e-11), although surface CD34 expression is not detected by flow cytometry. These findings were confirmed when we compared the canine PTCL gene expression programs to a canine single cell transcriptomic atlas of hematopoietic precursors and T cells across canine bone marrow, thymic, and lymph node tissues. Canine CD4+ PTCL cells were significantly (padj < 0.05) enriched for gene signatures associated with early thymic and bone marrow precursors (NES 1.3-2.1), and negatively enriched for gene signatures associated with naïve nodal CD4+ T cells (NES = -3.2, padj = 0.006). In conclusion, the gene expression profile of canine CD4+ PTCL resembles human PTCL-NOS, supporting its potential as a naturally occurring preclinical model, and a subset of these neoplasms may arise from a thymic precursor cell of origin. Citation Format: Eileen Owens, Adam Harris, Anne Avery. A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified [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 1772.

The genome of the colonial hydroid Hydractinia reveals that their stem cells use a toolkit of evolutionarily shared genes with all animals.

Hydractinia is a colonial marine hydroid that shows remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, Hydractinia symbiolongicarpus and Hydractinia echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell-type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from nonself.

The Role of Angiogenesis Mediators in the Mobilization of Early and Late Endothelial Progenitor Cells from the Bone Marrow in Coronary Heart Disease

Background. A severe form of coronary heart disease (CHD) is ischemic cardiomyopathy (ICMP), the pathogenesis of which has not been fully studied. Disturbances in the mobilization of endothelial progenitor cells (EPC) due to an imbalance of angiogenesis mediators may exacerbate ischemia in ICMP. The aim — to establish the peculiarities of changes in the balance of early and late EPС and subpopulations of VEGFR2+ cells in the blood and bone marrow in relation to the content of angiogenesis mediators and the number of desquamated endothelial cells (DEK) in the blood of patients with CHD, suffering and not suffering from ICMP. Methods. A single-stage, clinical, controlled (case-control) study was conducted from March 2019 to June 2022. 52 patients with CHD who had a history of myocardial infarction were examined: 30 people suffering from ICMP and 22 people not suffering from ICMP, as well as 15 healthy donors. The content of VEGFR2+, VEGFR2+CD34+CD14+ (early EPC), VEGFR2+CD34+CD14– (late EPC), VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells in patients with CHD in the blood (before surgery) and bone marrow (sampling was performed at the beginning of coronary bypass) and in healthy individuals in the blood, as well as (in both groups) the content of CD45–CD146+ DEC in the blood was determined by flow cytometry. The concentration of SDF-1, VEGF-A, MCP-1, GM-CSF, G-CSF in blood plasma was measured by multiplex immunofluorescence analysis. Results. The development of CHD without cardiomyopathy was accompanied by an increase in the content of VEGFR2+CD34+CD14+ and VEGFR2+ cells (0.74 [0.46; 1.23]% and 10.00 [8.20; 11.60]%, respectively, versus 0.19 [0.13; 0.32]%, p 0.001 and 5.40 [4.30; 6.50]%, p = 0.005) and concentrations of SDF-1, MCP-1, GM-CSF (respectively 60.00 [50.00; 81.00] pg/ml; 223.0 [180.0; 297.0] pg/ml; 2.10 [1.45; 3.40] pg/ml versus 30.00 [5.00; 45.00] pg/ml, p = 0.041; 175.1 [140.0; 204.0] pg/ml, p = 0.046; 0.96 [0.46; 1.41] pg/ml, p = 0.038) in the blood relative to the norm. No such changes were observed in patients with ICMP. Regardless of the presence of ICMP, the content of VEGFR2+CD34+CD14–, VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells, VEGF-A, G-CSF in the blood of patients with CHD varied within physiological values, and the number of DEC exceeded the norm (7.26 [5.43; 17.94]×105/l, p = 0.039). The number of VEGFR2+ cells and their immunophenotypes in the bone marrow of patients with ICMP did not differ from the parameters in patients with CHD without cardiomyopathy. Prolonged bleeding from the venopuncture area was registered in one CHD patient without cardiomyopathy. Conclusion. The development of ICMP is associated with the absence of a compensatory response to atherogenesis in the form of increased mobilization of early EPC from the bone marrow due to the absence of a reaction associated with hyperproduction of SDF-1, MCP-1, GM-CSF, which is characteristic of CHD without cardiomyopathy. The content of EPС, VEGFR2+CD34–CD14+ and VEGFR2+CD34–CD14– cells, VEGF-A and G-CSF in the blood in СHD corresponds to physiological values, regardless of the presence of ICMP. The generation of EPC in the bone marrow in ICMP is not impaired.

Fusion transcripts are present in early progenitor cells in KMT2A-rearranged B-ALL

Fusion transcripts are present in early progenitor cells in KMT2A-rearranged B-ALL

Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.

Thrombopoietin treats erythropoietin resistance by correcting EPO-induced progenitorcell depletion

Recombinant human erythropoietin (rHuEPO) is a prevalent treatment for anemia in patients with chronic kidney disease. However, up to 10% of these patients exhibit EPO resistance or hyporesponsiveness, which may be caused by the depletion of erythroid progenitor cells. Thrombopoietin (TPO) has the potential to promote the growth of early progenitor cells and correct the depletion. In this study, we investigate the efficacy and the underlying mechanism of the combination therapy of TPO and EPO to EPO resistance. First, the in vivo studies suggested that intensive EPO treatment induced progenitor cell depletion in the bone marrow, where the depletion was corrected by TPO. Then, colony assays showed that EPO and TPO synergistically enhanced the burst-forming unit-erythroid (BFU-E) production but antagonistically boosted the colony-forming units of megakaryocytes (CFU-MK) production. Also, we found TPO promoted hematopoietic stem and progenitor cells (HSPCs) production, while EPO drove HSPCs toward the erythroid lineage. Additionally, EPO induced more megakaryocytic-erythroid progenitors (MEPs) toward the erythroid output. Model-based simulations indicate the efficacy of this combination therapy for treating EPO-resistant anemia in rats. In conclusion, our study demonstrated the efficacy of combination therapy in addressing EPO-resistant anemia by correcting EPO-induced erythroid progenitor depletion.

Generation of a Dcx-CreERT2 knock-in mouse for genetic manipulation of newborn neurons.

A wide variety of CreERT2 driver lines are available for genetic manipulation of adult-born neurons in the mouse brain. These tools have been instrumental in studying fate potential, migration, circuit integration, and morphology of the stem cells supporting lifelong neurogenesis. Despite a wealth of tools, genetic manipulation of adult-born neurons for circuit and behavioral studies has been limited by poor specificity of many driver lines targeting early progenitor cells and by the inaccessibility of lines selective for later stages of neuronal maturation. We sought to address these limitations by creating a new CreERT2 driver line targeted to the endogenous mouse doublecortin locus as a marker of fate-specified neuroblasts and immature neurons. Our new model places a T2A-CreERT2 cassette immediately downstream of the Dcx coding sequence on the X chromosome, allowing expression of both Dcx and CreERT2 proteins in the endogenous spatiotemporal pattern for this gene. We demonstrate that the new mouse line drives expression of a Cre-dependent reporter throughout the brain in neonatal mice and in known neurogenic niches of adult animals. The line has been deposited with the Jackson Laboratory and should provide an accessible tool for studies targeting fate-restricted neuronal precursors.

Combinatorial Optimization of Chemotherapeutic Agents on T-Cell Acute Lymphoblastic Leukemia

Introduction: Up to now, treatment options for T-cell acute lymphoblastic leukemia (T-ALL) remain quite limited. This study aims to assess the efficacy of monotherapies and combination of chemotherapeutic agents for T-ALL. Jurkat, CCRF-CEM are late T-cell progenitor (LTP) cell lines, while Loucy is early T-cell progenitor (ETP) cell line characterized by an immature phenotype. The drugs under evaluation include venetoclax, a selective BCL-2 inhibitor; cytarabine, a cytosine analog with established antineoplastic activity; bendamustine, an alkylating agent; and azacitidine, a DNA methyltransferase inhibitor. Given the notable tumor heterogeneity observed in T-ALL, the comprehensive assessment of these therapeutic agents on Jurkat, CCRF-CEM, and Loucy may render valuable insights for treatment strategies in T-ALL. Methods: Cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. Venetoclax, bendamustine, and azacitidine were prepared as stock solutions in DMSO, while cytarabine was prepared in distilled water. Monotherapies (venetoclax, cytarabine, bendamustine and azacitidine) and combination therapies (venetoclax plus cytarabine, venetoclax plus bendamustine and venetoclax plus azacitidine) were administered. A control group received DMSO or distilled water only. Cell viability was assessed after 48 hours by using Trypan blue and colorimetric method (MTS assay) on cells treated with various drug concentrations. Statistical analysis was conducted using the SynergyFinder Plus and drc R package, with significance set at p < 0.05. Results: Our study demonstrated that T-ALL cell lines displayed a different pattern of drug sensitivities according to combination of chemotherapeutic agents. Loucy showed greater sensitivity to venetoclax while Jurkat and CCRF-CEM exhibited higher sensitivity to cytarabine, bendamustine, and azacitidine. When conducting drug combinations on Jurkat and CCRF-CEM, the addition of venetoclax to cytarabine, bendamustine, or azacitidine resulted in an additive effect, with Loewe synergic scores ranging from -10 to 10 for each combination. On the other hand, Loucy demonstrated a distinct pattern of response; the combination of venetoclax with cytarabine showed additivity (Loewe synergic score: 8.45 and 5.82 with MTS and trypan blue assays, respectively), while venetoclax combined with bendamustine or azacitidine showed a synergistic effect (Loewe synergic score > 10 with MTS assay). Notably, among these combination treatments on Loucy, the pairing of venetoclax with bendamustine showed the most significant synergistic effects, with a Loewe score of 16.6. Conclusion: Venetoclax-based chemotherapeutic combinations exhibited various responses in T-ALL cell lines. Of note, the combination of venetoclax plus bendamustine showed potent synergistic effects in all T-ALL cell lines. These findings warrant further study to develop fundamentally curative therapeutic strategies in T-ALL.

Elucidation of the Unique Function of GATA1s in the Development of Transient Abnormal Myeloproliferative Disorders

Transient abnormal myeloproliferative disorder (TAM) is a pre-leukemic condition that occurs at birth in children with Down syndrome. The responsible gene of TAM, GATA1, is a hematopoietic transcription factor integral to hematopoiesis. The GATA1 gene has two translation start sites, giving rise to two protein variants: GATA1-fl, a full-length protein, and GATA1s, an isoform lacking the N-terminal domain. Notably, TAM blasts express only the GATA1s due to a mutation in the GATA1 gene, as opposed to normal cells which express both proteins. This implies that the precancerous state is not triggered by the production of an aberrant protein butrather by the absence of the full-length protein and the exclusive expression of the minor isoform protein also present in normal cells. This is in stark contrast with the functions of many other oncogenes. Thus, the prevailing view is that TAM development involves a “loss of function” due to deficiency of GATA1-fl. However, some studies suggest that GATA1s may have unique functions that actively drive the TAM phenotype. Because of these conflicting findings reported, the precise pathomechanism of TAM development remains elusive, despite the identification of the responsible gene. To address this, we analyzed the function of GATA1s using an induced pluripotent stem cell (iPSC) model of TAM. We previously examined the effect of increasing GATA1s expression levels during early hematopoietic differentiation stage by stage using the TAM-iPSC model. We found that, in the absence of GATA1-fl protein, overexpression of GATA1s in early hematopoietic progenitor cells (HPC) shifted megakaryocytic differentiation toward myeloid lineage, intensifying TAM severity by expanding progenitor cells. These findings suggest that GATA1s has a unique function distinct from that of GATA1-fl. In this study, we investigated the function of GATA1s proteins as transcription factors using chromatin immunoprecipitation sequencing (ChIP-seq). Our results demonstrated that genes involved in hematopoiesis or cell proliferation belong to the gene cluster to which overexpressed GATA1s (O/E GATA1s) binds solely in the absence of GATA1-fl at the early HPC stage. Gene Ontology (GO) analysis further validated the significant enrichment of terms relevant to the TAM phenotype, including regulation of myeloid cell differentiation and negative regulation of cell differentiation. Intriguingly, the genes to which O/E GATA1s binds when GATA1-fl is present were unrelated to hematopoiesis or cell proliferation. These observations suggest that GATA1s uniquely binds to genes, instigating TAM onset under GATA1-fl deficient conditions, thus confirming TAM as a disorder hallmarked by the absence of GATA1-fl due to the GATA1 gene mutation. Our RNA-sequencing data indicate that, at the early HPC stage, O/E GATA1s only induces changes in binding to gene regions involved in TAM pathogenesis, without altering gene expression levels. In contrast, gene expression analyses of other later stage HPC fractions (megakaryocyte-erythroid progenitor-like cells: MEP-like cells), revealed increased STAT3 expression and enrichment of the JAK-STAT pathway and myeloid cell differentiation pathway. These results suggest that TAM development may be influenced by shifts in the gene-binding region of GATA1s in the early HPC, which, in turn, induces TAM in more differentiated MEP-like cells by skewing their differentiation towards the myeloid lineage. In conclusion, our study provides important insights into the pathological mechanisms of TAM pathogenesis, particularly with respect to the role of GATA1 in driving the TAM phenotype. the unique function of GATA1s and their specific binding to genes involved in TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis. Understanding these mechanisms may pave the way for targeted therapies aimed at modulating GATA1s activity and reducing the severity of TAM.

Ras Activity Is Significantly Elevated in Patients with Primary Myelofibrosis and Is Agnostic to the Presence of RAS and JAK pathwaymutations

Myelofibrosis (MF) is a myeloproliferative stem cell disorder arising from various clonal mutations in early myeloid-committed hematopoietic progenitor cells. The subsequent acquisition of additional mutations and cytokine signaling then heralds the recruitment of fibroblasts to the marrow space, leading to collagen deposition. MF disease behavior varies widely and hematopoietic stem cell transplantation remains the only curative therapeutic option. In non-transplant candidates, managing cytopenic disease is uniquely challenging as patients develop resistance to commonly-utilized Jak-inhibitors. Expanding the molecular landscape of the disease beyond Jak/Stat signaling with the goal of identifying new therapeutic targets has, accordingly, been an area of growing interest in MF research. Recently-published studies, including results from our own institution, have identified mutations that potentially activate Ras signaling as being associated with features of advanced MF (higher somatic mutation burden) as well as with shorter overall survival and progression to AML. The goals of our study is to expand on this prior data, characterize the activity of Ras signaling and determine its role in patients with MF. Peripheral blood mononuclear cells (PBMCs) from 27 patients with myelofibrosis (MF) at a single institution underwent integrated genomic-transcriptomic sequencing analyses using the University of Michigan MiOncoSeq platform. PBMCs were separated by Ficoll® followed by cell lysis and protein quantification. Ras activity was measured by the binding of effector Ras binding domain (RBD) via ELISA. Patients with MF were compared to healthy donors and to patients with either polycythemia vera (PV) or essential thrombocythemia (ET). Ras activity in MF was examined across different mutations and with respect to various clinical parameters. Multivariate analyses were performed to assess the impact of each individual mutation on Ras activity. Accompanying statistical analyses for all assessments were conducted using two-way unpaired t-tests. Overall, Ras activity in 21 assessable patients with MF was 1.64 fold higher than in PV/ET (12 patients, p=0.0462) and 1.74 fold higher than healthy donors (13 volunteers, p=0.0304). While the majority of patients in the MF cohort had driver mutations, including in JAK2 (73%) and CALR (15.4%), the individual types of driver mutations did not impact Ras activity. Within the Ras/MAP Kinase signaling pathway, mutations were present at a range of variant allele frequencies (VAF), including 2 patients with NRAS (one with VAF 42%, one <5%), 2 KRAS (both <5%), 2 NF1, 1 CBL and 1 RAF1 (VAFs not assessed). The individual impact of various driver mutations, epigenetic regulators and splicing factors as assessed by multivariate analysis identified a trend toward enhanced Ras activity with mutations in RAS, CALR, ASXL1, TET2 and TP53, suggesting that in addition to RAS, other mutations also activate Ras signaling in MF. With regards to clinical parameters, Ras activity was weakly correlated with spleen size and high molecular risk mutation (HMR) presence, although this did not reach statistical significance in this cohort due to a relatively small sample size. Next, we performed a differential enrichment of myeloid and lymphoid cells from PBMCs using EasySep™ and measured Ras activity in lymphoid and myeloid enriched cells. We observed that the ratio of myeloid:lymphoid activity in patients with MF with RAS/MAP Kinase pathway mutations is 3.8 times higher than in patients without such mutations (p=0.0012), suggesting that RAS pathway mutations have a predominant role in myeloid cells. Our results show that the majority of patients with MF have hyperactive Ras signaling compared to PV/ET or healthy donors, regardless of the presence of alterations in the Ras signaling pathway. This underscores the importance of functional assessment of Ras activity in patients with MF and provides evidence that targeting Ras signaling in MF may lead to therapeutic benefit. We are currently expanding our analysis to a larger cohort of MF patients and performing preclinical testing of Ras/MAP Kinase pathway inhibitors as a means of overcoming Jak-inhibitor resistance.

A Two-Part Study Evaluating the Combination of Tazemetostat and CPX-351 (Part 1) and Palbociclib with CPX-351 (Part 2) for the Treatment of Relapsed or Refractory Acute Myeloid Leukemia

BACKGROUND AND SIGNIFICANCE Acute myeloid leukemia (AML) is an aggressive cancer of early hematopoietic progenitor cells with only 25-30% of patients (pts) surviving 5 years from diagnosis. For fit pts, conventional chemotherapy with DNA-damaging agents remains the mainstay of treatment. Conventional chemotherapeutic agents cause DNA-damage by targeting cells during DNA replication. Chromatin structure is highly influential in the efficacy of DNA-damaging agents, as areas of compacted chromatin are more resistant to DNA-damaging effects. Areas of DNA marked with histone 3 lysine 27 trimethylation (H3K27me3) are transcriptionally repressed and exhibit reduced accessibility to DNA-damaging agents. Enhancer of Zeste Homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repressive complex 2 which methylates H2K27 to produce a di- or trimethylated state (H3K27me2/3). In preclinical studies, treatment of AML cell lines with the combination of an EZH2 inhibitor (EZH2i) and doxorubicin resulted in an increase in DNA damage and apoptosis compared to those treated with doxorubicin alone. Further, a significant decrease in leukemia burden was seen in mice injected with THP-1 (AML) cells and treated with an EZH2i and doxorubicin and cytarabine compared to doxorubicin and cytarabine alone. This effect was further enhanced by pre-treatment of AML cells with palbociclib (PALB), a CDK4/6 inhibitor, by significantly increasing the number of cells in S phase when exposed to the EZH2i and doxorubicin combination, resulting in an increase in DNA damage and apoptosis compared to cells that were not pre-treated with PALB. These findings were reproduced in in vivo studies with mice injected with the primary AML cells (Porazzi P, et al. Cancer Res., 2021). STUDY DESIGN AND METHODS We will perform a 2-part phase I dose escalation study utilizing EZH2i tazemetostat (TAZ) in combination with CPX-351 induction chemotherapy and of PALB pre-treatment followed by CPX-351 in pts with relapsed or refractory (R/R) AML (NCT05627232). All pts will receive CPX-351 at standard dose on days 1, 3 and 5. Part 1 evaluate the addition of TAZ to CPX-351 via traditional 3+3 design. Pts will be treated with TAZ on days -1 to 6 at a starting dose of 600 mg twice daily to open chromatin concurrent with CPX-351 administration. Part 2 evaluate the pre-treatment with PALB via traditional 3+3 design. Pts will be treated with PALB starting dose of 100 mg daily on days -3 to -1 to induce cell cycle G1. PALB will be held on day 1 to allow cells to then enter S phase prior to administration of CPX-351. ( Figure 1). The study will enroll pts with R/R AML treated at Sidney Kimmel Cancer Center. Pts must be ≥18 years of age with R/R AML (non-acute promyelocytic leukemia) and have received at least 1 prior line of therapy and have adequate performance status and organ function. Pts whose participation would result in a total cumulative dose of daunorubicin greater than 550 mg/m 2 (450 mg/m 2 if they previously received mediastinal radiation) and those with active central nervous system involvement of AML, unstable cardiac disease or other severe concurrent disease will be excluded. The primary endpoints of Parts 1 and 2 will be to determine the maximum tolerated dose of TAZ and of PALB in combination with CPX-351, respectively. Secondary endpoints for Parts 1 and 2 include preliminary assessment of efficacy. Correlative studies to evaluate the on-target effect of TAZ on H3K27me3 and DNA-damage and of PALB on cell cycling will be performed at protocol-specified time points. If these two 2-drug combinations prove to be safe, we will amend the protocol to add Part 3 which will evaluate the safety of the 3-drug combination of pre-treatment with PALB followed by co-treatment with TAZ and CPX-351.

TNFα induces Caspase-3 activity in hematopoietic progenitor cells CD34+, CD33+, and CD41 + of myelodysplastic syndromes

BackgroundCytopenia is the primary feature of Myelodysplastic Syndrome, even in the presence of hypercellular bone marrow. TNFα is recognized as both a proinflammatory, and proapoptotic cytokine with a well established role in promoting apoptosis in MDS. Therefore, TNFα has the potential to be a valuable biomarker for predicting the progression of cytopenia in MDS. This study aims to establish the role of TNFα exposure in triggering apoptosis through caspase-3 activity in CD34+, CD33+, and CD41 + cells in MDS.MethodsThis study is an in vitro comparative experimental research. Bone marrow mononuclear cells were isolated as the source of hematopoietic progenitor cells. Subsequently, CD34+, CD33+, and CD41 + cells were exposed to rhTNFα, and the caspase-3 activity was measured using flowcytometry.ResultsIn MDS CD33 + and CD41 + caspase-3 activity of rhTNFα exposed cells was significantly higher than without exposed cells. The opposite result was found in CD34 + cells, where the caspase-3 activity without rhTNFα exposed cells was significantly higher than rhTNFα exposed cells.ConclusionrhTNFα exposure led to an elevation in caspase-3 activity in MDS progenitor cells, especially in those that had differentiated into myeloid cell CD33 + and megakaryocyte cell CD41+, as opposed to the early progenitor cells CD34+.

Human cytomegalovirus induces significant structural and functional changes in terminally differentiated human cortical neurons.

Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that can cause serious neurological deficits in infants experiencing an in utero infection. Also, as a life-long infection, HCMV has been associated with several diseases in the adult brain. HCMV is known to infect early neural progenitor cells, but whether it also infects terminally differentiated neurons is still debated. Here, we differentiated human-induced pluripotent stem cells into neurons for 84-120 days to test the ability of HCMV to infect terminally differentiated neurons and assess the downstream functional consequences. We discovered that mature human neurons are highly permissive to HCMV infection, exhibited late replication hallmarks, and produced infectious virus. Moreover, infection in terminally differentiated neurons essentially eliminated neuron function. These results demonstrate that terminally differentiated human neurons are permissive to HCMV infection, which can significantly alter both structural and functional features of this mature neuron population.

Glomerular filtration rate predicts pro-vascular progenitor cell depletion: insights from the IPE-PREVENTION and ORIGINS-RCE studies

Abstract Background People living with cardiometabolic disease and impaired renal function are at increased risk of experiencing adverse cardiovascular outcomes(1,2). Recent evidence indicates that individuals living with atherosclerotic cardiovascular disease (ASCVD) and type 2 diabetes (T2D) exhibit a reduction in circulating vascular regenerative (VR) cells that are essential for maintaining blood vessel homeostasis(3). However, there is limited data on whether VR cell content changes in response to cardiometabolic and kidney disorders. Purpose We sought to determine if there is an association between estimated glomerular filtration rate (eGFR) and VR cell content in individuals with ASCVD and/or diabetes. Data were from participants enrolled in the IPE-PREVENTION and ORIGINS-RCE studies. Methods IPE-PREVENTION participants had moderately elevated triglycerides (1.5 - 5.65 mmol/L) with established ASCVD and/or T2D. ORIGINS-RCE participants had established ASCVD or T2D and at least one additional cardiovascular risk factor. Circulating mononuclear cells were isolated from peripheral blood and characterized using side scatter properties (SSC) and high aldehyde dehydrogenase activity to quantify pro-angiogenic early myeloid progenitor cells (ALDHhi), and primitive versus lineage-specific cell surface marker co-expression. Participants were stratified according to those with an eGFR <60 mL/min/1.73m², or ≥60 mL/min/1.73m². Results Median [IQR] eGFR of the pooled cohort (n=169) was 75 [59, 88] mL/min/1.73m²; 45% had an eGFR that was <60 mL/min/1.73m². The stratum with eGFR <60 mL/min/1.73m² had fewer circulating ALDHhiSSClow progenitor cells (0.037 vs. 0.051%; P<0.01) (Figure 1A) and ALDHhiSSClow cells expressing CD34+ and CD133+ (49 vs. 54%; P=0.03) (Figure 1B) than that with an eGFR ≥60 mL/min/1.73m². The predictive value of eGFR on ALDHhiSSClowCD34+CD133+ frequency was conserved even after controlling for age, sex, LDL-C, BMI, and HbA1c (P<0.01) (Table 1). The frequency of ALDHhiSSCmid monocyte precursor cells did not differ based on eGFR status. Conclusions Low eGFR was predictive for the depletion of ALDHhiSSClow primitive progenitor cells that co-express the stem cell markers CD133 and CD34 in individuals with ASCVD and/or T2D suggesting that diminished kidney function is associated with a decrease in vessel reparative pro-angiogenic progenitor cell content. Strategies that improve circulating VR cell content in individuals living with kidney and cardiometabolic disease may provide clinically meaningful benefits.Figure 1Table 1

Core clock gene BMAL1 and RNA-binding protein MEX3A collaboratively regulate Lgr5 expression in intestinal crypt cells

The intestinal epithelium is highly regenerative. Rapidly proliferating LGR5+ crypt base columnar (CBC) cells are responsible for epithelial turnover needed to maintain intestinal homeostasis. Upon tissue damage, loss of LGR5+ CBCs can be compensated by activation of quiescent +4 intestinal stem cells (ISCs) or early progenitor cells to restore intestinal regeneration. LGR5+ CBC self-renewal and ISC conversion to LGR5+ cells are regulated by external signals originating from the ISC niche. In contrast, little is known about intrinsic regulatory mechanisms critical for maintenance of LGR5+ CBC homeostasis. We found that LGR5 expression in intestinal crypt cells is controlled by the circadian core clock gene BMAL1 and the BMAL1-regulated RNA-binding protein MEX3A. BMAL1 directly activated transcription of Mex3a. MEX3A in turn bound to and stabilized Lgr5 mRNA. Bmal1 depletion reduced Mex3a and Lgr5 expression and led to increased ferroptosis, which consequently decreased LGR5+ CBC numbers and increased the number of crypt cells expressing +4 ISC marker BMI1. Together, these findings reveal a BMAL1-centered intrinsic regulatory pathway that maintains LGR5 expression in the crypt cells and suggest a potential mechanism contributing to ISC homeostasis.