Extracellular Matrix Functionalized Microcavities to Control Hematopoietic Stem and Progenitor Cell Fate

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk, a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability, clonogenic hematopoietic potential, and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells, together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel, the supportive cells self-assemble into a hypoxic stromal network, stimulating CD34+ CD38+ cell formation, while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks, in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly, the primary in vitro niche model supports HSPCs with no cytokine addition. Overall, the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches, in the context of normal hematopoiesis or blood-related diseases.

Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Hematopoiesis is the process that generates the cells of the blood and immune system from hematopoietic stem and progenitor cells (HSPCs) and represents the system with the most rapid cell turnover in a mammalian organism. HSPC differentiation trajectories, their underlying molecular mechanisms, and their dysfunctions in hematologic disorders are the focal research questions of experimental hematology. While HSPC transplantations in murine models are the traditional tool in this research field, recent advances in genome editing and next generation sequencing resulted in the development of many fundamentally new approaches for the analyses of mammalian hematopoiesis in situ and at single cell resolution. The current review will cover many recent developments in this field in murine models, from the bulk lineage tracing studies of HSPC differentiation to the barcoding of individual HSPCs with Cre-recombinase, Sleeping Beauty transposase, or CRISPR/Cas9 tools, to map hematopoietic cell fates, together with their transcriptional and epigenetic states. We also address studies of the clonal dynamics of human hematopoiesis, from the tracing of HSPC clonal behaviours based on viral integration sites in gene therapy patients to the recent analyses of unperturbed human hematopoiesis based on naturally accrued mutations in either nuclear or mitochondrial genomes. Such studies are revolutionizing our understanding of HSPC biology and hematopoiesis both under homeostatic conditions and in the response to various forms of physiological stress, reveal the mechanisms responsible for the decline of hematopoietic function with age, and in the future may advance the understanding and management of the diverse disorders of hematopoiesis.

Bone Marrow (BM) Delivery of Genetically-Modified (gm) Adult CD34+ Hematopoietic Stem and Progenitor Cells (HSPC) Improves Homing and Engraftment of Short-Term Progenitors over Long-Term Repopulating Hematopoietic Stem Cells

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.

INTRODUCTION: Hematopoietic stem and progenitor cell (HSPC) transfer during adoptive T cell immunotherapy (ACT) prolongs median survival and generates 30% long-term cures for malignant brain tumors. We recently demonstrated during ACT that intratumoral HSPCs differentiate into immune-activating dendritic cells (DCs) through T cell-released IFN-γ. We also determined that CCR2+ HSPCs are the key immune-activating progenitor population. We previously utilized syngeneic transfer of naive HSPCs that were not endemic to the recipient tumor-bearing host. This is different from the clinical paradigm of autologous transfers. This is important because in peripheral cancers tumor-bearing host (TB) HSPCs possess considerable immunosuppressive potential. It remains unknown if this is true in brain tumor-bearing hosts, and it remains unknown how this would impact HSPC+ACT immunotherapy. We therefore evaluated the immunologic function of naïve or TB HSPCs during immunotherapy. METHODS: We utilized KR158B and GL261 intracranial gliomas to generate TB HSPCs. HSPC culture experiments were performed in RPMI alone or conditioned with T cell supernatants to study HSPC differentiation. In vivo treatment models utilized intracranial gliomas and intravenous transfer of HSPCs with ACT. Data from the immgen.org database was utilized for hypothesis generation. RESULTS: Brain tumors promoted a 20% expansion of HSPCs including granulocyte monocyte precursors (GMP) but a loss of DC progenitors. The immgen database revealed that GMPs express high levels of IFN-γR1 while DC progenitors express high levels of IFN-γR2, CCR2, and CXCR4, the chemotactic receptor for HPSC homing. We next determined by flow cytometry that TB HSPCs express 40% more IFN-γR1 and 90% more IFN-γR2 on DC progenitors. To determine the differentiation preference of these cells, we performed in vitro culture. After a 3 day culture, naïve HSPCs differentiated into 30% myeloid-derived suppressor cells (MDSCs; CD11b+Ly-6G/6C+) while TB HSPCs differentiated into 60% MDSCs. When both cells types were cultured in activated tumor-specific T cell supernatants containing IFN-γ, both HSPC types differentiated into 30% MDSCs, 80% MHCII+ antigen-presenting cells, and 20% CD11c+MHCII+ DCs. In vivo, ACT rescued intratumoral TB HSPCs to prolong median survival and generate long-term cures. Additionally, TB HSPC-derived cells in brain tumors maintained higher IFN-γR2 and displayed non-inferior differentiation into DCs when compared naïve HSPCs. We are now investigating the impact of PD-1 blockade on TB HSPC function. CONCLUSIONS: Gliomas exert an immunosuppressive pressure on HSPCs. However, ACT can overcome dysregulated TB HSPC programming and promote generation of DCs instead of MDSCs. A phase I trial evaluating the impact of HSPC transfer on adoptive immunotherapy in pediatric high-grade gliomas is underway at our center (ACTION; NCT03334305). Citation Format: Tyler J. Wildes, Catherine T. Flores, Bayli DiVita Dean, Adam Grippin, Kyle Dyson, Duane A. Mitchell. Adoptive cellular therapy overcomes tumor-induced dysregulation of myelopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1194.

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that are primed to produce cDC1s and not through activation of cDC1 fate by other HSPCs. In particular, multi/oligo-potent clones selectively amplify their cDC1 output, without compromising the production of other lineages, via a process we term tuning. We then develop Divi-Seq to simultaneously profile the division history, surface phenotype and transcriptome of individual HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative 'early progenitor'-like state, leading to the selective expansion of multiple transitional cDC1-primed progenitor stages that are marked by Irf8 expression. These findings define the mechanistic action of Flt3L through clonal tuning, which has important implications for other models of 'emergency' haematopoiesis.

3020 – THE CLONAL AND MOLECULAR AETIOLOGY OF EMERGENCY DENDRITIC CELL DEVELOPMENT

Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells Using a Natural Product, Garcinol

IFN-γ Inhibitors and Ruxolitinib Therapy for Acquired Severe Aplastic Anemia in an Ex-Vivo Model

BCOR Loss Confers Increased Stemness and Partially Rescues RUNX1-Deficient Phenotypes in Human Hematopoietic Stem and Progenitor Cells

Reticular Dysgenesis-Associated Adenylate Kinase 2 Deficiency Impairs Purine Metabolism and Ribosomal Biogenesis during Myelopoiesis

Purpose: Adoptive T-cell immunotherapy (ACT) has emerged as a viable therapeutic for peripheral and central nervous system (CNS) tumors. In peripheral cancers, optimal efficacy of ACT is reliant on dendritic cells (DCs) in the tumor microenvironment. However, the CNS is largely devoid of resident migratory DCs to function as antigen-presenting cells during immunotherapy. Herein, we demonstrate that cellular interactions between adoptively transferred tumor-reactive T cells and bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) lead to the generation of potent intratumoral DCs within the CNS compartment.Experimental Design: We evaluated HSPC differentiation during ACT in vivo in glioma-bearing hosts and HSPC proliferation and differentiation in vitro using a T-cell coculture system. We utilized FACS, ELISAs, and gene expression profiling to study the phenotype and function of HSPC-derived cells ex vivo and in vivo To demonstrate the impact of HSPC differentiation and function on antitumor efficacy, we performed survival experiments.Results: Transfer of HSPCs with concomitant ACT led to the production of activated CD86+CD11c+MHCII+ cells consistent with DC phenotype and function within the brain tumor microenvironment. These intratumoral DCs largely supplanted abundant host myeloid-derived suppressor cells. We determined that during ACT, HSPC-derived cells in gliomas rely on T-cell-released IFNγ to differentiate into DCs, activate T cells, and reject intracranial tumors.Conclusions: Our data support the use of HSPCs as a novel cellular therapy. Although DC vaccines induce robust immune responses in the periphery, our data demonstrate that HSPC transfer uniquely generates intratumoral DCs that potentiate T-cell responses and promote glioma rejection in situClin Cancer Res; 24(16); 3955-66. ©2018 AACR.

Recent evidence suggests that the environment-sensing transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of hematopoiesis. Yet, the mechanisms and extent of AHR-mediated regulation within the most primitive hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), are poorly understood. Through a combination of transcriptomic and flow cytometric approaches, this study provides new insight into how the AHR influences hematopoietic stem and progenitor cells. Comparative analysis of intraphenotypic transcriptomes of hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells from AHR knockout (AHR KO) and wild type mice revealed significant differences in gene expression patterns. Notable among these were differences in expression of cell cycle regulators, specifically an enrichment of G2/M checkpoint genes when Ahr was absent. This included the regulator Aurora A kinase (Aurka, AurA). Analysis of AurA protein levels in HSPC subsets using flow cytometry, in combination with inducible AHR KO or in vivo AHR antagonism, showed that attenuation of AHR increased levels of AurA in HSCs and lineage-biased MPP cells. Overall, these data highlight a potential novel mechanism by which AHR controls HSC homeostasis and HSPC differentiation. These findings advance the understanding of how AHR influences and regulates primitive hematopoiesis.