Human Bone Marrow Niche Research Articles

Combined Single-Cell and Spatial Transcriptomics Unveil the Complex Organization of the Non-Immune Human Bone Marrow Microenvironment during Aging

Hematopoietic stem cells (HSCs), reside in a specialized bone marrow (BM) microenvironment known as the hematopoietic stem niche. Despite the critical role of the niche in tightly controlling the processes of normal and malignant hematopoiesis, its cellular composition remains only partially understood. Recent studies, including our own, using single-cell RNA sequencing (scRNA-seq) approaches have provided valuable insights into the profiling of the human BM niche. However, a comprehensive effort to describe the cellular heterogeneity and regulatory circuitry of the aged BM microenvironment in elderly humans is still lacking. As individual age, multiple systems and organs experience a progressive loss of anatomical and physiological integrity. Aging of the HSC niche is accompanied by a reduction in the numbers and function of its constituents and a decrease in the levels of HSC-supporting factors. Whether niche aging can contribute to the defects observed in aged hematopoiesis, such as the clonal shift towards myelopoiesis, the decrease in immune surveillance, or age-associated metabolic diseases, remains unresolved. To address this, we utilized scRNA-seq and spatial transcriptomics to provide a detailed characterization of the molecular landscape and stromal interactions in the aged non-immune BM microenvironment. First, we performed scRNA-seq profiling on fluorescence-activated cell sorting-purified endothelial cells (ECs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD31 +/CD9 +) and mesenchymal stromal cells (MSCs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD271 +) from human BM samples of young (n=4) and elderly healthy donors (n=5). We analyzed a total of 1514 ECs and 3848 MSCs from older adults, grouped into 7 and 10 subclusters, respectively, defining distinct functional cell states. ECs and MSCs cells from young individuals were annotated using SingleR, utilizing the gene signature of each functional state in the elderly as a reference. Our results revealed significant shifts in the distribution of the functional states of BM niche cells during aging. Regarding ECs, we noticed reduced pathways associated with the cell cycle and RNA transcription pathways, indicating impaired cell cycle activity, coupled with decreased antioxidant defense (Figure 1A). In contrast, there was an increase in subgroups related to the response to foreign molecules, immune system activation, and vascular remodeling, suggesting an inflammatory response and increased vascular remodeling processes associated with aging. For MSCs, we observed a decline in osteogenesis and a reduction in the proportion of the early mesenchymal group related to immunity and the extracellular matrix (Figure 1B). These changes could contribute to the decreased ability of MSCs in elderly individuals to maintain HSCs function. On the contrary, we noticed increased adipogenic differentiation to the detriment of osteogenesis, which could be responsible for osteoporosis problems. Next, and to further explore the location and the direct interactions between niche cells, we are currently integrating spatially resolved transcriptomics with our scRNA-seq data to accurately comprehend the spatial distribution and interactions among the BM niche cells, using for the first time 10x Genomics Visium Spatial Gene Expression on bone BM tissue in humans. As a final validation step, we are using a mouse model to validate further the mechanisms of age-associated changes in the BM microenvironment. In summary, our results provide valuable insights into age-related transcriptional alterations in human BM ECs and MSCs, suggesting altered behavior of these niche cells during the aging of the hematopoietic system. This deeper understanding of the architecture of the aged hematopoietic system and its microenvironment offers the potential for developing novel therapeutic strategies preventing the detrimental effects of aging.

Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids to Model Hematopoietic Development

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life and inherited bone marrow failure syndromes are often linked to dysfunction of the niche. Recently, methods for generating induced pluripotent stem cell (iPSC)-derived hematopoietic organoids (Motazedian et al., 2020) and bone marrow organoids (Khan et al., 2022) have been developed. We recently presented our approach to generate a model of a complex human bone marrow microenvironment in vitro (ASH annual meeting 2022 ). Here, we characterize our model in greater detail and analyse functionality of bone marrow organoid (BMO)-derived HSPCs. We devised a feeder- and serum-free protocol to generate a complex human iPSC-derived BMO within three weeks. In comparison to the protocol by Khan et al., we incorporated a sequential combination of Wnt activation and Activin/Nodal inhibition to induce mesoderm formation and patterning. We replicated the method with five different iPSC lines, including fibroblast- and PBMC-derived iPSCs, as well as iPSCs generated from renal epithelial cells isolated from urine samples. Using confocal and two-photon microscopy we visualized the spatial architecture of differentiated BMOs and detected CD45 + cells embedded into a network of CD31 + vascular cells and CD271 + stromal cells. PDGFRβ expressing pericytes were enwrapping the vascular cells. Moreover, stromal cells expressed CXCL12, Leptin receptor and Nestin. Electron microscopy, HE staining of organoid sections and 3D surface rendering showed lumen-forming vascular structures containing hematopoietic cells. To further elucidate the cell type composition, we performed single-cell RNA sequencing (scRNA-seq) of differentiated BMOs. Clustering of the scRNA-seq data by marker gene expression yielded three main populations: Cells in the endothelial cluster expressed canonical genes of arterial ECs. In the mesenchymal cluster we could distinguish pericytes, vascular smooth muscle cells and osteochondrogenic precursors. In the hematopoietic compartment, we identified cells expressing molecular signatures of granulocytes, monocytes and their progenitors, but also megakaryocytic-like cells and lymphoid progenitors. Remarkably, a cluster of cells expressed genes of fetal HSCs. This suggests that BMOs may recapitulate definitive hematopoiesis. To test T-cell differentiation potential in vitro, we sorted CD34 + HSPCs from dissociated BMOs and differentiated them within an artificial thymic organoid system (ATO). After three weeks we detected CD45 +CD4 +CD8 + cells, some of them expressing CD3 +TCRαβ +. Thus, we postulate that T-cells phenotypically resembling conventional T-cells emerge from BMO-derived HSPCs. To assess functional properties of BMO-derived HSPCs in vivo, we transplanted sorted CD34 + HSPCs into immunodeficient NSG mice and are currently analysing their engraftment potential. In summary, we provide a novel bone marrow niche model to study hematopoietic development in a complex three-dimensional organoid derived from human iPSCs.

Generation of Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life. Three dimensional in vitro models to study spatio-temporal control of intrinsic and/or extrinsic factors orchestrating hematopoietic fate decisions in health and disease are needed. Here we present a method to generate a complex bone marrow-like organoid (BMO) from human induced pluripotent stem cells (iPSCs), consisting of hematopoietic, mesenchymal and vascular cells. Our approach expands current co-culture-based models of isolated primary endothelial and stromal cells, or bioengineered microfluidic chips and overcomes some of their intrinsic limitations. We devised a protocol of stepwise mesodermal progenitor differentiation through hemogenic endothelium to generate a complex heterocellular organoid: After embryoid body formation, mesoderm was induced and patterned by a sequential combination of Wnt activation, Nodal inhibition, BMP4, VEGF, bFGF and SCF. Embryoid bodies were embedded into a three-dimensional matrix and stimulated with defined cytokine cocktails in a timely fashion to generate hematopoietic progenitor cells while maintaining endothelial cell generation. At day 10 of differentiation, sprouted embryoid bodies were separated and transferred into a 96-well plate to promote full organoid maturation until day 17. Flow cytometric analysis of dissociated BMOs revealed three main cellular compartments, consisting of hematopoietic (CD45+), mesenchymal (CD45-CD31-CD34-CD271+) and endothelial cells (CD45-CD31+). Analysis of the cellular composition yielded an average content of~40,6% hematopoietic cells, ~39,5% mesenchymal cells, ~6% endothelial cells, ~1,6% HSPCs and ~0,9% MSCs per BMO. Single-cell RNA sequencing of dissociated BMOs confirmed the presence of these distinct cell clusters. Sorted hematopoietic (CD45+CD11b-CD34+-HSPCs) and mesenchymal (CD45-CD31-CD271+CD105+CD90+CD73+-MSCs) stem and progenitor cells showed multilineage differentiation potential in colony-forming-unit (CFU) and trilineage-differentiation assays, respectively. Confocal imaging of whole organoids showed that CD31+ endothelial cells formed a vessel-like network covered by a Collagen IV+ basement membrane and PDGFR-β+ murals cells, resembling pericytes. Ultrastructural analysis by transmission electron microscopy (TEM) revealed capillary-like structures of endothelial cells enclosed by pericytes, which were connected by tight junctions. Importantly, we also detected CXCL12+- CAR-like cells in close association to endothelial cells in our BMOs. Whereas CD45 expressing cells were distributed throughout the organoid, Runx1 expressing cells were found to be localized in distinct clusters. We examined whether the BMO niche promotes maturation of blood progenitors into mature cells of the myeloid lineage. We identified neutrophil progenitor stages from ProNeu to matureNeus by flow cytometry, S100A8/A9 and MPO expressing cells by confocal imaging and cells with characteristic features of neutrophil granulocytes by TEM, showing that granulopoiesis proceeds without the addition of lineage-instructing cytokines as G-CSF or GM-CSF. Next, we tested whether BMOs can be used as a model system to recapitulate the phenotype of monogenic bone marrow failure syndromes. BMOs from iPSCs edited to express a hypomorphic variant of VPS45 showed increased formation of reticulin fibers and a higher abundance of SMA expressing pericytes, reminiscent of increased myelofibrosis in bone marrow biopsies of VPS45 deficient patients. We also started to show that BMOs can be transplanted into immunodeficient NSG-mice with further BMO growth in vivo for up to four months. In ongoing experiments we analyze the contribution of human hematopoietic cells to blood cell chimerism in the host mice. In summary, iPSC-derived BMOs offer a new tool to study hematopoietic development and disease evolution within a complex three-dimensional human environment exhibiting key cellular and structural features of the human bone marrow niche.

Alterations of Human Bone Marrow Niche in Clonal Hematopoiesis and AML

Acute myeloid leukemia (AML) is a heterogeneous disease, predominantly caused by acquisition of mutations in the hematopoietic stem and progenitor cells. Some of these mutations are acquired in a preleukemic state termed clonal hematopoiesis of indeterminate potential (CHIP). Recent studies show that alterations in the bone marrow niche can support progression and therapy resistance of myeloid neoplasia. In our study, we aim to elucidate alterations in the bone marrow niche, in particular in mesenchymal stem cells (MSC), during the evolution from healthy to CHIP and further to AML. We collected primary human bone marrow (BM) and peripheral blood (pB) samples from hematologically healthy individuals who underwent hip replacement surgery at the LMU University Hospital as well from AML patients treated at our institution (LMU University Hospital Munich). Individuals were assigned to non-CHIP or CHIP cohorts based on targeted mutation analyses on pB samples. So far, we have included 105 healthy individuals and 92 have undergone targeted sequencing, revealing 27 individuals (29%) with CHIP. While there was no difference between the gender of the non-CHIP and CHIP groups, individuals with CHIP tended to be of older age (median, 64 y non-CHIP vs. 73.5 y CHIP). We detected DTA gene mutations as the most common CHIP alteration and 1.4 mutations per individual were found on average. Variant allele frequencies varied between 1% and 17%. The amount of mutations per individual increased with age. In the non-CHIP and CHIP individuals, we analyzed various blood parameters such as hemoglobin, leukocyte counts, mean corpuscular volume and others, as well as various comorbidities such as myocardial infarction, hypertesia, previously treated cancer, etc. We found a trend for more frequent myocardial infarction in the CHIP individuals. We did not find any significant differences between CHIP and non-CHIP for the blood parameters. MSC from donor BM samples were isolated via plastic adherence over 10-14 days of in vitro culture. MSC were characterized regarding their surface marker expression (FACS), differentiation capacity (osteogenic, adipogenic, chondrogenic) and metabolic activity (Seahorse XF). Analysis of MSCs from non-CHIP (n=5) and CHIP (n=5) individuals revealed no difference in surface marker expression over 10 passages. Also, differentiation assays of MSCs (non-CHIP=4, CHIP= 2) showed no differences in adipogenic and chondrogenic differentiation after 21 days. For osteogenesis, we noticed a trend towards an increased differentiation potential in the MSCs of CHIP individuals. This experiment is ongoing with the goal to increase the sample number up to 10 per condition, as well as including the AML controls. The data will be shown at the ASH meeting. To gain deeper insights into the BM niche we sorted 4 non-hematopoietic and 6 hematopoietic cell populations from non-CHIP (n=3), CHIP (n=4) and AML (n=4) individuals and performed bulk-RNA sequencing on each population. So far, changes in the composition of the bone marrow niche were only detected in AML samples. Currently, in a series of pilot experiments, we established primary MSC and HSPC cross co-cultures from non-CHIP, CHIP and AML samples. A preliminary experiment with one non-CHIP and one CHIP sample showed that both non-CHIP and CHIP HSC produced less hematopoietic cells and less colony forming units (CFUs) after co-culturing with CHIP MSCs, than with non-CHIP MSCs. The results of the main experiment with 3 individuals per condition and including the AML samples will be shown at the conference.

Synergistic Immunostimulatory Effects and Therapeutic Benefit of Combined T Cell Engager and Cytotoxic Therapy in a Multiple Myeloma Preclinical Model

Introduction B-cell maturation antigen (TNFRSF17; BCMA) targeted immunotherapy has revolutionized treatment options for multiple myeloma (MM) patients. BCMA/CD3-targeting T cell engagers (TCE) are a recently developed immunotherapy that shows potent tumor killing activity in MM. While still under intense clinical development, TCE alone are not curative, and therapy relapse remains a clinical challenge. Our well-established, immunocompetent Vk*MYC mouse model provides an ideal controllable platform to model the full heterogeneity of the human bone marrow niche, identify mechanisms of relapse after TCE treatment, and test efficacy and toxicity of novel immunotherapy combinations. We recently published preclinical investigations using a murine BCMA/CD3-targeting TCE in the Vk*MYC model. Here, the TCE was safe and efficacious in most mice but failed in those with high tumor burden, consistent with clinical reports of TCE in leukemia. Our attempts to use pomalidomide with TCE, ultimately led to T cell exhaustion that compromised disease control. Alternatively, we reasoned that combination of TCE with a more effective cytotoxic therapy could debulk the tumor and help maintain immune function long-term. We found that a combination with cyclophosphamide was safe and allowed for a tempered proinflammatory response associated with long-lasting complete remission. Now we provide single cell RNA-seq data describing in granular detail the cellular flux underlying the preclinical success of TCE and cyclophosphamide combination. Methods We treated tumor bearing mice with BCMA/CD3 TCE or a negative control antibody alone, or with cyclophosphamide. We performed single cell 5’ RNA-sequencing (scRNAseq) of splenic tissue containing tumor at the peak of immunotherapy response. scRNA-seq is a powerful tool for examining complete gene expression profiles at an unprecedented level of granularity. We examined CellRanger-generated count matrices with the Seurat R package to generate subsets of homogeneous cells according to nearest neighbor clustering of principal components, then visualized with uniform manifold approximation and projection (UMAP). We derived cell clusters based on expression data as well as the correlation structure between a large set of custom reference samples derived from the ARCHS4 repository of over 500,000 mouse RNA samples. This less-biased method informs us about definitive, and sometimes lesser-known, immune typologies in our high dimensional analysis of microenvironmental remodeling. We tested for differential gene expression between cell clusters using Wilcoxon Rank Sum tests. We pair these scRNA-seq data with flow cytometry and immunohistochemistry. Results To characterize the features of the T cell subsets that contribute to the noted complete remission with TCE cyclophosphamide combination, we focused on the nine clusters of T cell subsets. Two clusters of T cells were enriched after cyclophosphamide lymphodepletion regardless of whether they were treated with TCE or TCE control: (i) a naïve CD8+ T cell cluster of stem-like cytotoxic cells expressing genes of recent thymic emigrants, and (ii) CD4+ T follicular helper cells. These cells help with germinal centers and memory, which may promote the protection and curative response we observed. Importantly regulatory T cells, as a ratio with total T cells, were reduced in mice treated with cyclophosphamide, TCE, and the combination compared to controls. Finally, we defined signatures of the T cell types activated by and proliferating in response to TCE and cyclophosphamide combination: (i) highly proliferative CD8+ T cells, and (ii) tumor infiltrating CD8+ T cells with high expression of cytotoxicity genes, cell migration genes, and genes associated with recent thymic emigrants. These data suggest that the combination therapy leads to an influx of supportive and highly functional T cell subsets that are optimal for redirection with TCE. Additional comparisons will be presented at the meeting. Conclusions We discovered a curative combination of BCMA/CD3 TCE with cyclophosphamide in the Vk*MYC mouse model. These data suggest that the unique ability of cyclophosphamide in combination with TCE to reshape the tumor microenvironment by depleting suppressive and exhausted T cells while replenishing with supportive and cytotoxic stem-like T cell subsets to provide a tempered and sustainable anti-MM therapy.

Targeting CD38 in acute myeloid leukemia interferes with leukemia trafficking and induces phagocytosis

Targeting the interaction between leukemic cells and the microenvironment is an appealing approach to enhance the therapeutic efficacy in acute myeloid leukemia (AML). AML infiltration induces a significant release of inflammatory cytokines in the human bone marrow niche which accelerates leukemogenesis. As the transmembrane glycoprotein CD38 has been shown to regulate cytokine release, we assessed the anti-leukemic potential of CD38 inhibition in AML. CD38 expression in AML cells proved to depend on microenvironmental cues and could be significantly enforced through addition of tretinoin. In fact, the anti-CD38 antibody daratumumab showed significant cytostatic efficacy in a 3D in vitro triple-culture model of AML, but with modest cell-autonomous cytotoxic activity and independent of CD38 expression level. In line with a predominantly microenvironment-mediated activity of daratumumab in AML, CD38 inhibition significantly induced antibody-dependent phagocytosis and showed interference with AML cell trafficking in vivo in a xenograft transplantation model, but overall lacked robust anti-leukemic effects.

Latest culture techniques: cracking the secrets of bone marrow to mass-produce erythrocytes and platelets ex vivo.

Since the dawn of medicine, scientists have carefully observed, modeled and interpreted the human body to improve healthcare. At the beginning there were drawings and paintings, now there is three-dimensional modeling. Moving from two-dimensional cultures and towards complex and relevant biomaterials, tissue-engineering approaches have been developed in order to create three-dimensional functional mimics of native organs. The bone marrow represents a challenging organ to reproduce because of its structure and composition that confer it unique biochemical and mechanical features to control hematopoiesis. Reproducing the human bone marrow niche is instrumental to answer the growing demand for human erythrocytes and platelets for fundamental studies and clinical applications in transfusion medicine. In this review, we discuss the latest culture techniques and technological approaches to obtain functional platelets and erythrocytes ex vivo. This is a rapidly evolving field that will define the future of targeted therapies for thrombocytopenia and anemia, but also a long-term promise for new approaches to the understanding and cure of hematologic diseases.

Development and Exploitation of a Fully Human and Modular Organotypic Bone Marrow Niche Model to Study the Role of Stroma-Produced Factors in Human MDS

Background: Myelodysplastic syndromes (MDS) are a heterogenous group of stem cell driven disorders primarily affecting the elderly and characterized by inefficient production of mature blood cells and a high risk (30%) of evolution to secondary acute myeloid leukemia. Despite tremendous progress in the past decade, treatment options for MDS patients remain limited, and primarily address disease symptoms, rather than altering disease course. This points to the urgent need to better understand the pathogenesis of this heterogenous group of syndromes to develop new therapies that address disease vulnerabilities. However, this effort has been largely hampered by the limited availability of model systems that allow the exploration of MDS biology in a fully humanized setting. In recent years, studies from our lab and others, have highlighted the crucial role niche cells play in human MDS, hence reinforcing the notion that MDS is a disease of a tissue rather than hematopoietic cells alone. Therefore, exploration of MDS biology requires the further development of fully human MDS models in which both constituents of the disease, namely hematopoietic and niche cells, are present. Methods: To address this issue we successfully isolated endothelial cells (ECs) and mesenchymal stromal cells (MSC) from bone marrow biopsies obtained from MDS patients or healthy age matched controls, and subsequently utilized them to develop fully human 2D and 3D organotypic niche models, which were successfully used to support normal and MDS HSPCs expansion ex-vivo. The 3D system makes use of a collagen scaffold, as this protein makes up for 90% of the matrix proteins in the bone. Importantly, MSC and EC cultures could be successfully established from several independent donors and immortalized to generate primary cell lines that can be used to reproducibly establish these ex-vivo systems in a robust manner. Moreover, we could show that these niche cells were easily amenable to genetic editing using CRISPR-Cas9 technology as well as modified to carry fluorescent reporter proteins for tracking cellular interactions using live cell imaging and confocal microscopy. Results: In this work, we successfully isolated human mesenchymal and endothelial cells, from primary bone marrow biopsies (MDS and healthy) and established fully human 2D and 3D organotypic co-cultures ex-vivo. Of note, although bone marrow ECs represent an essential component of the hematopoietic niche, they have so far been omitted in previously described human bone marrow niche models, owing to the notorious difficulties in isolating and expanding this cell type from primary bone marrow biopsies. Therefore, we established immortalized EC lines (iECs) that faithfully recapitulate the morphological, phenotypic and functional features of primary bone marrow ECs. When cultured at defined ratios and under defined conditions, MSCs instructed ECs and iECs to form of vessel-like structures that mimic the meshwork observed in vivo and are typically escheated by aSMA positive cells that stabilize the structures. Genetic manipulation of the cellular components of the niche also allowed to explore the functional relevance of a specific ECM protein, which we previously identified to be significantly upregulated in MSCs isolated from MDS patients, namely the Secreted Protein Acidic and Rich in Cysteine (SPARC). SPARC ablation triggered enhanced proliferation of MDS derived HSPCs and sensitized them treatment with 5-Azacytidine, a standard of care hypomethylating agent used for the treatment of MDS patients. Additional studies are underway to further understand the underlying molecular mechanisms and define a potential druggable target that could sensitize MDS cells to standard of care treatment. Besides gene targeting studies, these organotypic models are also being used to evaluate the relative fitness of MDS and healthy stem/progenitor cells in healthy versus patient derived niches, to explore the contribution of niche components to the establishment of the progressive clonal dominance observed in MDS. Disclosures Bönig: Terumo BCT: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kiadis: Honoraria; Bayer: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fresenius: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Uniqure: Research Funding; medac: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Healthineers: Current equity holder in publicly-traded company; Chugai: Honoraria, Research Funding; Erydel: Research Funding; Miltenyi: Honoraria, Research Funding; Polyphor: Research Funding; Sandor-Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stage: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Platzbecker:Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Götze:Celgene: Research Funding. Medyouf:Bergenbio: Consultancy, Research Funding.

Acute myeloid leukemia–induced remodeling of the human bone marrow niche predicts clinical outcome

AbstractMurine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However, little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here, we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly, their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact–dependent manner through inhibition of cytoplasmic β-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30% lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore, patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7% whereas patients with higher osteocalcin levels reached a survival rate of 66.8%. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients.

Abstract B24: A multicellular, microfluidic model of the human bone marrow niche in metastatic cancers

Abstract Bone metastases cause significant morbidity in solid tumors, with prostate, breast, lung, and kidney cancers representing the most common primary sources. Understanding the landscape of stromal interactions in bone and bone marrow would facilitate development of novel therapies targeting this unique metastatic niche. Mesenchymal stromal cells (MSCs) are multipotent cells in bone marrow that differentiate into osteoblasts, adipocytes, and fibroblasts and regulate immune and inflammatory responses. MSC recruitment into primary tumors enhances invasiveness of prostate adenocarcinoma, which suggests that bone marrow MSCs impact development of bone metastases in prostate cancer and promote treatment resistance. We hypothesize that multicellular interactions drive a permissive bone marrow environment for outgrowth of metastatic lesions. The goal of this study is to develop a multicellular bone stromal model from patients with solid tumors for functional and molecular analysis of cellular crosstalk. We have recently published a reconfigurable, microfluidic platform that enables culture of up to 6 different cell types in 3-dimensional matrices and multi-endpoint analysis of limited patient samples. In this study, we have modified this platform to culture patient-derived tissue, by using primary bone marrow cells from patients consented to provide bone marrow aspirate at time of radical prostatectomy. This technology allows spatiotemporal control of individual treatment conditions, enabling culture and differentiation of primary human cells in separate chips that can be reconfigured into defined microenvironments. Subsequently, specific compartments can be isolated for gene expression analysis and in-device imaging. Using this platform, we present two distinct bone marrow models. In the first model, we cultured primary human bone marrow MSCs that were induced into osteoblastic and adipocytic lineages in 3D microculture. MSCs induced invasion of prostate cancer cell lines, and paracrine crosstalk between MSCs and prostate cancer cells increased expression of IL-6, IL-8, CXCL12, and MMP-9 in MSCs. In the second model, we generated a multicellular mixed 3D culture in which osteoblasts and adipocytes spontaneously differentiate in coculture with stromal cells, osteoclasts, and leukocytes. The mixed 3D cultures generated spheroid structures that resemble stem cell components with self-renewal capability, previously called mesenspheres. Overall, we have recapitulated cellular compartments in the bone marrow niche using two complementary methods. The impact of defined molecular subtypes of cancer is now being tested to understand if specific genetic alterations promote development of invasive cancer in bone marrow. Future directions include analysis of paracrine signaling and functional interactions between disseminated cancer cells and bone marrow cellular stroma as potential therapeutic targets. Citation Format: Nan Sethakorn, David Kosoff, Erika Heninger, Jasmine Martinez Soto, Jiaquan Yu, Rahul Das, David Jarrard, Jacques Galipeau, Peiman Hematti, Scott Dehm, David J. Beebe, Joshua M. Lang. A multicellular, microfluidic model of the human bone marrow niche in metastatic cancers [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B24.

Induction of a Leukemic Bone Marrow Niche Mediated By TGFB1 of Leukemic Blasts in Pediatric Acute Mekaryoblastic Leukemia: Results of an in Vitro and In Vivo Analysis

Background: An increasing knowledge about the bone marrow niche demonstrates the high complexity of leukemogenesis. Mesenchymal stromal cells (MSC) are important members of the bone marrow niche and source of fibrosis. Further, the microenvironment seems to be regulated by megakaryocytes and platelets via cytokines, such as transforming growth factor beta 1 (TGFB1). Despite extensive research, the pathogenesis of the bone marrow niche in childhood leukemia and the therapeutic potential is still unclear. We focus on acute childhood megakaryoblastic leukemia (AMKL) as a disease model and include patients with (ML-DS) and without Down syndrome. Based on similar clinical progressions - myelofibrosis occurs as a side-effect of both leukemia subtypes; these two diseases suit to characterize the leukemic bone marrow niche. Methods: We performed a comprehensive characterisation of MSC from ML-DS (n=9), AMKL patients (n=5) and healthy donors (HD; n=6) via e.g. differentiation assays (adipogenic, osteogenic), gene expression profiles and western blot analysis. In addition, we established an in vivo model with humanized ossicles, representing a human bone marrow microenvironment (as described by Chen et al. 2012; Reinisch et al. 2015): We injected MSC mixed with pooled human umbilical vein endothelial cells (HUVEC) and Matrigel subcutaneously into NOD scid gamma (NSG) mice. After 8 weeks, the engrafted ossicles were injected with megakaryoblastic cells (CMK cell line); injected ossicles (n=16); uninjected ossicle (n=27), MSC from ML-DS (n=19 ossicles), AML M1 (n=15 ossicles) and HD (n=9 ossicles). After 4 more weeks, histopathology evaluation of fibrosis in the ossicles was performed in accordance with the European Consensus on Grading Bone Marrow criteria from an independent pathologist. Results: The detailed characterisation of MSC with ML-DS and AMKL demonstrated a high similarity to MSC of HD: morphology, osteogenic differentiation potential, colony forming unit-fibroblast assay, proliferation and gene expression profiles. However, two differences emerged in our analysis: MSC showed a decreased adipogenic differentiation potential in ML-DS and AMKL compared to HD (ML-DS vs. HD=0.26-fold, p<0.05; AMKL vs. HD=0.50-fold). Gene expression profiling identified an upregulation of IGF2BP3, an oncofetal RNA binding protein, in MSC of ML-DS compared to HD confirmed by qRT-PCR (2.6-fold, p<0.05). IGF2BP3 is known to be highly expressed in many cancers and seems to be associated with proliferation. The increased level of IGF2BP3 (protein: IF2B3) was confirmed at protein-level detected by western blot analysis (ML-DS vs HD: 37.3-fold, p<0.05 and AMKL-MSC vs HD: 13.1-fold, p<0.05). TGFB1 - known to be secreted by leukemic megakaryoblasts - induced a fibrotic state in MSC regardless of their origin indicated by decreased adipogenic differentiation potential (treated vs. untreated: ML-DS 0.22-fold; AMKL 0.08-fold; HD 0.06-fold, p<0.05) and increased expression of collagen genes (qRT-PCR; COL1A1: ML-DS=1.63-fold, AMKL=1.80-fold (p<0.01), HD=1.66-fold (p<0.05); COL3A1: ML-DS=1.31-fold, AMKL=1.52-fold (p<0.05), HD=1.24-fold). The humanized bone marrow niche in our mouse model demonstrated a development of myelofibrosis after injection of the megakaryoblastic cell line (CMK): Grade 1 or 2 in 81% of the ossicles. The induction was independent of the MSC entity (HD/ML-DS). Of note, a monocytic subpopulation, which engrafted unexpectedly in ossicle from HD-MSC (n=3 ossicle), did not induce fibrotic fibers. Conclusion: Our data impressively illustrate the mutual influence between MSC and leukemic blasts that leads to a fibrotic microenvironment. This correlation has been observed in vitro but also in a unique mouse model. The interaction of MSC and leukemic blasts seems to be the key factor for the development of the leukemic niche in AMKL mediated inter alia by the TGFB pathway. However, we could identify several disease specific characteristics of MSC. Our model offers a unique opportunity to fundamentally examine of the leukemic niche in order to subsequently evaluate the potential therapeutic use in further studies. Disclosures Reinhardt: Novartis: Other: Participation in Advisory Boards; CSL Behring: Research Funding; Jazz: Other: Participation in Advisory Boards, Research Funding; Roche: Research Funding.

Alterations of Bone Marrow Microenvironment in Acute Graft-Versus-Host Disease

Severe acute graft-versus-host disease (aGVHD) indicates a poor prognosis after allogeneic hematopoietic stem cell transplantation. In our previous study, we found that hematopoiesis was progressively suppressed during aGVHD, while the hematopoietic regenerative potential of donor-derived hematopoietic stem cells remains intact. It prompts us to investigate whether bone marrow niche is a major target of GVHD. We addressed this issue by studying the critical components in bone marrow microenvironment, including mesenchymal stem cell (MSC), osteoblasts and adipocytes in haplo-MHC-matched murine bone marrow transplantation model. By comparing confocal images of femurs from control and aGVHD SCF-GFP mice or Col2.3-GFP mice, we found that both MSCs and osteoblasts were significantly reduced during aGVHD development. In addition, anti-perilipin staining showed that adipocytes were also decreased in aGVHD mice. We found a defect in the differentiation potential of MSCs from aGVHD niche by in vitro culture of both murine and human bone marrow niche cells. qRT-PCR showed decreased gene expressions of PPAR-gamma, Adipoq, Runx1 and Col2a1, suggesting the potential of MSCs differentiation into adipocytes and osteoblasts was blocked. These data provide new insights into the pathogenesis of aGVHD and may improve the clinical management of aGVHD. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia Induced Remodelling of the Human Bone Marrow Niche Predicts Clinical Outcome

The hematopoietic stem cell (HSC) niche consists of different cellular and non-cellular constituents which regulate HSC maintenance and retention in the bone marrow. It has been shown in a number of murine models of myeloid neoplasia how leukemia infiltration alters the HSC niche to reinforce malignancy. Acute myeloid leukemia (AML) is characterized in human by a high relapse rate indicating that leukemia initiating cells are protected by its niche. However, despite our knowledge in murine models little is known about the bone marrow architecture in human and the impact of the leukemic niche on clinical outcome.In this study, we combined immunohistochemical stainings (IHC) with protein and global gene expression analyses together with clinical data to dissect the human bone marrow architecture in AML and assess its clinical impact. Human bone marrow was collected from AML patients at first diagnosis and matching non-leukemic donors. To evaluate the bone marrow architecture CD271+ mesenchymal stem and progenitor cells (MSPCs) were automatically quantified on bone marrow sections. In fact, AML patients showed 1.5-fold increase in bone marrow MSPCs compared to non-leukemic donors (Median (IQR), AML: 5.5% (2.8-9.5), n=36; control: 3.7% (2.1-5.7), n=58; p < 0.01). MSPCs proved to produce reticular fibers, an extracellular matrix protein frequently associated with different malignancies. In AML bone marrow these fibers were also found to be more abundantly expressed (Median (IQR), AML: 3.4% (1.8-4.5), n=37; control: 1.6% (1.1-3.3), n=19; p < 0.05).Next, to globally assess the gene expression profile of MSPCs in AML bone marrow we performed microarray analyses (ClariomTM S Human Assay) of freshly isolated uncultured lineage- CD146+ CD271+ MSPCs. Strikingly, HSC-regulating genes in particular CXCL12, ANGPT1 and VCAM1 showed lower expression in AML MSPCs which correlated with the degree of hematopoietic failure in AML patients. Along with the increased number of MSPCs, geneset enrichment analysis (GSEA) revealed higher proliferation of MSPCs in AML. In murine models loss of quiescence of MSPCs was previously found to be due to bone marrow sympathetic neuropathy. We therefore measured catecholamines and neurotrophic factor in the bone marrow extracellular fluid of AML patients and non-leukemic donors at first diagnosis. In fact, noradrenalin and brain-derived neurotrophic factor (BDNF) showed a 2-fold (p=0.26) resp. 4-fold (p<0.0001) lower expression in AML bone marrow. Importantly, BDNF is proved to be essential for sympathetic neuron proliferation and differentiation.In order to get an overview of alterations of canonical pathways in bone marrow MSPCs upon AML infiltration, we applied QIAGEN's Ingenuity® Pathway Analysis software. Several of the major differently regulated pathways proved to involve differentiation and mineralization of MSPCs. We therefore assessed bone metabolism in AML patients at first diagnosis and quantified serum osteocalcin levels. Notably, AML patients showed 30% lower osteocalcin levels than non-leukemic donors (Median (IQR), AML, 12.15ng/ml (7.53-16.28) n=58; control, 17.2ng/ml (12.5-23.45) n=31; p < 0.05). To evaluate if the deficiency in osteoblast mineralization is specifically due to AML infiltration we performed in vitro co-culture assays. Both MSPCs and an osteoblast-like cell line (SaOS2) showed significant impaired mineralization in presence of certain AML cell lines as well as primary human AML cells, while healthy mononuclear cells did not affect mineralization. Strikingly, this AML-induced defect in osteoblast mineralization proved to be of clinical significance. Patients with low osteocalcin levels (<11ng/ml) showed inferior overall survival with 1-year survival rate of 38.7% while patients with high osteocalcin levels reached 66.8% (n=58; median duration of follow-up 9.7 months).In summary, we globally characterized the bone marrow architecture in AML patients in comparison to non-leukemic donors and assessed its clinical significance. This increasing understanding of the human AML bone marrow microenvironment might open the window for new niche-targeted therapies to eradicate leukemic stem cells and eventually decrease the high relapse rate in AML. DisclosuresDuehrsen:Amgen: Research Funding; Roche: Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Janssen: Honoraria.

MicroRNA-141-3p Negatively Modulates SDF-1 Expression in Age-Dependent Pathophysiology of Human and Murine Bone Marrow Stromal Cells.

Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a cytokine secreted by cells including bone marrow stromal cells (BMSCs). SDF-1 plays a vital role in BMSC migration, survival, and differentiation. Our group previously reported the role of SDF-1 in osteogenic differentiation in vitro and bone formation in vivo; however, our understanding of the post-transcriptional regulatory mechanism of SDF-1 remains poor. MicroRNAs are small noncoding RNAs that post-transcriptionally regulate the messenger RNAs (mRNAs) of protein-coding genes. In this study, we aimed to investigate the impact of miR-141-3p on SDF-1 expression in BMSCs and its importance in the aging bone marrow (BM) microenvironment. Our data demonstrated that murine and human BMSCs expressed miR-141-3p that repressed SDF-1 gene expression at the functional level (luciferase reporter assay) by targeting the 3'-untranslated region of mRNA. We also found that transfection of miR-141-3p decreased osteogenic markers in human BMSCs. Our results demonstrate that miR-141-3p expression increases with age, while SDF-1 decreases in both the human and mouse BM niche. Taken together, these results support that miR-141-3p is a novel regulator of SDF-1 in bone cells and plays an important role in the age-dependent pathophysiology of murine and human BM niche.

Exposure of the Bone Marrow Microenvironment to Simulated Solar and Galactic Cosmic Radiation Induces Biological Bystander Effects on Human Hematopoiesis

The stem cell compartment of the hematopoietic system constitutes one of the most radiosensitive tissues of the body and leukemias represent one of the most frequent radiogenic cancers with short latency periods. As such, leukemias may pose a particular threat to astronauts during prolonged space missions. Control of hematopoiesis is tightly governed by a specialized bone marrow (BM) microenvironment/niche. As such, any environmental insult that damages cells of this niche would be expected to produce pronounced effects on the types and functionality of hematopoietic/immune cells generated. We recently reported that direct exposure of human hematopoietic stem cells (HSC) to simulated solar energetic particle (SEP) and galactic cosmic ray (GCR) radiation dramatically altered the differentiative potential of these cells, and that simulated GCR exposures can directly induce DNA damage and mutations within human HSC, which led to leukemic transformation when these cells repopulated murine recipients. In this study, we performed the first in-depth examination to define changes that occur in mesenchymal stem cells present in the human BM niche following exposure to accelerated protons and iron ions and assess the impact these changes have upon human hematopoiesis. Our data provide compelling evidence that simulated SEP/GCR exposures can also contribute to defective hematopoiesis/immunity through so-called "biological bystander effects" by damaging the stromal cells that comprise the human marrow microenvironment, thereby altering their ability to support normal hematopoiesis.

Modeling the human bone marrow niche in mice: From host bone marrow engraftment to bioengineering approaches.

Xenotransplantation of patient-derived samples in mouse models has been instrumental in depicting the role of hematopoietic stem and progenitor cells in the establishment as well as progression of hematological malignancies. The foundations for this field of research have been based on the development of immunodeficient mouse models, which provide normal and malignant human hematopoietic cells with a supportive microenvironment. Immunosuppressed and genetically modified mice expressing human growth factors were key milestones in patient-derived xenograft (PDX) models, highlighting the importance of developing humanized microenvironments. The latest major improvement has been the use of human bone marrow (BM) niche-forming cells to generate human-mouse chimeric BM tissues in PDXs, which can shed light on the interactions between human stroma and hematopoietic cells. Here, we summarize the methods used for human hematopoietic cell xenotransplantation and their milestones and review the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis.

Engineered humanized bone organs maintain human hematopoiesis in vivo

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) environment, the so-called HSC niche, that provides pivotal factors for their maintenance. Although the cellular and molecular components of the mouse BM HSC niche have been extensively studied using genetically modified animals, relatively little is known about the counterpart human BM niche components. We previously illustrated, with a developmental tissue engineering approach, that human adult BM-derived mesenchymal stromal cells (MSCs) can develop into human bone organs (so-called ossicles) through endochondral ossification in vivo and that these human ossicles are able to maintain functional mouse HSCs. We here report that human ossicles in immunodeficient mice maintain human immature and mature hematopoiesis in vivo. Moreover, a higher percentage of human stem and progenitor cells are kept in quiescence in human ossicles as compared with mouse BM. These findings indicate that the human MSC-derived ossicles function as a hematopoietic niche and may potentially serve as a re-engineerable platform to study normal and diseased human hematopoiesis in a physiologically optimized environment.

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

Establishing human leukemia xenograft mouse models by implanting human bone marrow–like scaffold-based niches

AbstractTo begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches.