Hematopoietic Stem Cell Niche Research Articles

Oncostatin M regulates hematopoietic stem cell (HSC) niches in the bone marrow to restrict HSC mobilization.

We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential.

Simple SummaryIn chronic myeloid leukemia (CML), a neoplasm brilliantly taken care of by a molecularly targeted therapeutic approach, the achievement of cure is nevertheless prevented by the maintenance of a small subset of treatment-resistant leukemia stem cells (LSCs), sustaining the so-called minimal residual disease of CML. The phenotypical and functional characterization of this LSC subset is, therefore, crucial to aim at the eradication of disease. Such a characterization includes the acquisition of information relative to the metabolic profile of treatment-resistant LSCs, which is functional to their maintenance in bone marrow. A number of metabolic features of LSCs were shown to determine their sensitivity or resistance to therapy. Glutamine metabolism emerged from this study as a potential target to overcome the persistence of therapy-resistant LSCs.This study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective.

Aplastic anemia, cellular and molecular aspects.

Aplastic anemia (AA) is an autoimmune disorder characterized by bone marrow and peripheral blood pancytopenia. Different environmental and genetical conditions could be effective in an outbreak of this disease. The exact pathogenesis of this disease, however, is still idiopathic. The present study is based on Pubmed database information (2002-2021) using the words "Aplastic Anemia," "Hematopoietic Stem Cells niche," "Signaling pathway," "Cytokines," and "Immuno cells." In this disease, both hematopoietic stem cells and mesenchymal stromal cells are impaired, which is associated with impaired hematopoiesis and decreased hematopoietic cells. Inflammatory cytokines increase, which changes the ratio of T lymphocytes and leads to disease progression. In addition, the most common mechanism of AA is damage by the immune system, which leads to increased apoptosis in progenitor cells. We have shown in this review that the disease involves quantitative defects in stem cell numbers and qualitative abnormalities in the function of these cells and the activity of many different cellular and molecular factors can damage hematopoietic cells and the protective substrate of these cells in this disease.

Hematopoiesis during Ontogenesis, Adult Life, and Aging.

In the bone marrow of vertebrates, two types of stem cells coexist—hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Hematopoiesis only occurs when these two stem cell types and their descendants interact. The descendants of HSCs supply the body with all the mature blood cells, while MSCs give rise to stromal cells that form a niche for HSCs and regulate the process of hematopoiesis. The studies of hematopoiesis were initially based on morphological observations, later extended by the use of physiological methods, and were subsequently augmented by massive application of sophisticated molecular techniques. The combination of these methods produced a wealth of new data on the organization and functional features of hematopoiesis in the ontogenesis of mammals and humans. This review summarizes the current views on hematopoiesis in mice and humans, discusses the development of blood elements and hematopoiesis in the embryo, and describes how the hematopoietic system works in the adult organism and how it changes during aging.

Dynamic Changes of the Bone Marrow Niche: Mesenchymal Stromal Cells and Their Progeny During Aging and Leukemia.

Mesenchymal stromal cells (MSCs) are a heterogenous cell population found in a wide range of tissues in the body, known for their nutrient-producing and immunomodulatory functions. In the bone marrow (BM), these MSCs are critical for the regulation of hematopoietic stem cells (HSC) that are responsible for daily blood production and functional immunity throughout an entire organism’s lifespan. Alongside other stromal cells, MSCs form a specialized microenvironment BM tissue called “niche” that tightly controls HSC self-renewal and differentiation. In addition, MSCs are crucial players in maintaining bone integrity and supply of hormonal nutrients due to their capacity to differentiate into osteoblasts and adipocytes which also contribute to cellular composition of the BM niche. However, MSCs are known to encompass a large heterogenous cell population that remains elusive and poorly defined. In this review, we focus on deciphering the BM-MSC biology through recent advances in single-cell identification of hierarchical subsets with distinct functionalities and transcriptional profiles. We also discuss the contribution of MSCs and their osteo-adipo progeny in modulating the complex direct cell-to-cell or indirect soluble factors-mediated interactions of the BM HSC niche during homeostasis, aging and myeloid malignancies. Lastly, we examine the therapeutic potential of MSCs for rejuvenation and anti-tumor remedy in clinical settings.

Botryllus schlosseri as a Unique Colonial Chordate Model for the Study and Modulation of Innate Immune Activity.

Understanding the mechanisms that sustain immunological nonreactivity is essential for maintaining tissue in syngeneic and allogeneic settings, such as transplantation and pregnancy tolerance. While most transplantation rejections occur due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity. Botryllus schlosseri, a colonial tunicate, which is the nearest invertebrate group to the vertebrates, is devoid of T- and B-cell-based adaptive immunity. It has unique characteristics that make it a valuable model system for studying innate immunity mechanisms: (i) a natural allogeneic transplantation phenomenon that results in either fusion or rejection; (ii) whole animal regeneration and noninflammatory resorption on a weekly basis; (iii) allogeneic resorption which is comparable to human chronic rejection. Recent studies in B. schlosseri have led to the recognition of a molecular and cellular framework underlying the innate immunity loss of tolerance to allogeneic tissues. Additionally, B. schlosseri was developed as a model for studying hematopoietic stem cell (HSC) transplantation, and it provides further insights into the similarities between the HSC niches of human and B. schlosseri. In this review, we discuss why studying the molecular and cellular pathways that direct successful innate immune tolerance in B. schlosseri can provide novel insights into and potential modulations of these immune processes in humans.

The Impact of Sedentary Lifestyle, High-fat Diet, Tobacco Smoke, and Alcohol Intake on the Hematopoietic Stem Cell Niches.

Hematopoietic stem and progenitor cells maintain hematopoiesis throughout life by generating all major blood cell lineages through the process of self-renewal and differentiation. In adult mammals, hematopoietic stem cells (HSCs) primarily reside in the bone marrow (BM) at special microenvironments called “niches.” Niches are thought to extrinsically orchestrate the HSC fate including their quiescence and proliferation. Insight into the HSC niches mainly comes from studies in mice using surface marker identification and imaging to visualize HSC localization and association with niche cells. The advantage of mouse models is the possibility to study the 3-dimensional BM architecture and cell interactions in an intact traceable system. However, this may not be directly translational to human BM. Sedentary lifestyle, unhealthy diet, excessive alcohol intake, and smoking are all known risk factors for various diseases including hematological disorders and cancer, but how do lifestyle factors impact hematopoiesis and the associated niches? Here, we review current knowledge about the HSC niches and how unhealthy lifestyle may affect it. In addition, we summarize epidemiological data concerning the influence of lifestyle factors on hematological disorders and malignancies.

Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells.

Currently, more than 30 000 allogeneic hematopoietic stem cell (HSC) transplantations have been performed for the treatment of hematological and nonhematological diseases using HSC from umbilical cord blood (CB). However, the wide utilization of CB as a source of HSC is limited by the low number of cells recovered. One strategy to expand ex vivo CB‐HSC is represented by the use of bone marrow mesenchymal stromal cells (BM‐MSCs) as a feeder to enhance HSC proliferation while maintaining HSC stemness. Indeed, BM‐MSCs have been recognized as one of the most relevant players in the HSC niche. Thus, it has been hypothesized that they can support the ex vivo expansion of HSC by mimicking the physiological microenvironment present in the hematopoietic niche. Due to the role of placenta in supporting fetal hematopoiesis, MSC derived from the amniotic membrane (hAMSC) of human term placenta could represent an interesting alternative to BM‐MSC as a feeder layer to enhance the proliferation and maintain HSC stemness. Therefore, in this study we investigated if hAMSC could support the ex vivo expansion of HSC and progenitor cells. The capacity of hAMSCs to support the ex vivo expansion of CB‐HSC was evaluated in comparison to the control condition represented by the CB‐CD34+ cells without a feeder layer. The coculture was performed at two different CD34+:MSC ratios (1:2 and 1:8) in both cell‐to‐cell contact and transwell setting. After 7 days, the cells were collected and analyzed for phenotype and functionality. Our results suggest that hAMSCs represent a valuable alternative to BM‐MSC to support: (a) the ex vivo expansion of CB‐HSC in both contact and transwell systems, (b) the colony forming unit ability, and (c) long‐term culture initiating cells ability. Overall, these findings may contribute to address the unmet need of high HSC content in CB units available for transplantation.

Distinct skeletal stem cell types orchestrate long bone skeletogenesis.

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

The Role of Cysteine Peptidases in Hematopoietic Stem Cell Differentiation and Modulation of Immune System Function.

Cysteine cathepsins are primarily involved in the degradation and recycling of proteins in endo-lysosomal compartments but are also gaining recognition as pivotal proteolytic contributors to various immune functions. Through their extracellular proteolytic activities within the hematopoietic stem cell niche, they are involved in progenitor cell mobilization and differentiation. Cysteine cathepsins, such as cathepsins L and S contribute to antigen-induced adaptive immunity through major histocompatibility complex class II antigen presentation whereas cathepsin X regulates T-cell migration. By regulating toll-like receptor signaling and cytokine secretion cysteine cathepsins activate innate immune cells and affect their functional differentiation. Cathepsins C and H are expressed in cytotoxic T lymphocytes and natural killer cells and are involved in processing of pro-granzymes into proteolytically active forms. Cytoplasmic activities of cathepsins B and L contribute to the maintenance of homeostasis of the adaptive immune response by regulating cell death of T and B lymphocytes. The expression pattern, localization, and activity of cysteine cathepsins is tightly connected to their function in immune cells. Furthermore, cysteine cathepsins together with their endogenous inhibitors, serve as mediators in the interplay between cancer and immune cells that results in immune cell anergy. The aim of the present article is to review the mechanisms of dysregulation of cysteine cathepsins and their inhibitors in relation to immune dysfunction to address new possibilities for regulation of their function.

Spatial transcriptome profiling by MERFISH reveals fetal liver hematopoietic stem cell niche architecture

The hematopoietic stem cell (HSC) niche has been extensively studied in bone marrow, yet a more systematic investigation into the microenvironment regulation of hematopoiesis in fetal liver is necessary. Here we investigate the spatial organization and transcriptional profile of individual cells in both wild type (WT) and Tet2−/− fetal livers, by multiplexed error robust fluorescence in situ hybridization. We find that specific pairs of fetal liver cell types are preferentially positioned next to each other. Ligand-receptor signaling molecule pairs such as Kitl and Kit are enriched in neighboring cell types. The majority of HSCs are in direct contact with endothelial cells (ECs) in both WT and Tet2−/− fetal livers. Loss of Tet2 increases the number of HSCs, and upregulates Wnt and Notch signaling genes in the HSC niche. Two subtypes of ECs, arterial ECs and sinusoidal ECs, and other cell types contribute distinct signaling molecules to the HSC niche. Collectively, this study provides a comprehensive picture and bioinformatic foundation for HSC spatial regulation in fetal liver.

Intraosseous Administration of Multipotent Mesenchymal Stromal Cells to Patients with Graft Failure after Allogeneic Hematopoietic Stem Cell Transplantation

IntroductionMultipotent mesenchymal stromal cells (MSCs) are involved in the formation of niches for hematopoietic stem cells and support hematopoiesis.There are several reasons for the graft failure in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). One of them could be the damage of hematopoietic microenvironment by chemotherapeutic drugs and tumor cells. As a result, the bone marrow (BM) stroma loses its ability to support hematopoiesis.Perhaps the donor's MSCs can serve as a source of hematopoietic niches recovery in these cases.The aim of the study was to investigate the possibility to restore the hematopoietic stromal microenvironment in patients with graft failure with donors MSCs implanted into trabecular bone.Patients and methodsA randomized study for acute graft versus host disease prophylaxis with MSCs has been conducted by the National Research Center for Hematology, Moscow, Russia. MSCs were developed for each patient from their individual BM donor and cryopreserved.There was long-term BM aplasia observed in 4 patients with GF following allo-HSCT. All of the patients received a second allo-HSCT with MSCs from the donor BM. The MSCs were injected under local anesthesia in the iliac crest that is, directly into the bone marrow in the trabecular bone area. All patients provided informed consent. The implanted MSCs from several passages were thawed and washed free of dimethyl sulfoxide. The cells were then suspended in 2 ml of 6% polyglucin. The cells (200-370 х106) were injected into the bone tissue in small portions (100-200 microliters) through 2 skin and multiple periosteal punctures.BM punctures were performed for patients 1, 2 and 4.5 months after donor MSC implantation simultaneously. BM (2-3 ml) was collected from 2-4 independent punctures of the iliac spine for each patient. From these BM samples MSCs were isolated and colony forming unit fibroblast (CFU-F) were analyzed according to standard protocols.The proportion of chimeric DNA was determined in the patients' MSCs at the 1-3rd passages and CFU-F. The cell DNA was isolated by standard methods of extraction and salt precipitation with isopropanol. The chimerism in MSCs was analyzed by the STR-PCR method. The protocol uses reagents for the multiplex analysis of 19 STR-markers and the human amelogenin locus. The informative loci were preselected to monitor the chimerism in the observed patients.ResultsIn all 4 cases, after intraosseous injection of MSCs and re-allo-HSCT from the same donor, the patients had a restoration of donor hematopoiesis.In stromal precursor cells - MSCs and CFU-F, obtained from the BM of patients at 1, 2 and 4.5 months after the implantation of MSCs and second allo-HSCT from the same donor, donors' markers were identified. The table presents the data on chimerism in the BM and studied MSCs and CFU-F in patients.The sites of BM punctures in patients only approximately coincided with the place of injection of donor MSCs, therefore the proportion of donor cells does not clearly reflect the content of donor stromal cells in patients. Nevertheless, it can be argued that implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. In fact, these cells survived 3 transfers: from the donor's organism to the culture, then again into the patient's body and repeatedly into the culture. The presence of donor CFU-F - descendants of MSCs proves the functional activity of these cells.ConclusionsIt can be assumed that MSCs caused the restoration of donor hematopoiesis, they probably performed a trophic function, participated in the restoration of niches for hematopoietic cells, or had an immunomodulating effect preventing re-failure of the hematopoietic cell transplant.Thus, the long-term existence of donor's MSCs in the patient's organism is shown for the first time. A strong potential of MSCs for repeated transfers was revealed. It has been shown that possibly intraosseous MSCs implantation could facilitate transplant engraftment.The materials are supported by grant from the Russian Science Foundation, Project № 16-15-00102 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hematologic Response to Romiplostim Treatment Is Associated with Stimulation of Primitive Stem/Progenitor Cells and Stromal Cells in Patients with Aplastic Anemia Refractory to Immunosuppressive Therapy: A 2-Year Interim Exploratory Analysis of a Phase 2 Clinical Trial

Introduction:Romiplostim (RMP) is a thrombopoietin-mimetic protein that enhances platelet production. RMP has already been approved in numerous countries for treatment of immune thrombocytopenia. We previously reported the efficacy and safety of RMP through a 1-year treatment period in patients with aplastic anemia (AA) that was refractory to immunosuppressive therapy (IST). In this study, we report the interim exploratory analysis of the influence of RMP after a 2-year treatment period (105 weeks) on the hematopoietic stem cells (HSCs) and progenitor cells, and on mesenchymal stromal cells, a specialized HSC niche, in patients with AA who were refractory to IST.Methods:This study is a multicenter, randomized, open-label, parallel, comparative, dose-finding study in South Korea (NCT02094417). Eligible patients with AA, refractory to prior IST, were randomized into 4 dose groups that received 1, 3, 6, or 10 μg/kg of RMP for the initial 8-week treatment. After the initial treatment, the dose was adjusted from 1 to 20 μg/kg to maintain their platelet response. After week 53, if the blood cell count improved and was maintained at a predefined range, the dose was tapered and the patient was considered to be in a “tapering-dose” status; if the dose was decreased to 0 µg/kg at this point, the patient was considered to be off the drug. The range was defined as follows: platelet count, > 50x109/L; hemoglobin concentration, > 10.0 g/dL; and neutrophil count, > 1.0x109/L. The blood cell response was defined based on the absolute cell count. To examine the association between cellular changes in the bone marrow in response to RMP and the clinical course of the patients with AA, the primitive HSC and progenitor cell subsets were analyzed by flow cytometry and the colony formation assay at the baseline and every 6 months after initiation of RMP. Further, changes in the mesenchymal stromal cells within the bone marrow were also analyzed by the CFU-F assay.Results:A total of 35 subjects were randomized; of these, 18 received RMP beyond 1 year, and 10 are still receiving it over 2 years.At week 105, the platelet response was observed in all 10 subjects, erythroid response in 9 subjects, neutrophil response in 5 subjects, and tri-lineage response in 5 subjects.Of the ongoing 10 subjects, 7 have achieved “tapering-dose” status, and 2 have achieved “off-the-drug” status. In addition, The RMP was suspended in 1 subject due to excessive increase in platelet-count (>400x109/L). This subject has also been exhibiting the tri-lineage response.At week 53, the median counts of all colony assays were higher in subjects with any blood cell response than in those without the response. When we compared the serial colony assays in the subjects with any blood cell response at week 53 up to week 105, the colony counts of BFU-E, CFU-E, CFU-M, and CFU-GM gradually increased until week 77 from the baseline, and then declined at week 105. Further, the colony counts of CFU-F also exhibited a similar pattern. The colony counts of CFU-G and CFU-MK increased until week 53, and then decreased. However, the CD34+CD38- cells steadily increased throughout up to week 105 from the baseline.Conclusion:These data indicate that RMP influences the stimulation of primitive HSC (CD34+CD38- cells) and bone marrow microenvironment (HSC niche), and enhances the differentiation of primitive HSCs to late progenitor cells resulting in hematologic response in the patients with AA.Although further follow up is ongoing the up to the end of 3-year study period, but this interim analysis may strongly implies that the RMP might be beneficial for patients with AA, refractory to IST. DisclosuresNo relevant conflicts of interest to declare.

The E3 Ligase Itch Is a Regulator of Bone Marrow Mesenchymal Stem Cells and the Hematopoietic Stem Cell Niche

Hematopoietic stem cells (HSCs) reside in a complex cellular microenvironment within the bone marrow. Bone marrow mesenchymal stromal/stem cells (BM-MSCs) have emerged as an important cellular component of this hematopoietic stem cell niche. BM-MSCs have been implicated in physiological hematopoiesis as well as in hematological malignancies. The intracellular regulatory network that governs BM-MSC homeostasis and their function within the HSC niche remains poorly understood. Posttranslational modification of proteins through ubiquitylation has been proven an integrative component in the regulation of quiescence, proliferation, self-renewal and differentiation of stem cells. We have previously demonstrated that the E3 ubiquitin ligase Itch is a negative regulator of HSC homeostasis and function (Rathinam et al., Nat.Immunol., 2011). Notably, patients suffering from a rare ITCH deficiency syndrome display morphologic abnormalities with dysmorphic features, suggesting a role for this E3 ligase within the skeletal tissue and mesenchymal lineage cells (Lohr et al., Am.J.Hum.Genet., 2010).In the present study, we identify Itch as a critical regulator of BM-MSCs. Itch-/- mice exhibit an at least two-fold reduction of MSCs (immunophenotypically identified as CD45-TER119-CD31-PDGFRα+Sca-1+CD90+) in the BM. Consistently, Colony Forming Unit-Fibroblasts (CFU-F) assays revealed a remarkable reduction of MSCs in the BM (p=0.0392). Moreover, in-vitro culture studies identified a hyperproliferative phenotype of Itch mutant MSCs. However, further studies revealed that Itch deficiency leads to reduced differentiation of MSCs into osteoblasts, both in-vivo and in-vitro, and therefore resulting in osteopenic bone phenotype. Reciprocal and Serial Bone marrow transplantation studies suggested an alteration of the niche for HSCs in Itch deficient BM. Interestingly, Itch-/- recipients exhibited a five-fold increase in their donor (wild-type) hematopoietic stem and progenitor cell compartment. Molecular and biochemical studies documented an increase of signals mediated by Notch in Itch deficient MSCs and co-localization of Itch and Notch-1 intracellular domain. Of note these findings gain importance, as Notch signaling has been previously implicated in the regulation of MSC differentiation into osteoblasts, as well as in the regulation of the HSC niche.In summary, our studies identified Itch as a positive regulator of BM-MSC homeostasis. Our results suggest that the Itch-Notch axis within the HSC niche may serve as a potential pharmaceutical target, especially in the setting of bone marrow transplantation in humans. Treatment of the transplant recipient with an Itch inhibitor may avoid graft failure or rescue poor hematopoietic recovery. DisclosuresSchünemann:German Academic Exchange Service: Other: scholarship; Boehringer Ingelheim Fonds: Other: travel grant.

Macrophage Involvement in the Response of Acute Myeloid Leukaemia to Chemotherapy

Acute myeloid leukemia (AML) is a clonally heterogeneous neoplasm with poor survival rates. Although chemotherapy often leads to clinical remission, the relapse rate of AML is very high and usually fatal. Chemotherapy is usually effective at debulking AML blasts however a small population capable of re-initiating the leukemia (LIC) survive ultimately causing relapse. Mouse models of AML suggest that LIC are protected from chemotherapy by a protective niche similar to hematopoietic stem cell (HSC) niches. As 1) bone marrow (BM) macrophages are key regulators of HSC niche function and 2) tumor-associated macrophages promote chemotherapy resistance in several models of solid tumors (e.g. breast, lung & ovarian) we investigated a potential role of BM non-malignant macrophages in promoting chemoresistance of AML blasts.Using a mouse model of AML driven by the MLL-AF9 oncogene in which mouse HSC are transduced with MSC-MLL/AF9-ires-GFP and transplanted into syngeneic recipients, we examined how AML progression alters the phenotype of non-malignant GFP-negative BM macrophages by flow cytometry. 18 days post-transplant, GFP+ leukemia burden in the BM was in the 40-60% range. In non-malignant GFPneg BM macrophages, cell surface expression of MHC class II, CD80, L-selectin and neuropilin-1 was significantly decreased suggesting that AML blasts alter the function of BM macrophages.To determine whether BM macrophages could have any role in AML response to chemotherapy, we first determine whether non-malignant BM macrophages were depleted in vivo by treatment with cytarabine (5 days, 100mg/kg) and doxorubicin (3 days, 1mg/kg). Two days after cytarabine+doxoR treatment, most CD11b+ F4/80- Ly6G+ granulocytes and CD11b+ F4/80+ CD169- monocytes were ablated from the BM with 0.1% granulocytes and 1.0 % monocytes surviving after treatment. In contrast CD11b- F4/80+ CD169+ VCAM1+ macrophages were the most resilient myeloid cells in the BM with 9.5% still present 2 days after chemotherapy.To further investigate a possible role of these chemotherapy-resilient CD169+ macrophages in promoting response of AML blasts to chemotherapy, we transplanted MLL-AF9 leukemia cells generated with wild-type HSC into CD169DTR/WT mouse recipients in which one CD169 allele is knocked-in with human diphtheria toxin receptor (DTR). This enables specific depletion of CD169+ macrophages in vivo by injecting diphtheria toxin (DT). In this model, DT treatment targets host CD169+ macrophages but not the AML blasts as they do not contain the DTR knock-in. In the absence of chemotherapy, depletion of recipient CD169+ macrophages did not alter leukemia burden in the BM, spleen or blood 48 hours post depletion and did not alter mouse survival (median survival 29-30 days in both CD169-DTR and WT recipients treated with DT or saline).However when cytarabine + doxoR were administered 48 hours after DT administration, median survival of CD169-DTR recipients was significantly increased to 39.5 days compared to wild-type recipients 35 days, p<0.001. Furthermore leukemia burden in both the blood and spleen was significantly reduced.To determine whether CD169 could by itself mediate lower response of AML to chemotherapy, we transplanted MLL-AF9 cells generated from WT HSC into CD169DTR/DTR mice which are knocked-out for both CD169 alleles. Deletion of both CD169 alleles in recipient mice did not change mouse survival in response to chemotherapy in the absence of DT. Therefore absence of CD169 is not sufficient to prolong mouse survival in response to chemotherapy.In conclusion, non-malignant CD169+ macrophages may protect MLL-AF9 leukemia blasts from the cytotoxic effects of cytarabine and doxorubicin. The mechanisms of this chemoprotection remain to be determined but do not involve a direct effect of CD169 on AML blasts. DisclosuresNo relevant conflicts of interest to declare.

Niche-Targeted Clinical Grade Immunohistochemistry Reveals Heterogeneous Bone Marrow Neuropathy and Novel Stromal Patterns in Primary Myelofibrosis Patients

Regulation of the hematopoietic stem cell (HSC) niche includes elements of the nervous system, immune system as well as the vasculature and associated stroma. Evidence in murine models demonstrate microenvironmental influence on HSC and hematopoiesis however correlative clinical and translational data remain scarce. The sympathetic nervous system may contribute to the pathogenesis of myeloproliferative neoplasms (MPNs) mechanistically through effects on nestin+ mesenchymal stromal/stem cells. Temporally, bone marrow neuropathy may even precede overt MPN by promoting aberrant premalignant HSC expansion, at least in genetically engineered MPN mouse models. We hypothesize that clinical characterization of the bone marrow niche via conventional immunohistochemistry (IHC) on routine diagnostic trephine biopsies is feasible and may yield novel stromal architectural data supplementing classification and/or prognostic stratification of BCR-ABL1 negative MPN. Herein, we describe our initial results on normal bone marrows and patients with primary myelofibrosis (PMF) with a focus on the pan-neuronal marker (PGP9.5) and the stromal marker smooth muscle actin (SMA). Results: Bone marrow neuropathy, as assessed by loss of punctate or linear perivascular PGP9.5 reactivity around arterioles is highly variable at initial PMF diagnosis. This suggests that in PMF, bone marrow neuropathy may not be universal and if present may only occur in a subset of patients or affect only focal regions of the marrow. SMA staining reproducibly yielded 3 patterns of reactivity in PMF: paratrabecular, perivascular and interstitial. While perivascular SMA staining was observed in all cases of PMF, the interstitial SMA pattern exhibited both inter-patient and intra-patient variability. Intriguingly, SMA was capable of highlighting “hot” and “cold” spots in PMF marrows otherwise appearing homogeneous by conventional reticulin staining. These SMA patterns are presently being investigated in the context of clinical information and molecular profiling to further assess their prognostic relevance. From longitudinal biopsies, we show resolution of the PMF interstitial SMA pattern as early as 2 months post allo-hematopoietic stem cell transplant, far earlier than any reduction in reticulin fibrosis which suggests that SMA signal may have important biological implications - including as a biomarker of early response to JAK inhibition, superior to standard reticulin/collagen assessment. In summary, visualization of the bone marrow niche by routine clinical grade IHC is feasible and reveals stromal heterogeneity within WHO-defined PMF of potential interest for classification refinement, prognostication and therapeutic monitoring. DisclosuresTsui:Novartis: Honoraria. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Research Funding.

Early Growth Response (EGR)-1 Is a Key Regulator of Human Primary Bone Marrow Stroma Cells with a Dual Role in Proliferation and Hematopoietic Stroma Support Function

Hematopoietic stem cell (HSC) niches are specialized local tissue microenvironments that maintain HSCs and regulate HSC functions. Bone marrow mesenchymal stroma cells (BMSCs) are essential constituents of the HSC niche and they give rise to skeletal lineage cells as well as the hematopoietic stroma. Despite recent progress in the identification of the primary bone fide BMSC in human bone marrow, knowledge regarding the regulation of BMSC proliferation, differentiation and hematopoietic support function is incomplete.We have previously shown that the expression of Early Growth Response 1 (EGR1) in primary BMSCs is substantially higher compared to the non-colony-forming cells and cultured stromal cells, respectively, indicating a possible role of EGR1 in BMSC regulation (Li et al., Stem Cell Reports, 3(6):965-74, 2014). EGR1 encodes a zinc finger transcription factor that exemplifies a group of immediate early response genes, which have diverse functions in cell growth, development and stress response. EGR1 regulates murine HSC proliferation and localization (Min et al., Cell Stem Cell, 2(4):380-91, 2008), but its role in stroma cell regulation has thus far not been investigated. This study therefore aimed to investigate the functional role of EGR1 in human primary BMSCs.Quantitative gene expression analysis demonstrated that EGR1 expression was 126.9 ± 9.1-fold and 2.8 ± 0.2-fold higher in highly CFU-F (colony-forming unit, fibroblast) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population and CD45neg/CD271pos/CD140apos cells with minimum CFU-F activity. Furthermore, we confirmed that EGR1 expression in cultured cells was down-regulated in comparison with primary BMSCs.Multi-lineage differentiation capacity and surface marker expression profiles of BMSCs were not affected by shRNA-induced EGR1 down-regulation. However, colony-forming capacity was enhanced (1.9 ± 0.1-fold) and population doubling times were reduced by EGR1 knockdown compared to scramble controls. Furthermore, the HSC stroma supporting function as measured by production of transplantable HSC in co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells) was reduced when EGR1 knockdown cells were used as stroma layer.On the other hand, whereas CFU-F formation was virtually absent in EGR1 overexpressing cells and population doubling times increased by 2.4 ± 0.3-fold, the hematopoietic stroma support function was considerably improved by EGR1 overexpression as indicated by increased percentages and absolute numbers of transplantable CD34posCD90pos cells produced in co-cultures with EGR1 overexpressing BMSCs as stromal cells (12,496 ± 887 for EGR1 overexpression vs 6,867 ± 1242 for GFP control). Accordingly, CD34pos cells expanded on EGR1 overexpressing stroma exhibited improved in-vivo long-term reconstitution levels in NSG mice compared with cells grown on control stroma.To investigate how EGR1 regulates proliferation and hematopoiesis support in BMSCs, transwell experiments were performed showing that both soluble factors and cell-to-cell contact were important for EGR1-mediated hematopoiesis support. Furthermore, proteomic analysis revealed that a group of proteins related to oxidative-reduction processes was downregulated whereas reactive oxygen species (ROS) levels were elevated in EGR1 knockdown cells. In addition, gene expression profiling analysis identified a number of hematopoietic support genes that were upregulated in EGR1 overexpressing cells compared with control cells.In summary, our data indicate that EGR1 is an important BMSC regulator with a dual role on proliferation and hematopoietic stroma support function. EGR1 expression negatively regulated BMSC proliferation while positively regulating hematopoietic support function, which points to its possible role in coordinating the specific functions of BMSC in their different biological contexts. DisclosuresNo relevant conflicts of interest to declare.

Arteriolar Neovasculogenesis and Sinusoidal Collapse Are Pathologic Features of the Hematopoietic Stem Cell Niche in Sickle Cell Disease (SCD) Mice

Sickle cell disease is a worldwide distributed hereditary red cell disorder, caused by pathologic hemoglobin S (HbS). The main clinical manifestations of SCD are chronic hemolytic anemia and acute vaso-occlusion, leading to multiple end-organ complications. To what extent, vaso-occlusion may affect the bone marrow microenvironments that regulate hematopoiesis in SCD is poorly defined. Here, we first used quantitative 2-D and 3-D imaging analyses to study the bone marrow parenchyma. In 8-12 week-old SCD (SS) mice (humanized Townes model, SS), we observed a disorganized vasculature with both sinusoidal (laminin+ CD105+ Sca-1- alpha smooth muscle actin- ) and arteriolar (laminin+ CD105- Sca-1+ alpha smooth muscle actin+) structural abnormalities compared to healthy (AA) or HbS- carrier (SA) mice. The majority of sinusoids have either small or collapsed lumens, often filled with large Ter 119+ red blood cells (RBC) clumps, suggesting RBC sickling in localized areas. The RBC clumps involve significant numbers of Gr-1+/CD11b+ myeloid cells, which also are concentrated perisinusoidally consistent with a local inflammatory process. These features are amplified under hypoxia-reoxygenation conditions, used to mimick acute SCD vaso-occlusive crisis. Most strikingly, is the dramatic increase in small, tortuous artery/arteriole vessel formation, particularly in the central portions, i.e. non-endosteal regions, of the bone marrow cavity. Using laser scanning cytometry (LaSC) of whole femur sections, we determined a four fold increase in arterioles in the central bone marrow. In support of hypoxia induced pathways responsible for neovasculogenesis, we show that HIF-1alpha staining is dramatically increased in the bone marrow of SCD (SS) mice compared to healthy (AA) mice, and interestingly the distribution pattern also is concentrated to the central, non-endosteal portions of the marrow. Moreover the bone marrow supernatant, only in SS mice, exhibits high levels of vascular endothelial growth factor, VEGF in agreement with increased levels of HIF-1alpha. Because both the sinusoidal and arteriolar vascular structures are recognized components of the hematopoietic stem cell (HSC) niche, we explored the early stages of hematopoiesis by multiparameter flow cytometry. We found that the long term HSC population (Lin- Sca-1+ c-Kit+ CD48- CD150+) is reduced by 50% in SCD mice. Taken together, we propose a model for SCD bone marrow whereby vaso-occlusive events are accompanied by defects in both sinusoidal and arteriolar vessels and resultant severe tissue hypoxia. Finally, we speculate that the abnormal vascular niche structures in SS mice affect hematopoietic stem cell homeostasis, which requires further study. These findings may have significant implications for gene therapy protocols utilizing autologous hematopoietic stem cell transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Defining the Transcriptional Code That Specifies Sinusoidal Endothelial Cells in the HSPC Niche

Hematopoietic stem and progenitor cell (HSPC) niches are specialized microenvironments that support the maintenance and self-renewal of HSPCs. In the fetal liver, bone marrow and spleen, vascular endothelial cells are thought to function as core components of the HSPC niche. In the bone marrow, sinusoidal endothelial cells are known to secrete soluble factors and express cell surface adhesion molecules that promote the recruitment, maintenance and function of HSPCs. Using tomo-seq (RNA tomography) - a technique that involves extracting and barcoding the RNA from sequentially collected cryosections and then performing RNA-seq - we identified ~300 genes enriched in the zebrafish caudal hematopoietic tissue (CHT; the hematopoietic equivalent of the mammalian fetal liver). We also used FACS with tissue-specific fluorescent reporter transgenes to isolate different cell populations from whole embryos and then performed RNA-seq. This included endothelial cells, macrophages, neutrophils and erythrocytes. By overlaying these datasets with the tomo-seq results, we determined the cell types in which many of the CHT-enriched genes were expressed. Unexpectedly, we found a set of ~20 genes that appeared to be enriched specifically in endothelial cells within the CHT. Whole embryo in situ hybridization revealed that these genes had similar spatial and temporal expression patterns, being specifically enriched in sinusoidal endothelial cells in the CHT but absent in other endothelial cells throughout the body. We cloned the upstream promoter regions for several of these genes, fused them to a GFP reporter and then injected them into embryos. Of note, we found that a 5.3 kb sequence upstream of selectin-e (sele ; a gene known to be expressed by bone marrow endothelial cells where it promotes HSPC homing) drove GFP expression specifically in sinusoidal endothelial cells in the CHT. By crossing this sele:GFP transgene to a pan-endothelial marker (kdrl:mCherry), we were able to use FACS to isolate CHT endothelial cells, as well as endothelial cells from tissues outside the CHT. We performed ATAC-seq - a transposon-based method used to detect regions of open chromatin - on these two cell populations and identified 6,710 peaks (regions of open chromatin) that were unique to sele:GFP+; kdrl:mCherry+ CHT niche endothelial cells. Several of these peaks were associated with CHT endothelial genes we had initially identified by tomo-seq, including mrc1a, gpr182, stab1, stab2 and exoc3l2a . Informatic analysis of the sequences under the 6,710 CHT endothelial peaks identified Ets, Sox, Nuclear Hormone and Gata sites as the most enriched transcription factor binding motifs compared to peaks from the non-CHT endothelial cells. These results suggested a transcription factor code involving members of these families might specify sinusoidal endothelial cells in the HSPC niche. Upon closer examination of the sele locus itself, we observed a narrow ATAC-seq peak within the 5.3 kb upstream sequence that was uniquely open in the sele:GFP+; kdrl:mCherry+ CHT niche endothelial cells. Promoter truncation experiments identified a short 158 bp sequence that perfectly aligned with this unique ATAC-seq peak and which was sufficient to drive GFP expression in CHT endothelial cells. This 158 bp sequence contained three Ets, three Sox and one RORA (nuclear hormone receptor) binding sites. Synthetic variants were made in which the different transcription factor binding sites within the 158 bp sequence were disrupted. Injection of these variants driving GFP demonstrated that the Sox and RORA sites were required for expression in CHT endothelial cells. Disruption of the Ets sites, in contrast, led to dramatically enhanced GFP expression, suggesting these sites could be repressive. Together these studies demonstrate that the transcriptional regulation of niche endothelial cells include transcription factors that bind to Ets, Sox, and RORA motifs. The results of this work has important implications for culturing HSPCs in vitro with endothelial cells or for modulating the HSPC niche in the context of human disease or transplantation. DisclosuresZon:Fate, Inc.: Consultancy, Equity Ownership; Marauder, Inc.: Consultancy, Equity Ownership; Scholar Rock, Inc: Consultancy, Equity Ownership; Stemgent: Consultancy.

Inducible SBDS Deficiency and Germline FANCC/FANCG Deficiency Differentially Impact the Capacity of Bone Marrow Niches to Engraft Hematopoietic Stem Cells after Transplantation

Strategies to optimize interactions between the recipient bone marrow (BM) microenvironment and donor hematopoietic stem cells (HSC) could improve hematopoietic stem cell transplantation (HSCT) outcomes by reducing graft failure rates, improving blood and immune reconstitution efficiency, and reducing intensive cytotoxic conditioning requirements. Such methods are particularly relevant for patients with inherited bone marrow failure syndromes (iBMFs), rare genetic disorders for which HSCT is required to cure bone marrow failure (BMF), but for which HSCT is also associated with a high incidence of delayed donor engraftment, graft failure and morbidity due to chemotherapy and radiation sensitivity.In previous studies of the MPL-/- model of congenital amegakaryocytic thrombocytopenia, we discovered that decreased numbers of megakaryocytes and/or long-term (LT)-HSC caused by absent thrombopoietin/MPL signaling resulted in diminished capacity of the MPL-/- BM niche microenvironment to efficiently engraft wildtype (WT) donor HSC after HSCT. This observation led us to hypothesize that pre-existing BMF, regardless of cause, may generally alter HSC niche elements critical for engraftment after HSCT. We now present data testing this hypothesis in mouse models of Fanconi Anemia (FA) and Shwachman-Diamond Syndrome (SDS).To model FA, we used double mutant FANCC-/-;FANCG-/- mice, which have previously been demonstrated to develop manifestations of BMF. In our facility, FANCC-/-;FANCG-/- mice did not develop evidence of BMF, as measured by blood counts, BM cellularity, and HSC quantitation out to 1 year of age even with induction of HSC cycling by polyinosinic-polycytidilic acid (pIpC). Using SCT assays in which GFP+ WT donor BM was transplanted into FA recipients (FANCC-/-, FANCG-/-, FANCC-/-;FANCG-/-) or WT controls after 800-1100 cGy total body irradiation (TBI), efficiency and durability of donor engraftment was assessed at 3 weeks and 5 months, respectively, after primary SCT by competitive secondary HSCT. Unexpectedly, all FA recipients demonstrated more rapid BM reconstitution and enhanced early hematopoietic progenitor engraftment of WT donor BM compared with WT controls. However, there was no significant difference in LT-HSC engraftment efficiency or durability in the FA recipient groups versus WT controls.To model SDS, we first attempted to generate a model of osteoblast-specific SBDS deletion by crossing Col1A1Cre mice with mice carrying floxed SBDS alleles (SBDSfl/fl). However, this Col1A1CreSBDSExc model proved to be embryonic lethal at E12-E15 due to profound developmental abnormalities. We thus generated an inducible model by crossing SBDSfl/fl mice with Mx1Cre+ mice, and inducing SBDS deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by pIpC administration. SBDS deletion induced by pIpC was confirmed with PCR methods.Within 4 weeks of pIpC induction, Mx1CreSBDSExc mice show evidence of BMF, including a 40% reduction in platelet counts, inverted myeloid/lymphoid cell ratio in blood, and up to 80% reduction in immunophenotypic BM LT-HSC. When we attempted HSCT with GFP+ WT donor BM in Mx1CreSBDSExc and littermate control recipients following 1100 cGy TBI, 90% of Mx1CreSBDSExc recipients died by day 9 after HSCT, whereas all littermate controls survived. BM sections from Mx1CreSBDSExc recipients demonstrated persistent aplasia out to 1 week post-HSCT, suggesting that Mx1CreSBDSExc died from primary graft failure. Competitive secondary HSCT performed from BM harvested 1 week after primary HSCT demonstrated severe deficits in GFP+ WT donor HSC engraftment in Mx1CreSBDSExc recipients. Our initial studies as to why donor engraftment fails in Mx1CreSBDSExc recipients have identified that compared to WT, Mx1CreSBDSExc mice show significantly reduced expansion of osteolineage niche cells after TBI, a response that we have previously shown to be critical for efficient donor engraftment after HSCT.Taken together, our data demonstrate that pre-existing BMF and SBDS deficiency in BM osteolineage cells, but not FANCC or FANCG deficiency without co-existent BMF, significantly reduce the capacity of the BM niche to engraft donor HSC. Future studies defining specific niche deficits in our inducible SBDS deficiency model will provide critical insights into cellular and molecular pathways required for efficient engraftment after HSCT. DisclosuresNo relevant conflicts of interest to declare.