Hematopoietic Stem Cell Microenvironment Research Articles

Identification of Cellular Compositions in Different Microenvironments and Their Potential Impacts on Hematopoietic Stem Cells HSCs Using Single-Cell RNA Sequencing with Systematical Confirmation.

Hematopoietic stem cells (HSCs) are stem cells that can differentiate into various blood cells and have long-term self-renewal capacity. At present, HSC transplantation is an effective therapeutic means for many malignant hematological diseases, such as aplastic hematological diseases and autoimmune diseases. The hematopoietic microenvironment affects the proliferation, differentiation, and homeostasis of HSCs. The regulatory effect of the hematopoietic microenvironment on HSCs is complex and has not been thoroughly studied yet. In this study, we focused on mononuclear cells (MNCs), which provided an important microenvironment for HSCs and established a methodological system for identifying cellular composition by means of multiple technologies and methods. First, single-cell RNA sequencing (scRNA-seq) technology was used to investigate the cellular composition of cells originating from different microenvironments during different stages of hematopoiesis, including mouse fetal liver mononuclear cells (FL-MNCs), bone marrow mononuclear cells (BM-MNCs), and in vitro-cultured fetal liver stromal cells. Second, bioinformatics analysis showed a higher proportion and stronger proliferation of the HSCs in FL-MNCs than those in BM-MNCs. On the other hand, macrophages in in vitro-cultured fetal liver stromal cells were enriched to about 76%. Differential gene expression analysis and Gene Ontology (GO) functional enrichment analysis demonstrated that fetal liver macrophages have strong cell migration and actin skeleton formation capabilities, allowing them to participate in the hematopoietic homeostasis through endocytosis and exocytosis. Last, various validation experiments such as quantitative real-time PCR (qRT-PCR), ELISA, and confocal image assays were performed on randomly selected target genes or proteins secreted by fetal liver macrophages to further demonstrate the potential relationship between HSCs and the cells inhabiting their microenvironment. This system, which integrates multiple methods, could be used to better understand the fate of these specific cells by determining regulation mechanism of both HSCs and macrophages and could also be extended to studies in other cellular models.

Immune System Influence on Hematopoietic Stem Cells and Leukemia Development.

Hematopoietic stem cells (HSCs) are the source for all blood cells, including immune cells, and they interact dynamically with the immune system. This chapter will explore the nature of stem cells, particularly HSCs, in the context of their immune microenvironment. The dynamic interactions between stem cells and the immune system can have profound implications for current and future therapies, particularly regarding a potential "immune-privileged" HSC microenvironment. Immune/stem cell interactions change during times of stress and injury. Recent advances in cancer immunotherapy have overturned the long-standing belief that, being derived from the self, cancer cells should be immunotolerant. Instead, an immunosurveillance system recognizes and eliminates emergent pre-cancerous cells. Only in the context of a failing immunosurveillance system does cancer fully develop. Combined with the knowledge that stem cells or their unique properties can be critically important for cancer initiation, persistence, and resistance to therapy, understanding the unique immune properties of stem cells will be critical for the development of future cancer therapies. Accordingly, the therapeutic implications for leukemic stem cells (LSCs) inheriting an immune-privileged state from HSCs will be discussed. Through their dynamic interactions with a diverse immune system, stem cells serve as the light and dark root of cancer prevention vs. development.

Emerging Evidence of the Significance of Thioredoxin-1 in Hematopoietic Stem Cell Aging.

The United States is undergoing a demographic shift towards an older population with profound economic, social, and healthcare implications. The number of Americans aged 65 and older will reach 80 million by 2040. The shift will be even more dramatic in the extremes of age, with a projected 400% increase in the population over 85 years old in the next two decades. Understanding the molecular and cellular mechanisms of ageing is crucial to reduce ageing-associated disease and to improve the quality of life for the elderly. In this review, we summarized the changes associated with the ageing of hematopoietic stem cells (HSCs) and what is known about some of the key underlying cellular and molecular pathways. We focus here on the effects of reactive oxygen species and the thioredoxin redox homeostasis system on ageing biology in HSCs and the HSC microenvironment. We present additional data from our lab demonstrating the key role of thioredoxin-1 in regulating HSC ageing.

Deconvolution of the hematopoietic stem cell microenvironment reveals a high degree of specialization and conservation

SummaryUnderstanding the regulation of normal and malignant human hematopoiesis requires comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Here, we develop a tailored bioinformatic pipeline to integrate public and proprietary single-cell RNA sequencing (scRNA-seq) datasets. As a result, we robustly identify for the first time 14 intermediate cell states and 11 stages of differentiation in the endothelial and mesenchymal BM compartments, respectively. Our data provide the most comprehensive description to date of the murine HSC-regulatory microenvironment and suggest a higher level of specialization of the cellular circuits than previously anticipated. Furthermore, this deep characterization allows inferring conserved features in human, suggesting that the layers of microenvironmental regulation of hematopoiesis may also be shared between species. Our resource and methodology is a stepping-stone toward a comprehensive cell atlas of the BM microenvironment.

3164 – A CELLULAR TAXONOMY OF MURINE BONE MARROW STROMAL STEM AND PROGENITOR CELLS UPON DEVELOPMENT

Mesenchymal stem cells (MSCs) in the bone marrow (BM) are thought to play pivotal roles in tissue (re)generation and provide a supportive microenvironment for hematopoietic stem cells (HSCs). MSCs have been well characterized in murine models and isolated using different combinations of markers. Yet, these markers define a broader and more heterogeneous population, of which MSCs are only a minority. Although several studies indicate that bona fide MSCs reside in the BM, further investigations are needed to define their identity and allow their prospective and effective isolation. Here, using a single cell RNA sequencing approach, we aimed to delineate the cellular heterogeneity of the MSC-enriched Sca-1+ CD51+ population throughout development. At the fetal stage (E17), the BMSC population appeared relatively homogeneous with subsets predicted to contain candidate MSCs based on marker expression and computational modeling. The frequency of putative MSC subsets declined upon aging. After birth, the BMSC population was more heterogeneous, and characterized by the emergence of clusters showing transcriptional wiring consistent with specialization towards differentiated (progenitor) subsets. This included the postnatal emergence of a Lepr+ population, expressing HSC regulatory genes and previously established as HSC niches. In the fetal BM, subsets of endothelial cells expressed HSC factors and may thus represent an initial supportive microenvironment for emerging hematopoiesis. Our results shed light on the cellular diversity of BMSCs from different developmental phases, providing a cellular taxonomy of this population at single cell resolution. The exploration of our dataset, and the comparison of transcriptional landscapes from different developmental stages, may provide a resource to identify and further validate factors with a predicted role in BM regeneration. Mesenchymal stem cells (MSCs) in the bone marrow (BM) are thought to play pivotal roles in tissue (re)generation and provide a supportive microenvironment for hematopoietic stem cells (HSCs). MSCs have been well characterized in murine models and isolated using different combinations of markers. Yet, these markers define a broader and more heterogeneous population, of which MSCs are only a minority. Although several studies indicate that bona fide MSCs reside in the BM, further investigations are needed to define their identity and allow their prospective and effective isolation. Here, using a single cell RNA sequencing approach, we aimed to delineate the cellular heterogeneity of the MSC-enriched Sca-1+ CD51+ population throughout development. At the fetal stage (E17), the BMSC population appeared relatively homogeneous with subsets predicted to contain candidate MSCs based on marker expression and computational modeling. The frequency of putative MSC subsets declined upon aging. After birth, the BMSC population was more heterogeneous, and characterized by the emergence of clusters showing transcriptional wiring consistent with specialization towards differentiated (progenitor) subsets. This included the postnatal emergence of a Lepr+ population, expressing HSC regulatory genes and previously established as HSC niches. In the fetal BM, subsets of endothelial cells expressed HSC factors and may thus represent an initial supportive microenvironment for emerging hematopoiesis. Our results shed light on the cellular diversity of BMSCs from different developmental phases, providing a cellular taxonomy of this population at single cell resolution. The exploration of our dataset, and the comparison of transcriptional landscapes from different developmental stages, may provide a resource to identify and further validate factors with a predicted role in BM regeneration.

Heterozygous lipoprotein lipase knockout mice exhibit impaired hematopoietic stem/progenitor cell compartment.

BackgroundHematopoietic stem cells (HSC) maintain the hematopoietic system homeostasis through self‐renewal and multilineage differentiation potential. HSC are regulated by the microenvironment, cytokine signaling, and transcription factors. Recent results have shown that lipid pathways play a key role in the regulation of HSC quiescence, proliferation, and division. However, the mechanism by which lipid metabolism regulates HSC proliferation and differentiation remains to be clarified. Lipoprotein lipase (LPL) is an essential enzyme in the anabolism and catabolism of very low‐density lipoprotein, chylomicrons, and triglyceride‐rich lipoproteins.MethodsThe percentage of hematopoietic stem/progenitor cells and immune cells were determined by fluorescence‐activated cell sorting (FACS). The function and the mechanism of HSCs were analyzed by cell colony forming assay and qPCR analysis. The changes in LPL+/− HSC microenvironment were detected by transplantation assays using red fluorescent protein (RFP) transgenic mice.ResultsTo explore the function of LPL in HSC regulation, heterozygous LPL‐knockout mice (LPL+/−) were established and analyzed by FACS. LPL+/− mice displayed decreased hematopoietic stem/progenitor cell compartments. In vitro single‐cell clonogenic assays and cell‐cycle assays using FACS promoted the cell cycle and increased proliferation ability. qPCR analysis showed the expression of p57KIP2 and p21WAF1/CIP1 in LPL+/− mice was upregulated.ConclusionsLPL+/− mice exhibited HSC compartment impairment due to promotion of HSC proliferation, without any effects on the bone marrow (BM) microenvironment.

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.

Human hematopoietic microenvironments.

Dormancy of hematopoietic stem cells and formation of progenitors are directed by signals that come from the bone marrow microenvironment. Considerable knowledge has been gained on the murine hematopoietic stem cell microenvironment, while less so on the murine progenitor microenvironment and even less so on these microenvironments in humans. Characterization of these microenvironments is decisive for understanding hematopoiesis and finding new treatment modalities against bone marrow malignancies in the clinic. However, it is equally challenging, because hematopoietic stem cells are difficult to detect in the complex bone marrow landscape. In the present study we are characterizing the human hematopoietic stem cell and progenitor microenvironment. We obtained three adjacent bone marrow sections from ten healthy volunteers. One was used to identify a population of CD34+/CD38- “hematopoietic stem cells and multipotent progenitors” and a population of CD34+/CD38+ “progenitors” based on immunofluorescence pattern/intensity and cellular morphology. The other two were immunostained respectively for CD34/CD56 and for CD34/SMA. Using the combined information we performed a non-computer-assisted quantification of nine bone marrow components (adipocytes, megakaryocytes, bone surfaces, four different vessel types (arteries, capillaries, sinusoids and collecting sinuses), other “hematopoietic stem cells and multipotent progenitors” and other “progenitors”) within 30 μm of “hematopoietic stem cells and multipotent progenitors”, “progenitors”, and “random cell profiles”. We show that the microenvironment of the “hematopoietic stem cells and multipotent progenitors” is significantly enriched in sinusoids and megakaryocytes, while the microenvironment of the “progenitors” is significantly enriched in capillaries, other “progenitors”, bone surfaces and arteries.

Role of mesenchymal stromal/stem cells in regulation of hemotoration in 3D <i>in vitro</i> culture

The article is devoted to studying the role of mesenchymal stromal cells in formation of microenvironment for hematopoietic stem cells under the conditions mimicking physiological bone remodeling in presence of artificial three-dimensional matrices (Ra = 2-3 μm). The study was carried out using experimental samples of artificial implants obtained in electrolyte from hydroxyapatite nanoparticles (HAP) produced at the Institute of Strength Physics and Materials Science (Siberian Branch of the Russian Academy of Sciences). The work included cultural and instrumental research techniques. Phenotypic profile of cells was assessed by flow cytometry. Determination of cytokine/chemokine levels from cell culture supernatants was assessed by flow fluorimetry. Detection of hematopoietic cells in the vision fields, as well as areas of extracellular matrix mineralization was carried out by means of cytomorphometry.It was revealed that the 3D matrices with a calcium phosphate coating initiate the in vitro formation of specific microenvironment of MSCs, resulting in the increased numbers of HSCs with the CD45+CD34+ phenotype (at 14 days), an increased number of cells with hematopoietic morphology and evolving foci of extracellular matrix mineralization of the (at 21 days). Changed numbers of hematopoietic cells per vision field occurred, mainly, due to indirect effect of hematopoietic factors (SCF and G-CSF), along with decrease of proapoptotic factor TRAIL. It was also found that MSCs reduce the level of proinflammatory cytokines (IFNγ, TNFα, IP-10, IL-2, IL-6) in culture medium in the presence of artificial 3D calcium-phosphate-coated matrices. The revealed features of MSC functioning under the conditions simulating physiological bone remodeling, upon co-cultures with three-dimensional matrices (Ra = 2-3 μm), have shown a significant effect of MSCs upon regulation of HSCs by local microenvironment, through distinct modulating effects of cytokines, chemokines, and growth factors that provide intercellular interactions. Development of extracellular matrix mineralization areas during MSC cultivation in the presence of 3D matrices imitating mineral substance of bone tissue also indicates the formation of osteoblastic niches under the in vitro cultivation conditions.The results obtained are important in order to assess functions of hematopoietic niches and the role of MSCs in their development and maintenance of the microenvironment.The results obtained may find practical application in development of new classes of medical devices able to provide effective osseointegration.

Osteoblastic Wntless deletion differentially regulates the fate and functions of bone marrow-derived stem cells in relation to age.

Although functional association between Wnt signaling and bone homeostasis has been well described through genetic ablation of Wntless (Wls), the mechanisms of how osteoblastic Wls regulates the fate of bone marrow stromal cells (BMSCs) and hematopoietic stem cells (HSCs) in relation to age are not yet understood. Here, we generated Col2.3-Cre;Wlsfl/fl mice that were free from premature lethality and investigated age-related impacts of osteoblastic Wls deficiency on hematopoiesis, BM microenvironment, and maintenance of BMSCs (also known as BM-derived mesenchymal stem/stromal cells) and HSCs. Ablation of osteoblastic Wls deteriorated BM microenvironment and bone mass accrual along with age-independent effects on functions of BMSCs. Osteoblastic Wls deletion impaired HSC repopulation and progeny with skewing toward myeloid lineage cells only at old stage. As proven by hallmarks of stem cell senescence, osteoblastic Wls ablation differentially induced senescence of BMSCs and HSCs in relation to age without alteration in their BM frequency. Our findings support that deletion of Wls in Col2.3-expressing cells induces senescence of BMSCs and impairs BM microenvironment in age-independent manner. Overall, long-term deterioration in BM microenvironment contributes to age-related HSC senescence with impaired progeny and hematopoiesis, which also suggests possible roles of osteoblastic Wls on the maintenance of BM HSCs.

Hematopoietic stem cells from pluripotent stem cells: Clinical potential, challenges, and future perspectives.

The generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) is an active and promising area of research; however, generating engraftable HSCs remains a major obstacle. Ex vivo HSC derivation from renewable sources such as iPSCs offers an experimental tool for studying developmental hematopoiesis, disease modeling, and drug discovery, and yields tremendous therapeutic potential for malignant and nonmalignant hematological disorders. Although initial attempts mostly recapitulated yolk sac primitive/definitive hematopoiesis with inability to engraft, recent advances suggest the feasibility of engraftable HSC derivation from iPSCs utilizing ectopic transcription factor expression. Strategic development for de novo HSC generation includes further investigations of HSC ontogeny, and elucidation of critical signaling pathways, epigenetic modulations, HSC and iPSC microenvironment, and cell‐cell interactions that contribute to stem cell biology and function.

Bmi1 Maintains the Self-Renewal Property of Innate-like B Lymphocytes.

The self-renewal ability is a unique property of fetal-derived innate-like B-1a lymphocytes, which survive and function without being replenished by bone marrow (BM) progenitors. However, the mechanism by which IgM-secreting mature B-1a lymphocytes self-renew is poorly understood. In this study, we showed that Bmi1 was critically involved in this process. Although Bmi1 is considered essential for lymphopoiesis, the number of mature conventional B cells was not altered when Bmi1 was deleted in the B cell lineage. In contrast, the number of peritoneal B-1a cells was significantly reduced. Peritoneal cell transfer assays revealed diminished self-renewal ability of Bmi1-deleted B-1a cells, which was restored by additional deletion of Ink4-Arf, the well-known target of Bmi1 Fetal liver cells with B cell-specific Bmi1 deletion failed to repopulate peritoneal B-1a cells, but not other B-2 lymphocytes after transplantation assays, suggesting that Bmi1 may be involved in the developmental process of B-1 progenitors to mature B-1a cells. Although Bmi1 deletion has also been shown to alter the microenvironment for hematopoietic stem cells, fat-associated lymphoid clusters, the reported niche for B-1a cells, were not impaired in Bmi1 -/- mice. RNA expression profiling suggested lysine demethylase 5B (Kdm5b) as another possible target of Bmi1, which was elevated in Bmi1-/- B-1a cells in a stress setting and might repress B-1a cell proliferation. Our work has indicated that Bmi1 plays pivotal roles in self-renewal and maintenance of fetal-derived B-1a cells.

Rolling Signal-Based Ripley's K: A New Algorithm to Identify Spatial Patterns In Histological Specimens

The spatial distribution, or pattern, of cells within a tissue underlies organ function. However, these are difficult to identify, making it challenging to evaluate how these patterns are established, or how diseases may disrupt and impair their function. To address this, we developed an algorithm that identifies spatial patterns within tissues and used it to study the bone marrow, a specialized microenvironment in which spatial patterning of regulatory cells influence the cell fate of hematopoietic stem cells. Using this algorithm, we discovered clusters of cells within the bone marrow that suggest an organization of micro-niches, which may form the basis of the hematopoietic stem cell microenvironment. This work provides a new tool for the identification of spatial patterns within tissues that can lead to a deeper understanding of tissue function, provide clues for the early onset of disease, and be used for studying the impact of pharmaceutics on tissue regeneration.

Peri-arteriolar megakaryopoietic microenvironment via reciprocal CLEC-2/PDPN axis in bone marrow

Bone marrow (BM), the tissue specializing in the production of hematopoietic cells, consists of multiple components (e.g., extracellular matrixes, vasculatures, and stromal cells) that generate a complex three-dimensional network and several localized microenvironment. These microenvironments regulate hematopoietic stem and progenitor cells, including megakaryocyte lineage cells. In this review, we first provide an overview of the microenvironment for hematopoietic stem cells as an introduction to bone marrow microenvironment and subsequently summarize the microenvironment for megakaryocyte differentiation and maturation (megakaryopoiesis). In the last portion, we describe megakaryocyte regulation by podoplanin-positive peri-arteriolar stromal cells in the mouse bone marrow.

Generation of Organotypic Multicellular Spheres by Magnetic Levitation: Model for the Study of Human Hematopoietic Stem Cells Microenvironment.

Background and ObjectiveThe characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mesenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC.Methods and ResultsHuman bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, Lonza®) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm3 and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells.ConclusionsWe developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.

Inhibition of Endothelial mTOR Drives Hematopoietic Stem Cell Aging

Aging leads to a drastic decline in hematopoietic stem cell (HSC) function. In addition to a loss of their self-renewal potential, old HSCs exhibit a myeloid biased differentiation and an increased propensity to develop hematologic malignancies. While some of these age-related changes reflect cell-intrinsic alterations within HSCs, recent findings suggest that signals from the bone marrow (BM) microenvironment, in particular the BM vascular niche, might play crucial roles in regulating HSC aging. In support of this idea, we recently discovered that physiological aging of BM endothelial cells leads to an altered molecular crosstalk between the endothelial niche and HSCs that instructs young HSCs to behave as aged HSCs. We have previously demonstrated that AKT/mTOR activation within young BM endothelial cells supports balanced HSC self-renewal and differentiation. Based on these observations, we hypothesized that deficient AKT/mTOR signaling within endothelium will deprive the hematopoietic system of proper endothelial-derived instructive signals resulting in HSC aging phenotypes. We have generated preliminary data demonstrating that aged endothelial cells, in contrast to aged HSCs, display decreased AKT/mTOR signaling. Furthermore, our data indicates that pharmacological inhibition of mTOR signaling using Rapamycin, a widely regarded rejuvenating agent and clinically utilized immunosuppressant, has deleterious effects on the hematopoietic system, in part due to its adverse impact on endothelium by abolishing both mTORC1 and mTORC2 signaling, in homeostatic conditions and following myelosuppressive therapy (i.e. radiation and chemotherapy). To formally determine whether endothelial-specific inhibition of mTOR signaling can promote hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that HSCs from mTOR(ECKO) mice displayed all the phenotypic and functional attributes of an aged HSC including loss of cell polarity, increased DNA damage, myeloid biased output and loss of engraftment potential during serial transplantation. Gene expression profiling of HSCs isolated from young mTOR(ECKO) mice revealed that their intrinsic gene expression signature resembled aged HSCs, both at steady state and following transplantation. Notably, these aging-like phenotypic and functional HSC alterations are only maintained in secondary transplants that were performed in an mTOR(ECKO) microenvironment confirming the role of EC-derived instructive signals in governing HSC aging. Furthermore, we assessed the function of BM endothelial cells from mTOR(ECKO) mice and revealed severe defects in overall endothelial health which included alterations in proliferation, wound healing ability, and their metabolic profile that displayed a hyperglycolytic phenotype at steady state as well as increased glycolytic capacity under stress conditions, compared to control BMECs. Moreover, mTOR(ECKO) BMECs showed high respiratory capacity, thus phenocopying the metabolic profile of aged and tumor-associated BMECs. Furthermore, the BM microenvironment of mTOR(ECKO) mice manifested an increase in a NF-kB dependent inflammatory signature. Interestingly, chronic inflammation driven by NF-kB signaling is the leading cause of vascular dysfunction and aging. In summary, we demonstrate that deletion of mTOR signaling within young endothelium drives premature aging of HSCs and the hematopoietic system. Utilizing our model system, we have now identified candidate pro-aging and pro-rejuvenation factors that will lay the foundation for designing therapeutic strategies to rejuvenate the HSC microenvironment, thereby restoring the functional properties of the hematopoietic system to youthful levels and improving overall health. DisclosuresNo relevant conflicts of interest to declare.

Novel peptides derived from neuropeptide Y prevent chemotherapy-induced bone marrow damage by regulating hematopoietic stem cell microenvironment

ABSTRACTChemotherapy-induced bone marrow damage is accompanied by acute nerve injury in the bone marrow (BM), resulting in sensory and autonomic neuropathy. Cisplatin, a popular chemotherapy drugs, induces the impairment of hematopoietic stem cells (HSCs) and bone marrow regeneration, leading to chronic bone marrow abnormalities. Previously, we reported the protective roles of neuropeptide Y (NPY) against cisplatin-induced bone marrow impairment. In this study, we identified novel peptides, generated from full-length NPY that rescued cisplatin-induced sensory neuropathy and HSC suppression by regulating cell survival in the BM microenvironment. One of these peptides, especially, showed a better protective property against these impairments compared to that seen in full-length NPY. Therefore, we suggest the NPY sequences most effective against the chemotherapy-induced bone marrow dysfunction that could be potentially useful as therapeutic agents for patients receiving chemotherapy.

241 - Three-dimensional tissue culture system by the magnetic levitation method for the study of the hematopoietic stem cells microenvironment

241 - Three-dimensional tissue culture system by the magnetic levitation method for the study of the hematopoietic stem cells microenvironment

Evaluation of proinflammatory and immunosuppressive cytokines in blood and bone marrow of healthy hematopoietic stem cell donors

IntroductionCytokine composition of bone marrow microenvironment in comparison to blood is poorly explored. The goal of this study was to investigate the levels of cytokines present in peripheral blood and bone marrow of healthy hematopoietic stem cells donors. The data obtained on this subject with addition to cytometric analysis can provide new insight into the hematopoietic stem cells microenvironment. MethodologyStudy consisted of cytokine concentration analysis performed by ELISA tests of peripheral blood of healthy peripheral blood stem cells donors and bone marrow of healthy bone marrow donors. Additionally we have tested the expression of CD47 and CD274 proteins on the surface of hematopoietic stem cells by the flow cytometry analysis. ResultsThe results has shown different composition of analyzed cytokines (IL-1 β, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-β1, IFN-γ and TNF-α) present in bone marrow and blood of stem cells donors. The hematopoietic stem cells in peripheral blood are subjected to higher levels of proinflammatory cytokines whilst the lower level of those cytokines in bone marrow with a very high level of TGF-β1 which possibly creates a more immunosuppressive environment. The IL-10 level was significantly higher in peripheral blood of PBSC donors after the administration of mobilizing factor (G-CSF). The percentage of CD47+HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors.

Low-Dose Irradiation Promotes Persistent Oxidative Stress and Decreases Self-Renewal in Hematopoietic Stem Cells

Despite numerous observations linking protracted exposure to low-dose (LD) radiation and leukemia occurrence, the effects of LD irradiation on hematopoietic stem cells (HSCs) remain poorly documented. Here, we show that adult HSCs are hypersensitive to LD irradiation. This hyper-radiosensitivity is dependent on an immediate increase in the levels of reactive oxygen species (ROS) that also promotes autophagy and activation of the Keap1/Nrf2 antioxidant pathway. Nrf2 activation initially protects HSCs from the detrimental effects of ROS, but protection is transient, and increased ROS levels return, promoting a long-term decrease in HSC self-renewal. Invivo, LD total body irradiation (TBI) does not decrease HSC numbers unless the HSC microenvironment is altered by an inflammatory insult. Paradoxically, such an insult, in the form of granulocyte colony-stimulating factor (G-CSF) preconditioning, followed by LD-TBI facilitates efficient bone marrow transplantation without myeloablation. Thus, LD irradiation has long-term detrimental effects on HSCs that may result in hematological malignancies, but LD-TBI may open avenues to facilitate autologous bone marrow transplantation.