Bone Marrow-derived Hematopoietic Progenitor Cells Research Articles

Mechanical loading intensities affect the release of extracellular vesicles from mouse bone marrow-derived hematopoietic progenitor cells and change their osteoclast-modulating effect.

Low-intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high-intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 ± 0.3 Pa, 5 Hz) or high intensity (2.9 ± 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa-Β ligand-induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000× g) obtained after low-intensity loading and induced by exosomes (100 000× g) obtained after high-intensity loading. These osteoclast-modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT-independent exosome biogenesis (high intensity), as well as by inhibition of dynamin-dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast-modulating effect.

Enhanced Engraftment of Hematopoietic Progenitor Cells in Mice Treated With Granulocyte Colony-Stimulating Factor Before Low-Dose Irradiation: Implications for Gene Therapy

Gene therapy for inherited disorders of blood cells will require both efficient methods for stable gene transfer and nonablative bone marrow conditioning regimens to allow engraftment of modified hematopoietic progenitor cells (HPCs). We have used a sensitive murine system for detecting HPC engraftment using congenic C57BL/6 mice that differ at the Ly5 locus, which encodes the leukocyte common antigen. The system relies on the ability of monoclonal antibodies with specificity for Ly5.1 and Ly5.2 (revised nomenclature: CD45.1 and CD45.2, respectively) to distinguish donor and recipient peripheral blood leukocytes after transplantation of purified Sca-1+ bone marrow-derived HPCs. No detectable engraftment occurred in nonirradiated recipient mice, even when as many as 2.0 x 10(6) SCa-1+HPCs were transplanted. However, in mice receiving total body irradiation (TBI), engraftment increased as a function of pretransplantation radiation dose, number of transplanted cells, and time after transplantation. Moreover, mice receiving either granulocyte colony-stimulating factor (G-CSF) or G-CSF+ stem cell factor before low-dose TBI (160 cGy) exhibited a marked increase in engraftment compared with mice receiving a vehicle control before low-dose TBI (18.9% and 20.6% v 5.6% at a 1 month, respectively; 29% and 35% v 15.1% at 4 months, respectively). Use of growth factor pretreatment even allowed TBI doses as low as 30, 70, or 120 cGy to achieve significant engraftment of donor progenitors (0.3%, 1.5%, and 6.8% at 1 month, respectively; 1.7%, 5.8%, and 13.9% at 4 months, respectively). All animals remained healthy during the observation periods. Thus, growth factor preconditioning of the recipient followed by low-dose TBI may provide an optimal balance between safety and efficacy in achieving required levels of engraftment for gene therapy of blood disorders.

Stromal Cell-Derived Factor-1α Promotes Endothelial Colony-Forming Cell Migration Through the Ca2+-Dependent Activation of the Extracellular Signal-Regulated Kinase 1/2 and Phosphoinositide 3-Kinase/AKT Pathways.

Stromal cell-derived factor-1α (SDF-1α) drives endothelial colony-forming cell (ECFC) homing and incorporation within neovessels, thereby restoring tissue perfusion in ischemic tissues and favoring tumor vascularization and metastasis. SDF-1α stimulates ECFC migration by activating the Gi-protein-coupled receptor, CXCR4, and then engaging the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Sporadic evidence showed that SDF-1α may also act through an increase in intracellular Ca2+ concentration ([Ca2+]i) in bone marrow-derived hematopoietic progenitor cells and fully differentiated endothelial cells. Of note, recent evidence demonstrated that intracellular Ca2+ signals play a key role in controlling the proangiogenic activity of ECFCs. The present investigation was, therefore, undertaken to assess whether and how SDF-1α induces ECFC motility by triggering intracellular Ca2+ signals. We found that SDF-1α caused a dose-dependent increase in [Ca2+]i that was inhibited by ADM3100, a selective CXCR4 antagonist. Pharmacological manipulation revealed that the Ca2+ response to [Ca2+]i was shaped by an initial intracellular Ca2+ release through inositol-1,4,5-trisphosphate receptors (InsP3Rs), followed by a sustained phase of extracellular Ca2+ entry through store-operated Ca2+ channels. InsP3-dependent Ca2+ release and store-operated Ca2+ entry (SOCE) were both necessary for SDF-1α-induced extracellular signal-regulated kinases 1/2 (ERK 1/2) and AKT phosphorylation. Finally, SDF-1α employed intracellular Ca2+ signals, ERK 1/2, and PI3K/AKT to promote ECFC migration in vitro and neovessel formation in vivo. These data, therefore, provide the first evidence that SDF-1α induces ECFC migration through the Ca2+-dependent activation of the ERK 1/2 and PI3K/AKT pathways.

The Role of HIV-1 in Affecting the Proliferation Ability of HPCs Derived From BM.

HIV-1 causes chronic infection characterized by the depletion of CD4+ T lymphocytes and the development of AIDS. Current antiretroviral drugs inhibit viral spread, but they do not lead to a full immune recovery. Hematopoietic stem cells (HSCs) and multipotent hematopoietic progenitor cells (HPCs) give rise to all blood and immune cells, and in HIV infection, hematological abnormalities frequently occur in patients. Here, we used bone marrow samples from HIV-1-infected people to study the relationship between the proliferation ability of HSCs/HPCs and peripheral CD4+ T lymphocytes. Three indexes were used to reflect the proliferation ability of HSCs and HPCs: (1) colony-forming units of bone marrow mononuclear cells (BMMCs), (2) amplification of CD34+ cells purified from bone marrow mononuclear cells, (3) expression of HOXB4 and HOXA9 in CD34+ cells. We observed a direct correlation between peripheral number of CD4+ T lymphocytes and the HSCs/HPCs proliferation ability in our study. We also compared HIV-infected patients with or without antiretroviral therapy (ART). Our results demonstrated that after antiretroviral therapy, CD4+ T-cell recovery and HPCs proliferation ability are correlated. Our findings have implications in understanding whether bone marrow-derived HPCs can supplement for the loss of CD4+ T lymphocytes during HIV-1 infection.

T15: Circulating tumor cells and bone marrow progenitor cells in the blood of breast cancer patients in the dynamics of neoadjuvant chemotherapy

Background Despite the abundance of theoretical evidences displaying the considerable role of the “soil” in metastasis progression, the majority of cancer research and cancer therapy pathogenetic methods focused mainly on the tumor cells. There is still no clear clinical data showing the role of “soil” in the metastasis. Identification of factors and mechanisms driving the conditions promoting tumor dissemination may lead to therapeutic strategy to detect and prevent metastases at the earliest step. It has been established that bone marrow-derived hematopoietic progenitor cells home to pre-metastatic sites before tumor cells infiltrate in these sites. These hematopoietic progenitor cells form regulatory cell cluster of stromal microenvironment (premetastatic niche) to promote secondary tumor growth. In this context, the aim of our study was to evaluate the level of different pools of circulating tumor cells and bone marrow progenitor cells in the blood of patients with breast cancer in the dynamics of neoadjuvant chemotherapy. Materials and methods Patients (prospective study) with newly diagnosed invasive breast cancer in the age range 18–50 years and tumor volume of 2.0(> or =) cm, referred at the Tomsk Cancer Research Institute for treatment were included into the study. Eligibility criteria were as follows: the patient’s informed consent to participate in research; morphologically verified diagnosis of invasive carcinoma of non-specific type; luminal B-1, -2, triple negative (basal-like subtype included), HER2 positive breast cancer; T2-4N0-3M0; preserved menstrual function; satisfactory performance status (on a scale ( Results The study showed that each succeeding course of NACHT increased blood levels of circulating tumor cells, and bone marrow progenitor cells with a phenotype characteristic of EPC (endothelial progenitor cells) (CD34 + CD45–VEGFR + CD133 + CD202 +) and MSC (mesenchymal progenitor stem cells fibroblasts) (CD34–CD90 + VEGFR1–CD45–CD202–). Influence of the NACHT on hematopoietic stem cells HSC (VEGFR1 + CD34 + CD45lowSD202–) and progenitor cells HPC – CD34 + CD45 + CD90–VEGFR1 + CD133 – was ambiguous. Conclusion Neoadjuvant chemotherapy increases blood levels of circulating tumor cells, endothelial progenitor cells and mesenchymal stem cells progenitor fibroblasts, thus promoting the formation of premetastatic niche. The study was conducted with financial support from the Council for Grants of the President of the Russian Federation (Russia) for the state support of young philosophy doctors, agreements of SC 114.120.14.168-MD Russian Scientific Foundation (Russia), Grant No. 14-15-00318 (sample collection) and Russian Foundation for Basic Research (Russia), Grant No. 15-34-20864 . We acknowledge support of this work by the Tomsk State University Competitiveness Improvement Program (Russia).

Mastocytosis in the skin in children and adults

Background Mastocytosis is a rare disease of bone marrow-derived hematopoietic progenitor cells, which is characterized by abnormal growth and excessive accumulation of mast cells in various tissues. Cutaneous Mastocytosis is a skin limited disease, whereas Systemic Mastocytosis usually involves bone marrow, spleen, liver, lymph nodes and gastrointestinal tract and may present with or without skin involvement. The skin is one of the most commonly affected organ both in children and adults. Mastocytosis in the skin (MIS) is defined by a typical exanthema and monomorphic mast cell infiltrate. MIS is an initial diagnosis which requires further diagnostic steps leading to the final diagnosis of Cutaneous Mastocytosis or Systemic Mastocytosis.

Hematopoietic progenitor cells (HPCs) in node-negative invasive breast carcinomas: Immunohistochemical analysis and clinico-pathological correlations

Using immunohistochemistry, we investigated 603 negative lymph nodes from 51 patients affected by invasive breast cancer (BC) to recognize bone marrow-derived hematopoietic progenitor cells (HPCs). HPC aggregates, revealed by CD34, CD133, VEGFR1, and CD117 antisera, were determined by an intensity-distribution score (ID). Cases with an ID-score >3 at least for one marker were considered to strongly express HPCs. Twenty-five of 51 (49%) high expressor patients were identified by CD34 antiserum, while 24/51 (47.1%), 17/51 (33.3%), and 15/51 (29.4%) were identified by CD117, CD133, and VEGFR1, respectively. No significant relationships were found between HPCs status and histotype, tumor grade, stage, and hormone receptors, as determined at the moment of the first diagnosis. A significant correlation was recorded for Ki-67 values, as well as for death from invasive BC. No statistical significance was achieved regarding HER2 status, although a tendency toward a statistically significant P value was obtained. A significant relationship ( P < 0.001) was found between high expressors of HPC and progression of disease, documented by the development of distant metastases. An equivalent P value was ascertained for osseous localizations, with a lesser value in other metastatic sites. Regarding the appearance of distant metastases, the greatest efficiency value was obtained by CD133 (85.7%). Overall survival (OS) and distant metastases-free survival (DMFS) revealed a high statistical significance for HPC expression, Ki-67 values, and HER2 status. By multivariate analysis, HPC expression and Ki-67 values emerged as the higher independent prognostic variables in the analysis of DMFS and OS, respectively.

Downregulation of the CXC chemokine receptor 4/stromal cell–derived factor 1 pathway enhances myocardial neovascularization, cardiomyocyte survival, and functional recovery after myocardial infarction

Although adequate numbers of hematopoietic progenitor cells reside in the human bone marrow, the extent of endogenous neovascularization after myocardial infarction remains insufficient. The aim of this study was to identify the role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis in the mobilization and homing of hematopoietic progenitor cells in the ischemic heart. Human bone marrow-derived hematopoietic progenitor cells or saline were injected systemically into athymic nude rats 48 hours after myocardial infarction. Myocardial and bone marrow expression of stromal cell-derived factor 1 and chemotaxis of hematopoietic progenitor cells were measured in vitro in the presence or absence of stromal cell-derived factor 1. The role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis was investigated by means of antibody blockade or systemic administration of granulocyte colony-stimulating factor. Morphologic analysis included measurement of the infarct area, capillary density, and apoptosis, whereas left ventricular function was measured by means of echocardiographic analysis. Expression of postinfarct stromal cell-derived factor 1 was increased by 67% in the bone marrow and decreased by 43% in myocardium. Disruption of bone marrow stromal cell-derived factor 1/CXC chemokine receptor 4 interactions by antibody blockade resulted in a redirection of human hematopoietic progenitor cells from the bone marrow to the ischemic heart and augmented neovascularization and cardiomyocyte survival. Similarly, systemic administration of granulocyte colony-stimulating factor to block CXC chemokine receptor 4/stromal cell-derived factor 1 interaction resulted in increased mobilization and homing of hematopoietic progenitor cells to the ischemic heart, which translated to augmented myocardial neovascularization, prevention of apoptosis, and improved cardiac function. Bone marrow stromal cell-derived factor 1 upregulation after myocardial ischemia prevents mobilization of endogenous hematopoietic progenitor cells. We provide evidence that disruption of stromal cell-derived factor 1/CXC chemokine receptor 4 interactions allows redirection of hematopoietic progenitor cells to ischemic myocardium and enhances recovery of left ventricular function.

Vascular endothelial growth factor receptor‐1 in human cancer

The human vascular endothelial growth factor receptor-1 (VEGFR-1, or Flt-1) is widely expressed in normal and pathologic tissue and contributes to the pathogenesis of both neoplastic and inflammatory diseases. In human cancer, VEGFR-1 mediated signaling is responsible for both direct tumor activation and angiogenesis. VEGFR-1 mediated activation of nonmalignant supporting cells, particularly stromal, dendritic, hematopoietic cells, and macrophages, is also likely important for cancer pathogenesis. VEGFR-1 is also hypothesized to enable the development of cancer metastases by means of activation and premetastatic localization in distant organs of bone marrow-derived hematopoietic progenitor cells, which express VEGFR-1. IMC-18F1 is a fully human IgG(1) antibody that binds to VEGFR-1 and has been associated with the inhibition of cancer growth in multiple in vitro and human tumor xenograft models. The preliminary results of phase 1 investigations have also indicated a favorable safety profile for IMC-18F1 at doses that confer antibody concentrations that are associated with relevant antitumor activity in preclinical models.

Priming the ‘Soil’ for Breast Cancer Metastasis: The Pre-Metastatic Niche

The long prevailing model of metastasis recognizes the importance of both "seed" and "soil" for metastatic progression [1]. Much attention has focused on understanding the molecular and genetic factors that confer an intrinsic metastatic advantage to certain tumor cells. Meanwhile, changes occurring within distant tissues, creating a "soil" conducive for tumor invasion, have been largely neglected. Bone marrow-derived hematopoietic progenitor cells (HPCs) recently emerged as key players in initiating these early changes, creating a receptive microenvironment at designated sites for distant tumor growth and establishing the "Pre-Metastatic Niche" [2]. This insight into the earliest stages in the metastatic cascade revises our concept of the metastatic "microenvironment" to include physiological cells recruited from the bone marrow. Moreover, the concept of pre-metastatic tissues as 'niches' similar to physiological stem cell niches establishes a paradigm in which disseminated tumor cells may reside within a highly defined microcosm, both supportive and regulatory, and which may confer specific functions on indwelling cells. Understanding the cellular and molecular cross-talk between "seed" and "soil" may further our understanding of the factors that govern both site-specific patterning in metastasis and the phenomenon of tumor dormancy. This may lead to therapeutic strategies to detect and prevent metastasis at its earliest inception.

Bone marrow cells in the ‘pre-metastatic niche’: within bone and beyond

Metastasis, the spread of invasive carcinoma to sites distant from the primary tumor, is responsible for the majority of cancer-related deaths (Weigelt, B., Peterse, J. L., & van 't Veer, L. J. (2005). Breast cancer metastasis: Markers and models. Nature Reviews. Cancer, 5, 591-602). Despite progress in other areas of cancer therapeutics, the complexities of this process remain poorly understood. Consequently, there are few successful treatments that directly target this stage of carcinogenesis. Particularly enigmatic is the tissue-specificity of different tumor types observed in metastatic spread. One example is the predilection of colon cancer to spread to liver whereas breast, prostate, and lung carcinomas have a particular affinity to target and proliferate in bone. In 1889, Stephen Paget observed that circulating tumour cells would only "seed" where there was "congenial soil". Since then, attention has focused on explaining the dynamic adhesive and migratory capabilities intrinsic to tumor cells. Meanwhile, the earliest changes occurring within distant tissues that prime the "soil" to receive incoming cancer cells have largely been neglected. Recent work characterizing the importance of bone marrow-derived hematopoietic progenitor cells (HPC) in initiating these early changes has opened new avenues for cancer research and chemotherapeutic targeting (Kaplan, R. N., Riba, R. D., Zacharoulis, S., Bramley, A. H., Vincent, L., Costa, C., et al. (2005). VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature, 438, 820-827). This review discusses the inextricable relationship between bone stromal components, metastasizing cells, and bone marrow-derived hematopoietic cells, and their roles in carcinogenesis and metastasis. Understanding these dynamics may help explain the tissue-specific tropism seen in metastasis. Moreover, exploring the earliest events promoting circulating cancer cells to engraft and establish at secondary sites may expose new targets for diagnostic and therapeutic strategies and reduce the morbidity and mortality from metastatic disease.

In Vitro Proliferation and Expansion of Hematopoietic Progenitors from Patients with Diffuse Large B-Cell Lymphoma Before and After Chemotherapy

We have previously demonstrated that when cultured in Dexter-type Long-Term Marrow Cultures (LTMC), hematopoietic progenitor cells (HPC) from patients with Diffuse Large B-Cell Lymphoma (DLBCL) show a defective proliferation, as compared to HPC from normal marrow. In that study it was also demonstrated that functional alterations were present in the hematopoietic microenvironment developed in culture; thus, it was not clear whether such a defective proliferation in vitro was due to an intrinsic defect in the HPC compartment of DLBCL patients, or to an altered microenvironment, or both. In order to address this question, in the present study we have assessed the proliferation and expansion potentials of HPC present in bone marrow from patients with DLBCL, in cytokine-supplemented liquid cultures initiated with a cell population enriched for CD34+ Lin- cells, in the absence of stromal cells and in the presence of reduced numbers of accessory cells. Our results demonstrated that bone marrow-derived HPC from patients with DLBCL, both before and right after chemotherapy, possessed reduced proliferation and expansion potentials in vitro, as compared to their normal counterparts. Interestingly, in patients analyzed 18 months after treatment the proliferation and expansion levels were similar to those of normal HPC, indicating a complete restoration of the hematopoietic function. Although the reason for these observations is not clear, our results suggest the possibility that primitive CD34+ progenitor cells present in bone marrow, which show deficient proliferation and expansion potentials in vitro, are involved in the origin/progression of DLBCL.

Overproduction of PU.1 in mast cell progenitors: its effect on monocyte- and mast cell-specific gene expression

The Ets family transcription factor PU.1 is required for development of various lymphoid and myeloid cell lineages, and regulates the expression of several genes in a cell type-specific manner. Mouse bone marrow-derived hematopoietic progenitor cells are programmed to differentiate into mast cells, when the cells are maintained in the presence of pokeweed mitogen-stimulated spleen-conditioned medium. However, by retroviral introduction of PU.1 cDNA, the progenitor cells expressed MHC class II, CD11b, CD11c, and F4/80, and acquired the ability to stimulate T cells. Furthermore, PU.1-overproducing cells exhibited the morphology, in part, similar to that of monocyte. These results indicate that the mast cell progenitors still have the ability to express monocyte-specific genes by increased expression of PU.1.

Efficient generation of human T cells from a tissue-engineered thymic organoid

Biocompatible inorganic matrices have been used to enhance bone repair by integrating with endogenous bone architecture. Hypothesizing that a three-dimensional framework might support reconstruction of other tissues as well, we assessed the capacity of a tantalum-coated carbon matrix to support reconstitution of functioning thymic tissue. We engineered a thymic organoid by seeding matrices with murine thymic stroma. Co-culture of human bone marrow-derived hematopoietic progenitor cells within this xenogeneic environment generated mature functional T cells within 14 days. The proportionate T-cell yield from this system was highly reproducible, generating over 70% CD3+ T cells from either AC133+ or CD34+ progenitor cells. Cultured T cells expressed a high level of T-cell receptor excision circles (TREC), demonstrating de novo T lymphopoiesis, and function of fully mature T cells. This system not only facilitates analysis of the T-lymphopoietic potential of progenitor cell populations; it also permits ex vivo genesis of T cells for possible applications in treatment of immunodeficiency.

Chemotactic Response Toward Chemokines and Its Regulation by Transforming Growth Factor-β1 of Murine Bone Marrow Hematopoietic Progenitor Cell-Derived Different Subset of Dendritic Cells

Dendritic cells (DCs) are highly specialized antigen-presenting cells that distribute widely in all organs. DCs initiate the primary immune response and activate naive T cells and B cells responsible for the acquired immunity. In this study, CCR7 mRNA was proved to be expressed in DCs and their precursors derived from murine bone marrow-derived hematopoietic progenitor cells (HPCs), whereas CCR1 mRNA was expressed in both CD11b-/dullCD11c+ and CD11b+hiCD11c+ DC precursors. CCR6 mRNA was not detected in any murine DC populations. In agreement with the chemokine receptor mRNA expression by each population in the DC differentiation pathway, SLC (also termed as MIP-3beta), one of the ligands for CCR7, strongly and selectively chemoattracted both CD11b-/dullCD11c+ and CD11b+hiCD11c+ DC precursors (days 6 to 7) and more mature DCs (days 13 to 14). We have recently found that transforming growth factor-beta1 (TGF-beta1), a cytokine that is essential for the appearance of Langerhans cells in the skin, polarizes murine HPCs to generate Langerhans-like cells through monocyte/macrophage differentiation pathway. We observed here that TGF-beta1 not only inhibited the expression of CCR7 in DCs and DC precursors derived from HPCs, but also inhibited the migration of these cells in response to SLC. This is the first report describing the chemokine and chemokine receptors responsible for murine DC migration and downregulation of DC migration by TGF-beta1.

TIBO R82913, A New HIV-1 Inhibiting Agent, Does Not Inhibit Hematopoietic Progenitor Cells

In progressive stages of infection with human immunodeficiency virus type 1 (HIV-1), the majority of patients develop a pathophysiologically not yet completely explainable bone marrow failure with anemia, leukopenia, and thrombocytopenia. The clinically most widely used HIV-inhibiting antiviral drugs azidothymidine (AZT) and dideoxyinosine (ddI) frequently are hematotoxic to the host, resulting in dose reduction or discontinuation of antiviral therapy. In recent studies, a novel series of benzodiazepine derivatives highly active against HIV-1 was synthesized. These antiviral compounds have a much more favorable therapeutical index than the well-known 2'3'-dideoxyribosides, like AZT. In the experiments presented here, the authors investigated the most promising derivative R82913 [(+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl- 6-(3-methyl-2-butenyl)-imidazo[4,5,1-jk] [1,4]-benzodiazepin-2(1H)-thione] (TIBO) with regard to its toxicity on bone marrow-derived hematopoietic progenitor cells from six HIV-1+ and HIV- persons, respectively. In methylcellulose assays for hematopoietic colony growth any hematotoxic effects of R82913 in vitro were excluded, as both groups showed no difference of progenitor cell growth with or without the TIBO derivative, even at concentrations 6.7 x 10(4) times higher than the 50% inhibitory concentration for cytopathicity by HIV-1.