Bone Marrow Hematopoiesis Research Articles

Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1.

KIT D816V–mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression

AbstractMultilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V–mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome.

Loss of c-Kit and bone marrow failure upon conditional removal of the GATA-2 C-terminal zinc finger domain in adult mice.

Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.

State of erythroid, granulocyte and platelet branches of hematopoiesis on stages of chemotherapy in children with acute lymphoblastic leukemia, who were exposed to ionizing radiation after the Chornobyl NPP accident

Evaluation of proliferation and differentiation processes of progenitor cells in bone marrow by the com position of elements of erythroid, granulocyte and platelet branches of hematopoiesis on the treatment stages in children with acute lymphoblastic leukemia (ALL), who were exposed to radiation from the Chornobyl NPP accident. The 46 children with ALL were studied, who lived in Kyiv, Zhytomyr and Chernihiv regions. Studies were conducted before the start of chemotherapy (ChT), on the 33 day of ChT (phase I), and after the com pletion of ChT (phase II). Exposure doses of patients, hemogram and myelogram parameters both with indices of mat uration of progenitor cells were evaluated. Signs of dysplasia of hematopoietic branch elements were revewed. The 46 patients were studied. They have had the B ALL, namely pro B ALL (n=5), «common type» (n=36), pre B ALL (n=3), and T ALL in 2 other cases. In a debut of ALL the bone marrow was represented by lymphoblasts. Along with ChT conduction the bone marrow hematopoiesis recovered by such types, as erythroid, granulocyte, gran ulocyte whith monocytes, and uniform, when the cells number of all branches was within a normal quantity. At the phase ІІ of ChT the number of patients with hematopoiesis recovery by erythroid type decreased and number of chil dren with activation of granulocyte branch of hematopoiesis increased. In children with pro B ALL the number of erythroid elements was higher than normative at both ChT phases. A direct correlation was established between the number of myelokaryocytes (Mkc) and megakaryocytes (Mgkc) in both phase І and phase ІІ of treatment (Rs = +0.72; Rs = +0.56, respectively). There was no correlation between the radiation dose in patients (3.73 ± 0.12 mSv) and studied parameters. Types of bone marrow recovery were established in ALL patients after the ChT indicating to the differ ent kinetic pathways of hematopoietic progenitor cells. Evaluation of reasons of prevalence of some hematopoietic branches will allow to reveal their role in leukemogenesis and to correct the treatment programs.

KIT D816V Mutation Positive Bone Marrow Mesenchymal Stem Cells in Indolent Systemic Mastocytosis Are Associated with Disease Progression

PURPOSEMultilineageinvolvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here we investigated the potential involvement of BM mesenchymal stem cells (MSC) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. METHODSThe KIT D816V mutation was investigated in highly-purified BM MSC and other BM cell populations from 83 ISM patients followed for a median of 116 months. MC clonality was further evaluated in female patients by the pattern of inactivation of the X chromosome (XCIP). RESULTSKIT D816V-mutated MSC were detected in 22/83 (27%) ISM patients. All MSC-mutated patients had multilineage KIT mutation (100% vs. 30%, p=0.0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P =.03) and a polyclonal XCIP of the KIT- mutated BM MC (64% vs 0%; P =0.01) vs other multilineage ISM cases. Moreover, presence of KIT D816V-mutated MSC was associated with more advanced disease features of ISM, a greater rate of disease progression (50% vs 17%; P =.04) and a shorter progression-free survival at 10, 20 and 30 years (P ≤.003). CONCLUSIONOverall, these results support the notion that ISM patients with mutated MSC may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSC and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome. DisclosuresNo relevant conflicts of interest to declare.

Physiologic Hematopoiesis Is Dependent on Stromal Expression of Laminin-γ1

OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels; 2) adjacent stromal cells; 3) hematopoietic cells; and 4) cytokines, growth factors, and structural support molecules. Injury to the bone marrow niche affects hematopoietic cells through loss of either physical contact with stromal cells or niche-derived growth factors. Extracellular matrix factors required to maintain the steady-state bone marrow perivascular niche and hematopoiesis have not been fully identified. Here, we examine the role of the extracellular matrix protein laminin-γ1 in adult bone marrow for maintaining the perivascular hematopoietic niche and hematopoiesis.METHODS: A global and inducible laminin-γ1 deficient mouse, hereafter referred to as mutant, was generated in which LAMC1 gene recombination could be monitored by a fluorescent reporter transgene. Tamoxifen was used to induce LAMC1 gene recombination and knock-down of laminin-γ1 protein expression in 8-12 week old mice. Tamoxifen-treated mice lacking the inducible Cre transgene were used as controls for all experiments. Analysis of mutant mice was performed 17-24 days following the first dose of tamoxifen. Bone marrow samples were examined by immunohistochemistry for the presence of laminin-1 protein, and hematopoietic tissues, including bone marrow, peripheral blood, spleen, thymus and lymph node were analyzed by flow cytometry for hematopoietic stem and progenitor cells, and mature hematopoietic cell populations, and by ex vivo hematopoietic progenitor cell colony forming assays. To examine the stromal cell contribution to laminin-γ1 mediated hematopoietic dysfunction, control and mutant mice that had not yet undergone LAMC1 gene recombination were then transplanted with wild type bone marrow. After full hematopoietic reconstitution, chimeric mice were induced with tamoxifen and hematopoietic stem and progenitor cells and mature hematopoietic cell populations were examined.RESULTS: LAMC1 gene recombination in the bone marrow of tamoxifen-treated mutant animals varied between 20-45%. With LAMC1 gene recombination, laminin-protein in the bone marrow is rapidly depleted. Bone marrow blood vessels dilate, and the bone marrow becomes hypocellular. Hematopoietic stem and progenitor cells are reduced in number, as are bone marrow B-cell progenitors, and thymic double-positive T-cells. Erythrocyte and thrombocyte numbers are not changed, nor were NK cell populations, the majority of myeloid cell subtypes, or mature B cells in the bone marrow and blood. CD8+ T-cells were increased within the bone marrow, as were CD11b+, Ly6Clo Ly6G+ myeloid cells in the peripheral blood. Examination of gene recombination within each of these cell types was not consistent with cell-intrinsic mechanism of hematopoietic alterations. Analysis of bone marrow chimeric animals identified loss of stromal cell-produced laminin-γ1 as a significant mediator of these hematopoietic alterations.CONCLUSIONS: The rapid depletion of laminin-γ1 from the bone marrow indicates a high basal turnover rate for the extracellular matrix in the bone marrow. The attendant hematopoietic dysfunction that follows loss of laminin-γ1 production in the bone marrow is specific for immature hematopoietic cell populations, and is dependent on stromal cell production of laminin-γ1. This study identifies laminin-γ1 as a niche-dependent regulator of hematopoietic stem and progenitor cell populations, and suggests that that regulation of its production and degradation may be new targets for the study of the hematopoietic niche. DisclosuresNo relevant conflicts of interest to declare.

Proteinase 3 Is Expressed in Stem Cells and Regulates Bone Marrow Hematopoiesis

Previously published data from our lab identified proteinase 3 (PR3) as a key protein in regulating spontaneous death of neutrophils. Our lab showed that PR3 could cleave caspase 3 in a caspase 9-independent manner in ageing neutrophils (Loison et al. JCI. 2014). Meanwhile, the maintenance of stem cell pool in the bone marrow and the development of mature blood cells are tightly regulated processes. In an effort to understand the hematopoietic consequences of absence of PR3, we analyzed stem and progenitor cell compartments in wild-type (WT) and PR3 knock-out (PR3-/-) mice.Of note, PR3 is thought to be a neutrophil-specific serine protease. Surprisingly, we observed PR3 expression in sorted Lin- Sca-1+ c-Kit+ (LSK) cells by gene expression analysis at mRNA level as well as Western blotting at protein level. Conventional flow cytometry methods also confirmed this expression.While bone marrow cellularity was similar between WT and PR3-/- mice, conventional flow cytometry studies showed that bone marrow from PR3-/- mice contained a higher frequency of LSK and Lin- Sca-1- c-Kit+ (LK) cells. Colony forming cell assays also suggested that both bone marrow cells and splenocytes from PR3-/- mice contained a higher number of progenitor cells compared to their WT counterparts. Histological analysis of bone marrow smears revealed that bone marrow from PR3-/- mice had a higher number of immature myeloid cells and a reduced number of mature leukoyctes and lymphocytes.Next, we asked if the enhanced stem and progenitor cell compartments in PR3-/- mice were functional in vivo. Toward this aim, we performed competitive reconstitution experiments using total bone marrow cells as well as sublethal irradiation experiments. PR3-/- donor-derived cells gave rise to a higher number of cells in the recipient mice compared to WT donor-derived cells in competitive reconstitution experiments. Also, after WT and PR3-/- mice were irradiated with 4 Gy, peripheral blood cell counts recovered faster in PR3-/- mice. Similarly, when mice were irradiated with 6 Gy, PR3-/- mice had a higher survival rate. These data suggest that enhanced stem and progenitor cell compartments in PR3-/- mice are functional. To understand if the enhanced hematopoietic activity in PR3-/- mice is an intrinsic feature of stem cells, another set of competitive reconstitution experiments were performed using equal numbers of sorted LSK cells. Again, PR3-/- donor-derived cells were more dominant than WT donor-derived cells in the recipient mice.Then, we wanted to understand the underlying mechanism. The rate of proliferation and senescence in LSK cells were analyzed by BrdU incorporation studies and C12FDG staining, respectively. No difference in proliferation or senescence was observed between WT and PR3-/- mice. We also looked at the apoptosis rate of LSK cells in vivo and in vitro. Untreated LSK cells from WT and PR3-/- mice exhibited similar levels of viability both in vivo and in vitro. However, when bone marrow macrophages responsible for clearance of apoptotic cells were depleted using clodronate liposomes, PR3-/- LSK cells exhibit enhanced viability compared to WT LSK cells as evidenced by annexin V and 7-AAD staining. Using a fluorogenic substrate of caspase 3, we also observed that the enhanced viability seen in PR3-/- LSK cells after depletion of macrophages is due to a reduction in caspase 3 activation.Additionally, we asked whether LSK cells from PR3-/- mice represent features associated with stem cells from aged mice such as an increase in DNA damage accumulation and reduced polarity. Immunofluorescent microscopy studies suggested that long-term hematopoietic stem cells from PR3-/- mice exhibit higher levels of p-gH2AX foci, a marker of DNA damage, and a reduced polarized distribution of a-tubulin. Finally, we saw a reduced lifespan in PR3-/- mice. These data indicate that PR3 regulates bone marrow hematopoiesis by contributing to the maintenance of a healthy stem cell pool in the bone marrow. DisclosuresNo relevant conflicts of interest to declare.

A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Erythrocytes Sickling in Human Erythroid Culture Model

Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with "antisickling" globin genes. Hemoglobin A2 (HbA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid substitute for hemoglobin A in β-thalassemia, as well as an "anti-sickling" agent in sickle cell disease. We have previously demonstrated that two Eklf-GATA1 fusion proteins, which were recruited to the GATA1 binding motif at the δ-globin promoter, can significantly activate δ-globin expression in K562 cells and hematopoietic stem CD34+ cells. Here, we report that enforced expression of Eklf-GATA1 fusion protein in sickle trait CD34+ cells significantly increased δ-globin expression, as determined by quantitative PCR and HPLC. Upon deoxygenation, the percentage of sickling cells was lower in Eklf-GATA1-transduced red blood cells as compared with mock-transduced cells. By a series of flow cytometry analyses, which includes BRDU incorporation assay, CD71/GPA, and thiazole orange staining, we found that erythroid cells proliferation, differentiation and enucleation were not affected by Eklf-GATA1 expression. To assess the potential off-target effects of our fusion protein constructs, we analyzed differentially expressed genes (and proteins) in vector-only and Eklf-GATA-1 transduced-CD34+ cells by microarray and proteomic changes by liquid chromatography-mass spectrometry (LC-MS). We found that over-expression of Eklf-GATA1 resulted in a less than 2-fold change in the gene expression profile related to bone marrow hematopoiesis, and there were no significant changes that were detected in proteomic profiling. These results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. DisclosuresNo relevant conflicts of interest to declare.

Extramedullary hematopoiesis: Elucidating the function of the hematopoietic stem cell niche (Review).

Extramedullary hematopoiesis (EMH) occurs under various circumstances, including during embryonic/developmental periods, pathological status secondary to insufficient bone marrow function or ineffective hematopoiesis, in hematological disorders, for example malignancies, as well as stromal disorders of the bone. EMH is characterized by hematopoietic cell accumulations in multiple body locations. Common EMH locations observed in clinical and pathological practice include the spleen, liver, lymph nodes and para‑vertebral regions. Among the various organs associated with EMH, the spleen offers a unique site for evaluation of hematopoietic stem cell (HSC)/niche interactions, as this organ is one of the most common sites of EMH. However, the spleen does not have a major role in embryonic/developmental hematopoiesis. A recent study by our group revealed that circulating HSCs may be trapped by chemokine (C‑X‑C motif) ligand 12 (CXCL12)‑positive cells at the margin of sinuses near CXCL12‑positive endothelial cells, resulting in the initiation of the first step of EMH, which is a similar mechanism to bone marrow hematopoiesis. The present review briefly discusses the environment of EMH in extramedullary spaces in order to investigate the mechanisms underlying HSC maintenance, and aid the elucidation of the niche‑stem cell interactions that occur in the bone marrow.

Mouse Resistin (mRetn): cloning, expression and purification in Escherichia coli and the potential regulative effects on murine bone marrow hematopoiesis.

BackgroundResistin (Retn) is a cytokine which has a controversial physiological role regarding its involvement with obesity and type II diabetes mellitus. Recently, murine Retn was found to be a possibly potential regulator of hematopoiesis in mice shown in the screening results of a set of gene chips which mapped the expression level of murine genes during regeneration of impaired bone marrow (BM) by 5-fluorouracil.ResultsRecombinant mice Retn was expressed in Escherichia coli and purified using ion exchange chromatography. Totally 11.4 mg rmRetn was obtained from 500 ml culture with endotoxin level less than 1.0 EU/ug. The purity of recombinant murine Resistin reached to at least 97.6 % via SDS-PAGE analysis and HPLC. The protein possessed chemotaxis effects in the mouse aortic endothelial cells in vitro in transwell analysis. In vitro, rmRetn could up regulate the CFU number of mice BM and after rmRetn was administered, the cell number of murine bone marrow was significantly increased in vivo after chemotherapy. Finally, rmRetn was found able to protect mice from the chemotoxicity of 5-fluorouracil.ConclusionsThe discovery demonstrated a new function of murine Retn and suggested that it could potentially accelerate bone marrow regeneration post chemotherapy.

A perisinusoidal niche for extramedullary haematopoiesis in the spleen.

Haematopoietic stresses mobilize haematopoietic stem cells (HSCs) from the bone marrow to the spleen and induce extramedullary haematopoiesis (EMH). However, the cellular nature of the EMH niche is unknown. Here, we assessed the sources of the key niche factors, SCF and CXCL12, in the mouse spleen after EMH induction by myeloablation, blood loss, or pregnancy. In each case, Scf was expressed by endothelial cells and Tcf21+ stromal cells, primarily around sinusoids in the red pulp, while Cxcl12 was expressed by a subset of Tcf21+ stromal cells. EMH induction markedly expanded the Scf-expressing endothelial cells and stromal cells by inducing proliferation. Most splenic HSCs were adjacent to Tcf21+ stromal cells in red pulp. Conditional deletion of Scf from spleen endothelial cells or Scf or Cxcl12 from Tcf21+ stromal cells severely reduced spleen EMH and reduced blood cell counts without affecting bone marrow haematopoiesis. Endothelial cells and Tcf21+ stromal cells thus create a perisinusoidal EMH niche in the spleen, which is necessary for the physiological response to diverse haematopoietic stresses.

The role of Trib1 in myeloid leukaemogenesis and differentiation.

Tribbles homolog 1 (Trib1) was identified as a common integration site of the Homeobox a9 (Hoxa9)/murine ecotropic virus integration site 1 (Meis1) retrovirus in acute myeloid leukaemia (AML). Trib1 is by itself a transforming gene for myeloid cells but also significantly accelerates progression of Hoxa9/Meis1-induced AML. The strong transforming activity of Trib1 depends on its bi-directional function in CCAAT/enhancer-binding protein (C/EBPα) degradation and MAPK/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) activation. TRIB1 is also involved in a certain typeof human AML and a TRIB1 somatic point mutation R107L was identified in a case of Down syndrome (DS)-related acute megakaryocytic leukaemia. Although Trib1 knockout (KO) did not suppress haematopoiesis in mouse bone marrow significantly, increase in mature granulocytes was observed and promotion of myeloid differentiation was associated with the increased C/EBPα protein. Trib1 thus plays an important role in myeloid cell development and transformation.

Hemorrhage Exacerbates Radiation Effects on Survival, Leukocytopenia, Thrombopenia, Erythropenia, Bone Marrow Cell Depletion and Hematopoiesis, and Inflammation-Associated microRNAs Expression in Kidney.

Exposure to high-dose radiation results in detrimental effects on survival. The effects of combined trauma, such as radiation in combination with hemorrhage, the typical injury of victims exposed to a radiation blast, on survival and hematopoietic effects have yet to be understood. The purpose of this study was to evaluate the effects of radiation injury (RI) combined with hemorrhage (i.e., combined injury, CI) on survival and hematopoietic effects, and to investigate whether hemorrhage (Hemo) enhanced RI-induced mortality and hematopoietic syndrome. Male CD2F1 mice (10 weeks old) were given one single exposure of γ- radiation (60Co) at various doses (0.6 Gy/min). Within 2 hr after RI, animals under anesthesia were bled 0% (Sham) or 20% (Hemo) of total blood volume via the submandibular vein. In these mice, Hemo reduced the LD50/30 for 30-day survival from 9.1 Gy (RI) to 8.75 Gy (CI) with a DMF of 1.046. RI resulted in leukocytopenia, thrombopenia, erythropenia, and bone marrow cell depletion, but decreased the caspase-3 activation response. RI increased IL-1β, IL-6, IL-17A, and TNF-α concentrations in serum, bone marrow, ileum, spleen, and kidney. Some of these adverse alterations were magnified by CI. Erythropoietin production was increased in kidney and blood more after CI than RI. Furthermore, CI altered the global miRNAs expression in kidney and the ingenuity pathway analysis showed that miRNAs viz., let-7e, miR-30e and miR-29b that were associated with hematopoiesis and inflammation. This study provides preliminary evidence that non-lethal Hemo exacerbates RI-induced mortality and cell losses associated with high-dose γ-radiation. We identified some of the initial changes occurring due to CI which may have facilitated in worsening the injury and hampering the recovery of animals ultimately resulting in higher mortality.

Ontogeny of Tissue-Resident Macrophages.

The origin of tissue-resident macrophages, crucial for homeostasis and immunity, has remained controversial until recently. Originally described as part of the mononuclear phagocyte system, macrophages were long thought to derive solely from adult blood circulating monocytes. However, accumulating evidence now shows that certain macrophage populations are in fact independent from monocyte and even from adult bone marrow hematopoiesis. These tissue-resident macrophages derive from sequential seeding of tissues by two precursors during embryonic development. Primitive macrophages generated in the yolk sac (YS) from early erythro-myeloid progenitors (EMPs), independently of the transcription factor c-Myb and bypassing monocytic intermediates, first give rise to microglia. Later, fetal monocytes, generated from c-Myb+ EMPs that initially seed the fetal liver (FL), then give rise to the majority of other adult macrophages. Thus, hematopoietic stem cell-independent embryonic precursors transiently present in the YS and the FL give rise to long-lasting self-renewing macrophage populations.

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis.

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.

Progression of anemia and its relationship with renal function, blood pressure, and erythropoietin in rats with chronic kidney disease.

In chronic kidney disease (CKD), anemia and hypertension are significant co-morbidities that contribute to cardiovascular and renal disease progression. The purpose of the study was to identify correlations between changes in hematologic variables against markers of renal function, blood pressure, and erythropoietin (EPO) in a naturally occurring hypertensive model of CKD, the Lewis polycystic kidney (LPK) rat. Complete blood count, systolic blood pressure, urea and creatinine concentration, urinary protein to creatinine ratio, and plasma EPO concentration were determined in control Lewis (n = 51) and LPK rats (n = 56) aged 6-24 weeks. Renal EPO gene expression and RBC osmotic fragility were also documented. Hematopoiesis in spleen and bone marrow were assessed. Lewis polycystic kidney rats had increasing urea and creatinine concentrations, concurrent with the development of a nonregenerative normocytic/normochromic anemia and hypertension, with a significant negative correlation between both HGB and HCT with urea concentration and blood pressure (P < .01). HCT was also significantly negatively correlated with creatinine concentration (P = .014). WBC was significantly negatively correlated with urea (P < .01). Plasma EPO concentration was increased and renal EPO mRNA expression was significantly upregulated in LPK animals. The former was significantly positively correlated with blood pressure and platelet count (P < .05). RBC osmotic fragility was normal in LPK rats and there was no evidence for increased RBC elimination or extramedullary hematopoiesis. Marked anemia in the LPK CKD rodent model in the presence of elevated EPO suggests inefficient erythropoiesis that is correlated with plasma urea concentration and blood pressure.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)—with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)—as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

Effect of Norcantharidin on Hematopoietic Function in Leucopenia Model Rat Induced by Cyclophosphamide

To explore the effect and mechanism of norcantharidin (NCTD) on hematopoiesis function in leucopenia model rat induced by cyclophosphamide (CTX). Leucopenia model was replicated in SD rat with cyclophosphamide(CTX) and model animal was treated with NCTD. Peripheral blood and bone marrow tissue samples were collected from the rats in each experimental group. Peripheral white blood cells (WBC) were counted and analyzed by automatic blood cell analyzer. Histopathologic changes of the biopsied bone marrow tissues were observed by histopathological techniques. The cell cycle and apoptosis rate of bone marrow cells were detected by flow cytometry. Immunohistochemical method was applied to observe the expression of apoptosis-related proteins BCL-2 and BAX in bone marrow. After NCTD treatment in model rats, the WBC count of peripheral blood obviously increased, the cell structure of bone tissue significantly recovered, NCTD could promote the cell proliferation and cycle changes of bone marrow cells, inhibit the bone marrow cell apoptosis and necrosis induced with CTX, up-regulate the expression of apoptosis-related protein BCL-2 and downregulated the BAX. NCTD can stimulate the bone marrow hematopoiesis and promote recovery of peripheral white blood cell level in the leukopenia model induced by CTX, and its mechanism may be related with NCTD regulating bone marrow cell cycle and with NCTD inhibiting cell apoptosis.

Localized SCF and IGF-1 secretion enhances erythropoiesis in the spleen of murine embryos.

Fetal spleen is a major hematopoietic site prior to initiation of bone marrow hematopoiesis. Morphologic analysis suggested erythropoietic activity in fetal spleen, but it remained unclear how erythropoiesis was regulated. To address this question, we performed flow cytometric analysis and observed that the number of spleen erythroid cells increased 18.6-fold from 16.5 to 19.5 days post-coitum (dpc). Among erythropoietic cytokines, SCF and IGF-1 were primarily expressed in hematopoietic, endothelial and mesenchymal-like fetal spleen cells. Cultures treated with SCF and/or IGF-1R inhibitors showed significantly decreased CD45−c-Kit−CD71+/−Ter119+ erythroid cells and downregulated Gata1, Klf1 and β-major globin expression. Administration of these inhibitors to pregnant mice significantly decreased the number of CD45−c-Kit−CD71+/−Ter119+ cells and downregulated β-major globin gene expression in embryos derived from these mice. We conclude that fetal spleen is a major erythropoietic site where endothelial and mesenchymal-like cells primarily accelerate erythropoietic activity through SCF and IGF-1 secretion.

ROS-mediated iron overload injures the hematopoiesis of bone marrow by damaging hematopoietic stem/progenitor cells in mice.

Iron overload, caused by hereditary hemochromatosis or repeated blood transfusions in some diseases, such as beta thalassemia, bone marrow failure and myelodysplastic syndrome, can significantly induce injured bone marrow (BM) function as well as parenchyma organ dysfunctions. However, the effect of iron overload and its mechanism remain elusive. In this study, we investigated the effects of iron overload on the hematopoietic stem and progenitor cells (HSPCs) from a mouse model. Our results showed that iron overload markedly decreased the ratio and clonogenic function of murine HSPCs by the elevation of reactive oxygen species (ROS). This finding is supported by the results of NAC or DFX treatment, which reduced ROS level by inhibiting NOX4 and p38MAPK and improved the long-term and multi-lineage engrafment of iron overload HSCs after transplantation. Therefore, all of these data demonstrate that iron overload injures the hematopoiesis of BM by enhancing ROS through NOX4 and p38MAPK. This will be helpful for the treatment of iron overload in patients with hematopoietic dysfunction.