Homing Of Hematopoietic Progenitor Cells Research Articles

RGS1 and CREB5 are direct and common transcriptional targets of ZNF384-fusion proteins.

ZNF384-fusion (Z-fusion) genes were recently identified in B-cell acute lymphoblastic leukemia (B-ALL) and are frequent in Japanese adult patients. The frequency is about 20% in those with Philadelphia chromosome-negative B-ALL. ZNF384 is a transcription factor and Z-fusion proteins have increased transcriptional activity; however, the detailed mechanisms of leukemogenesis of Z-fusion proteins have yet to be clarified. We established three transfectants of cell lines expressing different types of Z-fusion proteins, and analyzed their gene expression profile (GEP) by RNA-seq. We also analyzed the GEP of clinical ALL samples using our previous RNA-seq data of 323 Japanese ALL patients. We selected upregulated genes in both Z-fusion gene-expressing transfectants and Z-fusion gene-positive ALL samples, and investigated the binding of Z-fusion proteins to regulatory regions of the candidate genes by ChIP-qPCR. We selected six commonly upregulated genes. After the investigation by ChIP-qPCR, we finally identified CREB5 and RGS1 as direct and common target genes. RGS1 is an inhibitor of CXCL12-CXCR4 signaling that is required for the homing of hematopoietic progenitor cells to the bone marrow microenvironment and development of B cells. Consistent with this, Z-fusion gene transfectants showed impaired migration toward CXCL12. We identified CREB5 and RGS1 as direct and common transcriptional targets of Z-fusion proteins. The present results provide novel insight into the aberrant transcriptional regulation by Z-fusion proteins.

Endothelial SIRPα signaling controls VE-cadherin endocytosis for thymic homing of progenitor cells.

Thymic homing of hematopoietic progenitor cells (HPCs) is tightly regulated for proper T cell development. Previously we have identified a subset of specialized thymic portal endothelial cells (TPECs), which is important for thymic HPC homing. However, the underlying molecular mechanism still remains unknown. Here, we found that signal regulatory protein alpha (SIRPα) is preferentially expressed on TPECs. Disruption of CD47-SIRPα signaling in mice resulted in reduced number of thymic early T cell progenitors (ETPs), impaired thymic HPC homing, and altered early development of thymocytes. Mechanistically, Sirpa-deficient ECs and Cd47-deficient bone marrow progenitor cells or T lymphocytes demonstrated impaired transendothelial migration (TEM). Specifically, SIRPα intracellular ITIM motif-initiated downstream signaling in ECs was found to be required for TEM in an SHP2- and Src-dependent manner. Furthermore, CD47 signaling from migrating cells and SIRPα intracellular signaling were found to be required for VE-cadherin endocytosis in ECs. Thus, our study reveals a novel role of endothelial SIRPα signaling for thymic HPC homing for T cell development.

Thymic Egress Is Regulated by T Cell-Derived LTβR Signal and via Distinct Thymic Portal Endothelial Cells.

Thymic blood vessels at the perivascular space (PVS) are the critical site for both homing of hematopoietic progenitor cells (HPCs) and egress of mature thymocytes. It has been intriguing how different opposite migrations can happen in the same place. A subset of specialized thymic portal endothelial cells (TPECs) associated with PVS has been identified to function as the entry site for HPCs. However, the cellular basis and mechanism underlying egress of mature thymocytes has not been well defined. In this study, using various conventional and conditional gene-deficient mouse models, we first confirmed the role of endothelial lymphotoxin beta receptor (LTβR) for thymic egress and ruled out the role of LTβR from epithelial cells or dendritic cells. In addition, we found that T cell-derived ligands lymphotoxin (LT) and LIGHT are required for thymic egress, suggesting a crosstalk between T cells and endothelial cells (ECs) for thymic egress control. Furthermore, immunofluorescence staining analysis interestingly showed that TPECs are also the exit site for mature thymocytes. Single-cell transcriptomic analysis of thymic endothelial cells suggested that TPECs are heterogeneous and can be further divided into two subsets depending on BST-1 expression level. Importantly, BST-1hi population is associated with thymic egressing thymocytes while BST-1lo/− population is associated with HPC settling. Thus, we have defined a LT/LIGHT-LTβR signaling–mediated cellular crosstalk regulating thymic egress and uncovered distinct subsets of TPECs controlling thymic homing and egress, respectively.

Bone Marrow (BM) Delivery of Genetically-Modified (gm) Adult CD34+ Hematopoietic Stem and Progenitor Cells (HSPC) Improves Homing and Engraftment of Short-Term Progenitors over Long-Term Repopulating Hematopoietic Stem Cells

Gene therapy/editing of CD34+ HSPC ex vivo, followed by their transplantation, can cure a variety of hematologic diseases. However, a substantial loss of HSPC occurs from collection to transplant. Losses occur during processing for HSPC enrichment, ex vivo genetic manipulation and culture, formulation, and testing prior to transplant. Further, HSPC are lost to peripheral organs during homing when delivered intravenously (IV), reducing the effective gm HSPC dose; a loss compounded by the lack of helper cells that aid in the homing and engraftment process which are removed during enrichment. Direct BM delivery of gm HSPC can overcome some of these limitations. This has been tried previously, with non-enriched whole cord blood (CB) and non-gm HSPC, with conflicting results. We hypothesized that BM delivery of a limited dose of gm adult HSPC would improve long-term repopulation over that of IV delivery by bypassing HSPC loss during homing. Using bioluminescent imaging, we determined that CB HSPC transduced with a luciferase lentiviral vector (LV) delivered by intra-femoral (IF) injection localized to the injected femur, validating our injection method. Next, we delivered mobilized peripheral blood (MPB) HSPC transduced with a GFP LV into irradiated NOD.LtSz-scid IL2rg -/- (NSG) mice via IV or IF injection in limiting dilution. Total human engraftment (hCD45+ cells), transduced human engraftment (hCD45+GFP+ cells), and multi-lineage engraftment were measured in the BM at 3- and 6-months post-transplant. HSPC gave rise to a bi-lineage (B-myeloid) graft at 3 months, suggesting hematopoietic progenitor cell (HPC) engraftment, and a multi-lineage graft (hCD33+, hCD19+, hCD3+, and hCD34+ cells) at 6 months, suggesting engraftment from a long-term repopulating cell or hematopoietic stem cell (HSC). At 3 months, IF delivery of HSPC resulted in significantly higher total and transduced human cell engraftment, measured in the non-injected femur (Table 1). The engraftment was bi-lineage. At 6 months, IF delivery of HSPC no longer significantly increased engraftment over IV delivery (Table 1). However, a multi-lineage graft was present, indicating full hematopoietic repopulation. There was no significant difference in the lineage output between either delivery method at 3 or 6 months. These data suggest that HPC homed and engrafted more efficiently than HSC, when delivered IF. Alternatively, IF delivery altered the BM microenvironment, allowing preferential homing of HPC. However, CD34- cells injected IF, to simulate pressure and passage of cells through the BM with IF delivery, followed by IV delivery of CD34+ cells (sham IF with IV HSPC delivery) resulted in similar homing patterns to CD34+ cells delivered IV (p=0.1, Figure 1A), suggesting that differences between IV and IF delivery were likely due to cell-intrinsic rather than cell-extrinsic differences between HPC and HSC. To study the mechanism of preferential engraftment of HPC over HSC with IF delivery, we analyzed expression of the major homing receptors CXCR4 and VLA-4 on HPC and HSC. CXCR4 (Figure 1B) and VLA-4 were both expressed at significantly higher levels on HPC than on HSC (CXCR4 p<0.01; VLA-4 p<0.05) and their expression increased with increasing culture time and with HSPC cycling. However, VLA-4 expression was significantly increased in GFP+ (MFI 65313 ± 4750) compared to GFP- (MFI 48969 ± 2099; p<0.01) HSPC. CXCR4 expression was similar in both GFP+ (MFI 4261 ± 189) and GFP- (MFI 5245 ± 1186) HSPC, mimicking the in vivo engraftment pattern of GFP+ and GFP- cells, suggesting that CXCR4 may be the molecule responsible for enhancing HPC homing and engraftment with BM delivery. An initial experiment shows that when we remove the high CXCR4 expressing CD34+38+ HPC and deliver HSC-enriched CD34+38- cells IV or IF, IF delivery results in higher long-term engraftment (additional experiments ongoing, Figure 1C, D). These data support the hypothesis that cell-intrinsic differences in the homing behavior of HSC and HPC is likely due to their differential expression of CXCR4. Studies underway on blockade of CXCR4 or VLA-4 on gm HPC and/or gm HSC followed by their IF or IV delivery will be presented. Overall, we show IV delivery of gm HSPC is comparable to BM delivery. However, as HSC-enriched cells become clinically available for genetic therapies, BM delivery of enriched gm HSC may result in superior engraftment. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

The thymus is relevant in the migration of mature lymphocytes.

The thymus is a primary lymphoid organ where T lymphocyte proliferation and selection takes place. The different subsets of lymphocytes leave the thymus as recent thymic emigrants. Peripheral dendritic cells migrate to the thymus. In addition to the homing of hematopoietic progenitor cells to the thymus, there is evidence for lymphocyte entry from peripheral lymphoid tissues mainly into the medulla. The entry sites are the venules in the medullary part near to the cortex with a higher endothelium. Furthermore, there are also B lymphocytes in the thymus. The thymus is not only a primary lymphoid organ but is well integrated in lymphocyte traffic as shown in several different species.

Tracking embryonic hematopoietic stem cells to the bone marrow: nanoparticle options to evaluate transplantation efficiency

BackgroundAs the prevalence of therapeutic approaches involving transplanted cells increases, so does the need to noninvasively track the cells to determine their homing patterns. Of particular interest is the fate of transplanted embryonic stem cell-derived hematopoietic progenitor cells (HPCs) used to restore the bone marrow pool following sublethal myeloablative irradiation. The early homing patterns of cell engraftment are not well understood at this time. Until now, longitudinal studies were hindered by the necessity to sacrifice several mice at various time points of study, with samples of the population of lymphoid compartments subsequently analyzed by flow cytometry or fluorescence microscopy. Thus, long-term study and serial analysis of the transplanted cells within the same animal was cumbersome, making difficult an accurate documentation of engraftment, functionality, and cell reconstitution patterns.MethodsHere, we devised a noninvasive, nontoxic modality for tracking early HPC homing patterns in the same mice longitudinally over a period of 9 days using mesoporous silica nanoparticles (MSNs) and magnetic resonance imaging.ResultsThis approach of potential translational importance helps to demonstrate efficient uptake of MSNs by the HPCs as well as retention of MSN labeling in vivo as the cells were traced through various organs, such as the spleen, bone marrow, and kidney. Altogether, early detection of the whereabouts and engraftment of transplanted stem cells may be important to the overall outcome. To accomplish this, there is a need for the development of new noninvasive tools.ConclusionsOur data suggest that multifunctional MSNs can label viably blood-borne HPCs and may help document the distribution and homing in the host followed by successful reconstitution.

Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

PurposeWe investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)–derived cells to the neurosensory retina (NSR) and RPE layer.MethodsGFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 μm) with 30 applications were placed 50 to 100 μm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed.ResultsWithin 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle–dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid.ConclusionsSRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle–dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.

Erratum to: Endogenous developmental endothelial locus-1 limits ischaemia-related angiogenesis by blocking inflammation.

We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of β2-integrin–dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1–deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1–proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted β2-integrin–dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the β2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1–dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases. Supplementary Material to this article is available online at www.thrombosis-online.com.

Endogenous developmental endothelial locus-1 limits ischaemia-related angiogenesis by blocking inflammation.

We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of β2-integrin-dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1-deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1-proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted β2-integrin-dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the β2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1-dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.

Nitrogen-containing bisphosphonate induces a newly discovered hematopoietic structure in the omentum of an anemic mouse model by stimulating G-CSF production

We previously reported that the injection of nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. Our previous study established a severely anemic mouse model that was treated with a combination of NBP with phenylhydrazine (PHZ), which induced newly discovered hematopoietic organs in the omentum. No reports have shown that new hematopoietic organs form under any condition. We characterized the structures and factors related to the formation of these new organs. Splenectomized mice were treated with NBP to inhibit erythropoiesis in the BM and then injected with PHZ to induce hemolytic anemia. The mice showed severe anemia and wine-colored structures appeared in the omentum. Some hematopoietic cells, including megakaryocytes, and well-developed sinuses were observed in these structures. Numerous TER119-positive erythroblasts were located with cells positive for PCNA, a cell proliferation marker. C-kit-positive cells were detected and mRNAs related to hematopoiesis were expressed in these structures. Moreover, TER119-positive erythroblasts emerged and formed clusters and hematopoiesis-related factors were detected in the omentum of mice treated with NBP and PHZ. The levels of G-CSF in the serum and hematopoietic progenitor cells (HPCs) in the peripheral blood were increased upon treatment with both NBP and PHZ. These results suggest that the induced hematopoietic structures act as the sites of erythropoiesis and that NBP-induced G-CSF production causes HPC mobilization, homing and colonization in the omentum because they constitutively express some factors, including SDF-1; thus, the newly discovered hematopoietic structure in this study might be formed.

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.

Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD-HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.

Thymic stromal lymphopoietin and IL-33 modulate migration of hematopoietic progenitor cells in patients with allergic asthma

Thymic stromal lymphopoietin (TSLP) and IL-33 are considered important initiators of type 2 immunity. In asthmatic patients allergic inflammatory responses are associated with increased lung homing of bone marrow-derived CD34(+) hematopoietic progenitor cells (HPCs), which include eosinophil lineage-committed progenitor cells. In this study we investigated the role of TSLP and IL-33 in the recruitment of progenitor cells to the airways in asthmatic subjects. We sought (i) to examine the effect of allergen inhalation challenge on expression levels of receptors for TSLP (thymic stromal lymphopoietin receptor [TSLPR] and CD127) and IL-33 (ST2) and (ii) investigate the functional effects of these cytokines on HPCs. Consenting patients with mild atopic asthma (n= 19) with an FEV1 of 70% or greater and methacholine PC20 of 16 mg/mL or less were recruited. Blood- and sputum-extracted progenitors were phenotyped by flow cytometry before and 24 hours after allergen challenge. Functional responses, including cytokine production and migration to TSLP and IL-33, were assessed invitro. Significant increases in mature eosinophil, HPC, and eosinophil lineage-committed progenitor cell counts in sputum were observed 24 hours after allergen and were associated with a significant allergen-induced increase in HPCs expressing TSLPR, CD127, and ST2. Pre-exposure to TSLP and IL-33 primed the migration of HPCs to a potent progenitor cell chemoattractant, stromal cell-derived factor 1α (CXCL12). Incubation with TSLP and IL-33 stimulated significant production of IL-5 and IL-13, but not IL-4, by HPCs. This priming effect was inhibited by blocking antibodies to TSLPR and ST2, respectively, and IL-13 receptor α1 in both scenarios. In allergic asthmatic responses increased lung homing of HPCs may be orchestrated by TSLP and IL-33 through an IL-13-dependent axis.

Arhgap21+/- Mice Show Impaired Adhesion, Migration, Homing and Short Term Reconstitution of Hematopoietic Progenitor Cells

The main signaling pathway involved in migration, adhesion and homing of hematopoietic progenitor cells (HPC) is the CXCL-12, chemokine produced by bone marrow (BM) stromal cells that bind to its main receptor CXCR-4, expressed by HPC. ARHGAP21 is a RhoGAP member, negative regulators of RhoGTPAses. It was described that ARHGAP21 is involved in cellular migration and adhesion but its role in hematopoiesis is unknown.Researching Arhgap21 role in hematopoiesis, Arhgap21+/- mice were generated using Embryonic Stem cell obtained from GeneTrap consortium. Hematologic parameters were investigated by blood cell count, colony formation assay in methylcellulose medium and immunophenotipic characterization of hematopoietic populations. Transwell migration and adhesion of Lin- cells assay in the presence of CXCL12 were also performed.In vivo analysis of Arhgap21+/- HPC was achieved with homing and CFU-S assays in sublethally irradiated mice and hematopoiesis stress was generated by 5-fluoracil treatment.Arhgap21+/- mice presented more than 50% reduction in Arhgap21 expression (0.47 ± 0.13) in BM when compared to WT (1 ± 0.03) and live in normal conditions. On the other hand, Arhgap21 mice showed a reduction in BFU-E colonies and erythroid (Terr119+) committed cells in BM together with decreased peripheral blood (PB) Terr119+ cells and red blood cell number and increased medium corpuscular volume.In myeloid compartment, Arhgap21+/- mice presented less GM colonies and myeloid (Gr1/Mac1+) cells in BM followed by an increase of these cells in PB suggesting myeloid mobilization. Corroborating to this, whenArhgap21+/- BM was challenged for hematopoiesis stress with 5FU, the animals showed increased neutrophil number in peripheral blood 14 days (WT: 1887/mm3 ±721.9; Arhgap21+/-:3325/mm3 ±1640, p=0.02) and 21days (WT: 1264/mm3 ±635; Arhgap21+/-: 2182/mm3 ±854, p=0.01) after treatment, together with increased number of LSK cells in the BM (WT: 1.3% ±0.4; Arhgap21+/-: 1.9% ±0.3, p=0.006) 28 days after 5FU infusion.Erythroid and myeloid compartments reductions were observed together with an increase of Arhgap21+/- BM short term LSK( Arhgap21+/- : 0.34% ±0.13; WT: 0.18% ±0.07, p=0.03) and long term LSK (Arhgap21+/-: 0.003% ±0.001; WT: 0.002 ±0.0007, p=0.02) suggesting an attempt to restore normal hematopoietic levels.Arhgap21+/- HPC showed reduced CXCL-12-induced migration compared to WT (WT: 100%, Arhgap21+/-: 54.73% ± 13.57, p=0.01) in addition to decreased adhesion to fibronectin (Arhgap21+/-: 15% ± 3.8; WT: 27% ± 3.5, p=0.003) and a4b1 integrin expression (WT: 83.76 ± 4.35; Arhgap21+/-: 71.06 ± 7.00, p=0.008).In homing assay, the percentage of donor Lin- HPC from Arhgap21+/- mice that homed to BM (6.32 ± 2.41) or spleen (3.48 ± 1.57), were lower than those from WT mice (BM=10.09 ± 1.81;p=0.004; spleen=6.9 ± 1.48; p=0.0007) together with higher frequency of these cells in peripheral blood (WT: 6.39 ± 3.38; Arhgap21+/-: 13.95 ± 5.33, p=0.003), suggesting a retention of Arhgap21+/- HPC in the bloodstream, which inefficiently home to BM and spleen. Arhgap21+/- CFU-S capacity decline was also observed (Arhgap21+/-: 20.86 ± 2; WT: 29.29 ± 5.4 p=0.002).This is the first study showing that ARHGAP21 is involved in hematopoiesis and also shows that ARHGAP21 is an important protein for chemotaxis, adhesion, homing and short term reconstitution of HPC.TableHematological ParametersWTArhgap21+/-Hemoglobin (g/dL) *16.19 ± 0.3515.62±0.32RBC (10^6/uL) *10.91 ± 0.310.46±0.25Hematocrit (%)54.78 ± 1.653.75± 1.3MCV (fL) *50 ±0.450.08±0.74WBC (10^3/Ul) *10.08 ±212.9±1.8Platelets(10^3/uL)1793 ±114.31770±96.5Myeloid, Gr1+Mac1+ in BM (%) *47.27 ±4.740.88 ±6.23Erythroid, Terr119+ in BM (%) *9.98 ±4.16.4 ±1.5Short Term HSC, LSK in BM (%) *0.18 ±0.070.34 ±0.13Long Term HSC, LSK CD150+CD48- in BM (%) *0.002 ±0.00070.003 ±0.001Myeloid, Gr1+Mac1+ in PB (%) *7.7 ±1.513.1 ±2.6Erythroid, Terr119+ in PB (%) *38.7 ±4.428.4 ±8.7Myeloid Colonies, CFU-GM (number)66.7 ±14.748.07 ±12.5Erythroid Colonies, BFU-E (number)5.5 ±2.23.2 ±1.5Blood samples were collected from 9 WT and 8 Arhgap21+/- mice. RBC: red blood cell count; MCV: mean cell volume; WBC: White blood cell count. Hematopoietic populations were analyzed in flow cytometry and colony formation in methylcellulose assay. Data are expressed as mean ± standard deviation, analyzed by Student t test and considered statistically significant (*) if p ≤ 0.05. DisclosuresNo relevant conflicts of interest to declare.

Pleiotrophin Regulates the Retention and Self-Renewal of Hematopoietic Stem Cells in the Bone Marrow Vascular Niche

The mechanisms through which the bone marrow (BM) microenvironment regulates hematopoietic stem cell (HSC) fate remain incompletely understood. We examined the role of the heparin-binding growth factor pleiotrophin (PTN) in regulating HSC function in the niche. PTN(-/-) mice displayed significantly decreased BM HSC content and impaired hematopoietic regeneration following myelosuppression. Conversely, mice lacking protein tyrosine phosphatase receptor zeta, which is inactivated by PTN, displayed significantly increased BM HSC content. Transplant studies revealed that PTN action was not HSC autonomous, but rather was mediated by the BM microenvironment. Interestingly, PTN was differentially expressed and secreted by BM sinusoidal endothelial cells within the vascular niche. Furthermore, systemic administration of anti-PTN antibody in mice substantially impaired both the homing of hematopoietic progenitor cells to the niche and the retention of BM HSCs in the niche. PTN is a secreted component of the BM vascular niche that regulates HSC self-renewal and retention invivo.

Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice

Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking.

R-Ras and Rac GTPase Cross-talk Regulates Hematopoietic Progenitor Cell Migration, Homing, and Mobilization

Adult hematopoietic progenitor cells (HPCs) are maintained by highly coordinated signals in the bone marrow. The molecular mechanisms linking intracellular signaling network of HPCs with their microenvironment remain poorly defined. The Rho family GTPase Rac1/Rac2 has previously been implicated in cell functions involved in HPC maintenance, including adhesion, migration, homing, and mobilization. In the present studies we have identified R-Ras, a member of the Ras family, as a key signal mediator required for Rac1/Rac2 activation. We found that whereas Rac1 activity is up-regulated upon stem cell factor, integrin, or CXCL12 stimulation, R-Ras activity is inversely up-regulated. Expression of a constitutively active R-Ras mutant resulted in down-regulation of Rac1-activity whereas deletion of R-Ras led to an increase in Rac1/Rac2 activity and signaling. R-Ras(-/-) HPCs displayed a constitutively assembled cortical actin structure and showed increased directional migration. Rac1/Rac2 inhibition reversed the migration phenotype of R-Ras(-/-) HPCs, similar to that by expressing an R-Ras active mutant. Furthermore, R-Ras(-/-) mice showed enhanced responsiveness to G-CSF for HPC mobilization and exhibited decreased bone marrow homing. Transplantation experiments indicate that the R-Ras deficiency-induced HPC mobilization is a HPC intrinsic property. These results indicate that R-Ras is a critical regulator of Rac signaling required for HPC migration, homing, and mobilization.

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.

Hematopoietic progenitor cells regulate their niche microenvironment through a novel mechanism of cell-cell communication

Cellular communication within a larger microenvironment is critical for a number of physiological processes. Within the bone marrow niche, direct cell communication between hematopoietic progenitor cells (HPCs) and osteoblasts provides essential cues for their proliferation and survival. While contact-dependent communication between HPCs and osteoblasts is known to be critical, the molecular pathways that govern this interaction are largely unclear. Moreover, the downstream events occurring at the HPC/osteoblast contact site remain uncharacterized, despite their major role in signaling and remodeling within the niche microenvironment. Using live cell imaging approaches, we found that intercellular transfer is a novel mode of cell communication within the bone marrow niche microenvironment. HPCs made prolonged contact with the osteoblast surface via a specialized membrane domain enriched in prominin 1, CD63 and rhodamine PE. At the contact site, portions of the HPC specialized domain containing these molecules were taken up by the osteoblast and internalized into long-lived, SARA-positive, signaling endosomes. This resulted in the downregulation of Smad signaling by the osteoblasts and a subsequent increase in the production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HPC homing to bone marrow. These findings identify a novel mechanism involving intercellular transfer to signaling endosomes for targeted regulation of signaling and remodeling events within the osteoblastic niche microenvironment.

The role of endothelium in the regulation of hematopoietic stem cell migration

Mobilization of hematopoietic progenitor cells appears to be a multifactorial process which is at least partially regulated at the level of bone marrow microvascular endothelium (BMEC). In order to study the regulation of progenitor cell migration by endothelium in vitro, methods have been developed to isolate BMEC from bone marrow aspirates. In addition, immortalized BMEC cell lines have been generated. Using an in vitro model of migration across bone marrow endothelium, we demonstrate that only a small number of more mature, committed progenitors migrate spontaneously. In this model, adhesion molecules of the beta2-integrin family and the corresponding endothelial ligands are involved. The low spontaneous migratory capacity suggests that, in addition to adhesion molecules which mediate direct cellular contacts, paracrine cytokines and chemokines may play a role in progenitor migration across endothelium. Growth-factor-stimulated hematopoietic cells can produce cytokines which act on endothelial cells (e.g., vascular endothelial growth factor, VEGF), modifying their motility, growth, permeability, and fenestration. Therefore, VEGF might be involved in the mobilization and homing of hematopoietic progenitor cells. Furthermore, transendothelial migration of progenitors in vitro is substantially enhanced by the chemokine stromal-cell-derived factor-1 (SDF-1), which is produced by bone marrow stromal cells. More primitive progenitors, which do not migrate spontaneously, also respond to this chemokine. We conclude that transendothelial progenitor cell migration is regulated by adhesion molecules, paracrine cytokines, and chemokines. Mobilizing hematopoietic growth factors stimulate proliferation of hematopoietic cells, which may indirectly result in changes of the local cytokine and chemokine milieu, adhesion molecule expression, and eventually the mobilization of hematopoietic progenitor cells.