Tie-2 Activation Is Required for Regeneration of Marrow Vasculature, Supporting Hematopoietic Reconstitution.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1297-1297
Author(s):  
Hans-Georg Kopp ◽  
Scott T. Avecilla ◽  
Rafael Tejada ◽  
Ronald G. Crystal ◽  
Neil R. Hackett ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Chimeric Mice ◽  
Hematopoietic Progenitor ◽  
Platelet Recovery ◽  
Decoy Receptor ◽  
Hematopoietic Reconstitution ◽  
Lacz Staining ◽  
Functional Regeneration ◽  
Beta Galactosidase

Abstract Accumulating evidence from our lab (Nature Med.2004; 10(1): 64–71) and others have shown that myelosuppression not only results in apoptosis of the hematopoietic cells but also in regression of bone marrow (BM) sinusoidal vessels. Remarkably, we have shown that restoration of hematopoiesis is dependent on the regeneration of BM sinusoidal neo-vessels. However, the mechanism whereby vascular reconstitution is regulated is unknown. Because Tie2/Angiopoietins are critical in the remodeling of neo-vessels, we hypothesized that activation of Tie2 plays a role in regeneration of sinusoidal neo-vessels and restoration of hematopoiesis. To this end, we took advantage of transgenic mice where the Tie2 promoter drives the expression of beta-galactosidase (LacZ) in order to follow the expression of Tie2 in hematopoietic progenitor cells after myelosuppression. Under steady-state conditions, only few LacZ/Tie-2+ cells were localized in the endosteal/osteoblastic region, without any expression within the BM sinusoidal neo-vessels. However, after a myelosuppressive dose of 5-Fluorouracil (5-FU) there was a robust expression of Tie2 expression in the regenerating BM sinusoidal neo-vessels. The expression of Tie2 on the regenerating neo-vasculature reached a maximum at day 10 and reverted back to steady-state by day 20 post 5-FU, the time in which the majority of the neo-vessels were functionally assembled. Blocking angiopoietin/Tie2 signaling with the soluble decoy-receptor, Tie2Fc, after 5-FU inhibited platelet recovery as well as vascular reconstitution. There was a paradoxical accumulation of megakaryocytes in the bone-marrow during the prolonged thrombocytopenic phase, which we have previously shown to be due to vascular niche disruption and inability of megakaryocytes to release platelets. To examine the possibility that Tie2+ progenitors can reconstitute BM sinusoids in the recipient mice, lethally irradiated wildtype mice were transplanted with the BM of Tie2-lacZ knock-in mice. After full recovery, the chimeric mice were challenged with a myelosuppressive dose of 5-FU and Tie2+ neo-vessels were detected histologically on day 5, 10, and 14 by LacZ staining. Remarkably, the reconstituted bone marrow showed the presence of Tie2+ vessels, which unambiguously demonstrates the contribution of donor-derived endothelial progenitors to the reconstitution of the regressed vasculature. Together, our data support the hypothesis that Tie2/angiopoietin signaling is essential for functional regeneration of BM sinusoidal neo-vessels and contributes to the reconstitution of hematopoiesis, specifically to thrombopoiesis. In addition, the use of Tie2-LacZ mice provides an invaluable model to quantify the number of regenerating BM neo-vessels. Angiopoietins may be used in clinical setting in conjunction with other lineage specific cytokines to enhance hematopoiesis after myelosuppression.

scholarly journals Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 581-591 ◽  
Author(s):  
AM Farese ◽  
F Herodin ◽  
JP McKearn ◽  
C Baum ◽  
E Burton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Pharmacokinetic Analysis ◽  
Pharmacokinetic Parameters ◽  
Platelet Recovery ◽  
Hematopoietic Reconstitution ◽  
Radiation Induced ◽  
Complete Blood Counts ◽  
60Co Gamma Radiation

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3, while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1, cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] 500/microL) and thrombocytopenia (THROM; platelet count 20,000/microL) were assessed. Synthokine significantly (P .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered, the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine, SC55 94, administered therapeutically post-TBI, significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host.

Hematopoietic Specific GATA-1-Improved Cre Mouse for Erythroid-Specific Gene Modification.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1291-1291
Author(s):  
Donghoon Yoon ◽  
Bumjun Kim ◽  
Myunghi Kwon ◽  
Josef T. Prchal
Keyword(s):  
Bone Marrow ◽  
Transgenic Mouse ◽  
Erythropoietin Receptor ◽  
Specific Gene ◽  
Hematopoietic Tissue ◽  
Knock Out ◽  
Tissue Sections ◽  
Lacz Staining ◽  
Splenic Cells ◽  
Beta Galactosidase

Abstract Animal models of erythropoiesis related genes have been limited by the fact that some of these genes have non-erythroid expression and other functions in addition to erythropoiesis and thus their knock-out may be embryonic lethal. Tissue specific knock-out or knock-in mice models employing GATA-1-Cre and other constructs showed that these promoters are also active in non-hematopoietic tissues, i.e. GATA-1 has activity in early embryonic development and in neuronal tissue. Suzuki et al (Blood, 2002, 100; 2279) isolated the GATA-1 locus hematopoietic regulatory domain (GATA-1-HRD) and demonstrated that the expression of a transgene under its control is limited to the hematopoietic tissue. We generated a transgenic mouse expressing an improved Cre (iCre) under GATA-1-HRD promoter control. This mouse was crossbred with ROSA 26 mouse and the progeny was examined for tissue specificity of iCre expression using beta-galactosidase staining. Brain, spleen, kidney, heart, thymus, liver, lung and ovary were examined for whole organ LacZ staining. All tested organs were negative except kidney and spleen where some positivity was observed. Subsequently, we prepared tissue sections from kidney, spleen and bone marrow and stained with LacZ and anti-beta-galactosidase antibody. Only the bone marrow EpoR expressing cells were positive; the kidney and the spleen cells were negative. Although Suzuki et al previously showed expression of the GATA-1-HRD driven erythropoietin receptor in spleen using RT-PCR, we were not able to find iCre expression in the splenic cells using these approaches. We demonstrate that our transgenic mouse (GATA-1-HRD-iCre) showed a restricted iCre expression in hematopoietic tissue that differs from previous studies of other hematopoiesis specific cre mouse. We conclude that this mouse model should be useful in studies of function of erythroid specific genes.

MT1-MMP and RECK Inversely Regulate Hematopoietic Progenitor Cell Egress.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1259-1259
Author(s):  
Abraham Avigdor ◽  
Yaron Vagima ◽  
Polina Goichberg ◽  
Shoham Shivtiel ◽  
Melania Tesio ◽  
...  
Keyword(s):  
Steady State ◽  
Progenitor Cell ◽  
Scid Mice ◽  
Hematopoietic Progenitor Cell ◽  
Chimeric Mice ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.

Murine Hematopoietic Progenitor Cells With Colony-Forming or Radioprotective Capacity Lack Expression of the β2-Integrin LFA-1

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Johannes F.M. Pruijt ◽  
Yvette van Kooyk ◽  
Carl G. Figdor ◽  
Roel Willemze ◽  
Willem E. Fibbe
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Hematopoietic Progenitor Cells ◽  
Colony Stimulating Factor ◽  
Hematopoietic Progenitor ◽  
Β2 Integrin ◽  
Stimulating Factor

Recently, we have demonstrated that antibodies that block the function of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) completely abrogate the rapid mobilization of hematopoietic progenitor cells (HPC) with colony-forming and radioprotective capacity induced by interleukin-8 (IL-8) in mice. These findings suggested a direct inhibitory effect of these antibodies on LFA-1–mediated transmigration of stem cells through the bone marrow endothelium. Therefore, we studied the expression and functional role of LFA-1 on murine HPC in vitro and in vivo. In steady state bone marrow ± 50% of the mononuclear cells (MNC) were LFA-1neg. Cultures of sorted cells, supplemented with granulocyte colony-stimulating factor (G-CSF)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-1/IL-3/IL-6/stem cell factor (SCF) and erythropoietin (EPO) indicated that the LFA-1neg fraction contained the majority of the colony-forming cells (CFCs) (LFA-1neg 183 ± 62/7,500 cells v LFA-1pos 29 ± 17/7,500 cells,P < .001). We found that the radioprotective capacity resided almost exclusively in the LFA-1neg cell fraction, the radioprotection rate after transplantation of 103, 3 × 103, 104, and 3 × 104 cells being 63%, 90%, 100%, and 100% respectively. Hardly any radioprotection was obtained from LFA-1pos cells. Similarly, in cytokine (IL-8 and G-CSF)–mobilized blood, the LFA-1neg fraction, which comprised 5% to 10% of the MNC, contained the majority of the colony-forming cells, as well as almost all cells with radioprotective capacity. Subsequently, primitive bone marrow-derived HPC, represented by Wheat-germ-agglutinin (WGA)+/Lineage (Lin)−/Rhodamine (Rho)− sorted cells, were examined. More than 95% of the Rho− cells were LFA-1neg. Cultures of sorted cells showed that the LFA-1neg fraction contained all CFU. Transplantation of 150 Rho− LFA-1neg or up to 600 Rho−LFA-1pos cells protected 100% and 0% of lethally irradiated recipient mice, respectively. These results show that primitive murine HPC in steady-state bone marrow and of cytokine-mobilized blood do not express LFA-1.

Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2008-2013 ◽  
Author(s):  
Hiroshi Kawada ◽  
Makio Ogawa
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mononuclear Cells ◽  
Chimeric Mice ◽  
Hematopoietic Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Hematopoietic Reconstitution ◽  
Bone Marrow Origin ◽  
Murine Skeletal Muscle

It has been reported that mononuclear cells harvested from murine skeletal muscle are capable of hematopoietic reconstitution of lethally irradiated mice. First, the nature of the hematopoietic progenitors in the muscle of C57BL/6–Ly-5.1 mice was examined by means of methylcellulose culture. The types and incidences of colonies grown from muscle mononuclear cells were different from those cultured from bone marrow (BM) or peripheral blood mononuclear cells. The next step was to examine the origin of the hematopoietic progenitors and stem cells in the muscle with the use of Ly-5.2 mice that had been made chimeric by transplantation of Ly-5.1 BM cells. The percentages of Ly-5.1 cells cultured from the muscle of the chimeric mice correlated with those cultured from BM, indicating BM origin of hematopoietic progenitors in the muscle. Long-term hematopoietic engrafting cells in the muscle of the chimeric mice were also derived from BM. However, mobilization of progenitors into circulation by granulocyte colony-stimulating factor did not change the population of hematopoietic progenitors in the muscle. It is proposed that hematopoietic progenitors and stem cells in the muscle tissue are of BM origin but their transition from BM to muscle may be a slow process.

scholarly journals Accelerated reconstitution of platelets and erythrocytes after syngeneic transplantation of bone marrow cells derived from thrombopoietin pretreated donor mice

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3308-3313 ◽  
Author(s):  
WE Fibbe ◽  
DP Heemskerk ◽  
L Laterveer ◽  
JF Pruijt ◽  
D Foster ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Direct Effect ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Platelet Recovery ◽  
Colony Assay ◽  
Cell Numbers ◽  
Colony Forming Unit

The recent cloning of the ligand of the c-Mpl hematopoietin receptor has indicated a major role for this cytokine in the development of megakaryocytes. In this study we have applied c-Mpl ligand (thrombopoietin [TPO]) in the setting of syngeneic transplantation in an attempt to accelerate the reconstitution of platelets. Donor mice were treated with 20 kilounits (kU)/d TPO intraperitoneally (ip) for 5 days. This resulted in a 2.5-fold increment in platelet counts from 1,119 x 10(9)/L to 2,582 x 10(9)/L (mean, n = 7). Total numbers of hematopoietic progenitor cells in bone marrow (BM) and spleen, as assessed in a colony-forming unit-granulocyte erythroid monocyte macrophage (CFU-GEMM) colony assay (55.3 v 38.6 x 10(3) CFU/femur; 27.3 v 16.3 x 10(3) CFU/spleen, mean, n = 7) as well as total numbers of burst-forming unit-erythroid (BFU-E) (24.0 v 16.4 x 10(3)/femur; 10.2 v 1.9 x 10(3)/spleen, mean, n = 7), were significantly higher in TPO- treated donors than in saline-treated controls. Female Balb-C mice were lethally (8.5 Gy) irradiated and transplanted with 10(5) BM cells. After transplantation, groups of mice were treated with recombinant murine TPO at a dose of 20 to 30 kU/d ip or subcutaneously (SC) for 5 to 14 days. Using this dose and schedule, TPO did not stimulate the recovery of platelets in comparison with control animals transplanted with equal cell numbers but given vehicle alone. In other experiments, 10(5) BM cells were procured from TPO-treated donor mice and transplanted into lethally irradiated recipient mice. In comparison with animals transplanted with an equal number of BM cells derived from saline-treated controls, recipients of TPO-treated BM cells had significantly faster platelet recovery and higher platelet nadir counts (88 v 30 x 10(9)/L, mean, n = 20). Transplantation of TPO-treated BM cells also resulted in an accelerated recovery of erythrocytes and increased erythrocyte nadir counts (7.2 v 5.0 x 10(12)/L, mean, n = 20). At the day of platelet nadir (day 12 after transplantation) these animals had higher numbers of BFU-Es (770 v 422, mean, n = 5) in the marrow and also had higher reticulocyte counts (44 / 1000 v 8 / 1000 mean, n = 5) in the blood. Therefore, the accelerated recovery of erythrocytes may be a direct effect of TPO on erythropoiesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine Hematopoietic Progenitor Cells With Colony-Forming or Radioprotective Capacity Lack Expression of the β2-Integrin LFA-1

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Johannes F.M. Pruijt ◽  
Yvette van Kooyk ◽  
Carl G. Figdor ◽  
Roel Willemze ◽  
Willem E. Fibbe
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Hematopoietic Progenitor Cells ◽  
Colony Stimulating Factor ◽  
Hematopoietic Progenitor ◽  
Β2 Integrin ◽  
Stimulating Factor

Abstract Recently, we have demonstrated that antibodies that block the function of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) completely abrogate the rapid mobilization of hematopoietic progenitor cells (HPC) with colony-forming and radioprotective capacity induced by interleukin-8 (IL-8) in mice. These findings suggested a direct inhibitory effect of these antibodies on LFA-1–mediated transmigration of stem cells through the bone marrow endothelium. Therefore, we studied the expression and functional role of LFA-1 on murine HPC in vitro and in vivo. In steady state bone marrow ± 50% of the mononuclear cells (MNC) were LFA-1neg. Cultures of sorted cells, supplemented with granulocyte colony-stimulating factor (G-CSF)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-1/IL-3/IL-6/stem cell factor (SCF) and erythropoietin (EPO) indicated that the LFA-1neg fraction contained the majority of the colony-forming cells (CFCs) (LFA-1neg 183 ± 62/7,500 cells v LFA-1pos 29 ± 17/7,500 cells,P &lt; .001). We found that the radioprotective capacity resided almost exclusively in the LFA-1neg cell fraction, the radioprotection rate after transplantation of 103, 3 × 103, 104, and 3 × 104 cells being 63%, 90%, 100%, and 100% respectively. Hardly any radioprotection was obtained from LFA-1pos cells. Similarly, in cytokine (IL-8 and G-CSF)–mobilized blood, the LFA-1neg fraction, which comprised 5% to 10% of the MNC, contained the majority of the colony-forming cells, as well as almost all cells with radioprotective capacity. Subsequently, primitive bone marrow-derived HPC, represented by Wheat-germ-agglutinin (WGA)+/Lineage (Lin)−/Rhodamine (Rho)− sorted cells, were examined. More than 95% of the Rho− cells were LFA-1neg. Cultures of sorted cells showed that the LFA-1neg fraction contained all CFU. Transplantation of 150 Rho− LFA-1neg or up to 600 Rho−LFA-1pos cells protected 100% and 0% of lethally irradiated recipient mice, respectively. These results show that primitive murine HPC in steady-state bone marrow and of cytokine-mobilized blood do not express LFA-1.

Dissection of the Role of Connexin-43 in Hematopoietic Progenitors and Stromal Cells Unveils Distinct Hematopoietic Intrinsic and Extrinsic Regulatory Functions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 607-607
Author(s):  
Lina Li ◽  
Bhuvana Murali ◽  
Dealma N. Worsham ◽  
Susan K. Dunn ◽  
Jose A. Cancelas
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Connexin 43 ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression

Abstract Bone marrow (BM) stromal cells seem to be crucial in the establishment of the hematopoietic niches in bone marrow. BM stromal cells can communicate through gap junctions, which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM stromal cells and upon adhesion to stroma, by hematopoietic stem cells and progenitors (HSC/P). Cx43 has been shown to be essential in controlling osteoblast and fibroblast function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC/P in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration in Mx1-Cre-Tg;Cx43KO mice (Presley C, et al., Cell Comm. Adh., 2005). We have also previously shown that after 5-FU administration, Cx43 is predominantly expressed in the endosteum and the deficiency of Cx43 in stroma of Collagen I (ColI)-Cre;Cx43KO and chimeric mice impairs their hematopoiesis by impairing the homing of wild-type (WT) hematopoietic progenitors and after 5-FU administration, the hematopoietic progenitor cycling inducing a ∼30% expansion of the long-term stem cell compartment in BM (Li L et al., ASH 2006). Interestingly, stromal Cx43-deficient mice contain around twice as many CFU-F as wild-type (WT) mice. Now, we have further investigated the role of stromal Cx43 expression in the regulation of hematopoietic progenitor adhesion to stroma, trans-stromal migration and mobilization. Cx43-deficient stromal cells display complete absence of intercellular communication as assayed by calcein dye transfer which can be reverted by retroviral transduction of Cx43. Trans-stromal migration of hematopoietic progenitors through Cx43-deficient irradiated stroma is impaired (7.8% vs 13.8% in WT stroma, p=0.015) but primary adhesion to Cx43-deficient irradiated stroma and in vivo mobilization response to G-CSF in ColI-Cre;Cx43KO mice were similar to WT controls, suggesting that stromal Cx43 plays a role in the regulation of the post-adhesion migration of HSC/P. On the other hand, Cx43-deficient HSC/P from Vav1-Cre;Cx43KO primary and chimeric mice show severe impairment of blood cell formation during the recovery phase after 5-FU administration (day +14) compared to wild-type controls (ANC: 0.23±0.12 vs 1.40±1.25 x 109 neutrophils/L; Platelet count: 135±91 vs 572±205 x 109 platelets/L; p < 0.05). Cx43 deficiency in hematopoietic progenitors did not significantly impair their homing ability in wild-type mice. Taken together, these studies indicate that Cx43 expression plays distinct roles in the regulation of hematopoietic intrinsic and extrinsic mechanisms. While Cx43 expression in stroma seems to be crucial in the regulation of the stromal progenitor and HSC pool content as well as HSC/P trans-stromal migration and homing, deficiency of Cx43 in either hematopoietic cells or stromal cells independently induce a significant impairment in the post-chemotherapy blood formation in vivo, suggesting that, under stress, hematopoietic regeneration depends on complete Cx43 channels communicating HSC/P and stromal cells.

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