Correction of the Phenotype in Canine Leukocyte Adhesion Deficiency Following Non-Myeloablative, Matched Littermate Transplant.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2143-2143
Author(s):  
Yuchen Gu ◽  
Thomas R. Bauer ◽  
Laura M. Tuschong ◽  
Robert A. Sokolic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Leukocyte Adhesion ◽  
Disease Phenotype ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Complete Reversal ◽  
Low Levels

Abstract Canine leukocyte adhesion deficiency (CLAD) represents the canine counterpart of the human disease leukocyte adhesion deficiency (LAD). Children with LAD and puppies with CLAD suffer life-threatening bacterial infections as a result of the failure of their leukocytes to adhere to the endothelial surface and migrate to the site of infection. Molecular defects in the leukocyte integrin CD18 molecule are responsible for both LAD and CLAD. Although myeloablative hematopoietic stem cell transplantation can correct the disease phenotype in LAD, this therapy is accompanied by considerable toxicity. Moreover, it is not clear that full donor chimerism is required for reversal of the disease phenotype. To assess the role of mixed chimerism in reversing the disease phenotype in CLAD, we used a non-myeloablative conditioning regimen consisting of 200 cGy total body irradiation preceding matched littermate allogeneic transplant, and followed by a brief post-transplant regimen consisting of cyclosporine and mycophenolic acid. Six dogs received bone marrow cells, three dogs received CD34+ bone marrow stem cells, and four dogs received mobilized peripheral blood stem cells. Eleven of 13 transplanted CLAD dogs achieved mixed donor-host chimerism resulting in complete reversal of the disease phenotype. Donor-derived CD18+ cells measured by flow cytometric analysis in the peripheral blood of the transplanted CLAD dogs correlated closely with donor chimerism measured by DNA analysis of microsatellite repeats in the peripheral blood leukocytes. The 11 dogs with reversal of the CLAD phenotype have been followed for over one year from the time of transplant and displayed levels of donor leukocyte chimerism ranging from 4 to 95%. Since engraftment, all eleven dogs have been free from infection and live in runs with other dogs. Three dogs with very low levels of donor leukocyte chimerism post-transplant displayed evidence of selective egress of CD18+ donor leukocytes into extravascular sites, indicating that the level of CD18+ donor cells measured in the periperal blood may underestimate the total number of CD18+ donor leukocytes. In the two dogs who did not have complete reversal of the CLAD phenotype post-transplant, one dog died at 3 weeks following transplant from a subcapsular hemorrhage of the liver secondary to thrombocytopenia, and one dog had donor microchimerism following transplant with partial reversal of the phenotype. Three dogs who did not have a matched littermate donor, and did not receive a transplant, died of infection at 2, 4, and 6 months of age, respectively. The fact that correction of the CLAD phenotype was achieved in 11 of 13 dogs with mixed donor-host chimerism and the absence of graft-versus-host disease has implications for allotransplant in LAD when a matched sibling donor exists. The observation that very low levels of donor CD18+ leukocytes reversed the disease phenotype supports the use of the CLAD model for testing the ability of autologous, CD18 gene-corrected hematopoietic stem cells to reverse the CLAD phenotype, since low levels of gene correction are anticipated with gene therapy.

Retroviral-Mediated Gene Transfer of CD18 into Hematopoietic Stem Cells in Dogs with Canine Leukocyte Adhesion Deficiency Reverses the Severe Deficiency Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3169-3169
Author(s):  
Mehreen Hai ◽  
Thomas R. Bauer ◽  
Yu-chen Gu ◽  
Laura M. Tuschong ◽  
Robert A. Sokolic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Leukocyte Adhesion ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Post Infusion ◽  
Low Levels

Abstract Background: Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific, large-animal model for the human disease leukocyte adhesion deficiency (LAD). Puppies with CLAD, like children with LAD, experience recurrent life-threatening bacterial infections due to the inability of their leukocytes to adhere and migrate to sites of infection. Mutations in the gene encoding the leukocyte integrin CD18 are responsible for both CLAD and LAD. Allogeneic bone marrow or hematopoietic stem cell transplantation is currently the only curative therapy for LAD. We recently reported the results of non-myeloablative allogeneic transplants in CLAD dogs and showed that very low levels of CD18+ donor-derived neutrophils (less than 300/microliter) were sufficient to reverse the CLAD disease phenotype. These results indicated that CLAD dogs may be amenable to treatment using gene therapy, where there are frequently low numbers of transduced cells. We report the results of retroviral- mediated transduction in autologous hematopoietic stem cells with the canine CD18 gene. Method: Bone marrow was harvested and CD34+ selected from four dogs with CLAD at approximately 3–4 months of age. The purified CD34+ cells were either used immediately or were frozen and subsequently thawed. Cells were pre-stimulated with cSCF, hFlt3-L, hTPO and cIL-6 for approximately 24 hours, then exposed to two rounds of supernatant from the retroviral vector PG13/MSCV-cCD18 for 24 hours each on recombinant fibronectin. At the end of the transduction, the cells were infused back into the animal that had been conditioned with 200 cGy total body irradiation. Post-transplant immunosuppression consisted of cyclosporine given at a dose of 30 mg/kg from day -1 to day 35, then 15 mg/kg from day 36 to day 60, and mycophenolate mofetil at a dose of 20 mg/kg from day 0 to day 28. Peripheral blood samples, as well as pus samples from one animal, were analyzed by flow cytometry at designated time points post-transplant. Results: The four dogs who received autologous, gene-corrected cells have been followed for 7–12 weeks post-infusion. The number of CD18+ CD34+ cells infused per dog ranged from 0.2 to 0.55 x 106 cells/kg. The post-infusion percentage of CD18+ neutrophils in each dog was 0.09%, 0.13%, 0.62% and 0.02% at 12, 10, 8 and 6 weeks respectively. Clinically all four treated CLAD dogs are alive with marked improvement of their CLAD disease. These dogs are now 6–7 months of age. These results contrast with those seen in untreated CLAD dogs who uniformly die or are euthanized within the first few months of life. The reversal of the severe CLAD phenotype despite the very low levels of CD18+ neutrophils in the peripheral blood is likely due to the selective egress of CD18+ neutrophils into the tissue since one treated CLAD dog who had less than 1% CD18+ neutrophils in the blood had nearly 10% CD18+ neutrophils in pus collected from an inflammatory dental lesion. Conclusion: These data suggest that a non-myeloablative conditioning regimen coupled with a minimal immunosuppressive regimen may enable sufficient CD18+ autologous gene-corrected cells to engraft and result in reversal of the severe CLAD phenotype.

In Utero Hematopoietic Cell Transplantation Using Haploidentical Parental Donors Reverses the Lethal Phenotype in Dogs with Canine Leukocyte Adhesion Deficiency.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 624-624 ◽  
Author(s):  
William H. Peranteau ◽  
Yuchen Gu ◽  
Susan Volk ◽  
Laura M. Tuschong ◽  
Thomas R. Bauer ◽  
...  
Keyword(s):  
Cell Transplantation ◽  
Genetic Disease ◽  
Leukocyte Adhesion ◽  
In Utero ◽  
Large Animal ◽  
Disease Phenotype ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Low Levels

Abstract Juvenile dogs with the genetic disease leukocyte adhesion deficiency or CLAD, like children with leukocyte adhesion deficiency or LAD, experience recurrent life threatening bacterial infections due to the inability of leukocytes to migrate to sites of infection. Both CLAD and LAD result from defects in the leukocyte integrin CD18 molecule. We have used the CLAD model to develop new therapeutic approaches to children with LAD. Previous studies demonstrate that low levels of donor chimerism following matched littermate transplant reverses the disease phenotype in CLAD. However, most children with LAD lack a matched sibling donor. In utero hematopoietic stem cell transplantation (IUHCT) has been shown to result in low levels of allogeneic chimerism in the normal mouse model. In the current study we evaluate IUHCT in the CLAD model using a haploidentical paternal donor. A previously transplanted CLAD female was mated with a paternal CLAD carrier. IUHCT was performed at gestational day 50 by ultrasound guided intraperitoneal injection of 1.7E+08 paternal CD34+ enriched BM cells/kg estimated fetal weight reconstituted with nonenriched paternal BM to provide 2.4% CD3+ cells. Seven fetuses were injected. One pup was still born and one died on day 2 from maternal neglect. Flow cytometry for CD18 expression in PB, spleen, liver, thymus and BM from the two deceased pups confirmed the diagnosis of CLAD with donor cell engraftment (PB:1.3–3%, spleen:3.5–4%, liver:3.4–4.2%, thymus:1.7–4.4%, BM:3.3–21.3%). Histology demonstrated no evidence of GVHD. Of the 5 surviving pups, 3 are CLAD carriers (Louie, Miles, Ella) and 2 are CLAD offspring (Billie, Duke). Currently all 5 pups are alive at 5 months of age. Engraftment analysis in those in which it is possible by CD18 expression (Billie and Duke) or the presence of the Y chromosome (Billie and Ella) demonstrates donor cells in all analyzable pups at 5 months. The PB levels of CD18+ donor cells in CLAD offspring are low but stable and contribute to multiple lineages (Fig 1A). Clinically, Billie and Duke are alive and active at 5 months compared to historical controls with 4 and 6 month mortalities of 75% and 100%. Both have had mild leukocytosis (Billie:17.6–23.8K/uL, 21.4K/uL @ 5 months; Duke:23.4–39.5K/uL, 23.8K/uL @ 5 months) compared to historical CLAD controls of 50–100K/uL. Billie has had no clinical episodes consistent with the CLAD phenotype. Duke has experienced five CLAD phenotypic episodes which have resolved without the need for intensive care (Fig 1B). There was no evidence of GVHD in any injected animals. This study highlights the ability to safely achieve levels of haploidentical donor CD18+ leukocytes following IUHCT which markedly improve the lethal disease phenotype in a disease-specific large-animal model of a human genetic disease. It supports the potential therapeutic value of IUHCT for diseases, such as LAD, which can be successfully treated with low levels of hematopoietic chimerism. Figure Figure

Homing and Engraftment of Mobilized Hematopoietic Stem Cells: Involvement of VLA-5.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4943-4943
Author(s):  
Pieter K. Wierenga ◽  
Gerald de Haan ◽  
Bert Dontje ◽  
Ellen Weersing ◽  
Ronald van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Hematopoietic Reconstitution

Abstract VLA-5 has been implicated in the adhesive interactions of stem and progenitor cells with the bone marrow extracellular matrix and stromal cells and is therefore considered to play an important role in the hematopoietic reconstitution after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3% of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-GSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 38±3%. Despite this low frequency of VLA-5+ cells, however, even when equal numbers of progenitor cells are transplanted MPB cells provide a much faster hematopoietic recovery compared to BM cells. To shed more light on the role of VLA-5 in the process of homing and engraftment, we investigated whether differences in homing potential of the stem cell subsets might be responsible for this enhanced reconstitution. At 3 hours post-transplant, however, no differences in homing efficiency of progenitor and stem cells from MPB and BM grafts in both bone marrow and spleen could be detected. It should be realized that MPB and BM grafts demonstrate different ratios of stem/progenitor cells which might be another explanation for the observed differences in repopulation potential. Furthermore, MPB cells migrating in vitro towards SDF-1α showed potent reconstitution while VLA-5 expression was reduced on these cells. In fact, in vitro treatment with SDF-1α showed further decrease in VLA-5 expressing cells (from 38% to 4%) in the lin- fraction. When equal numbers of MPB were transplanted with and without SDF-1α pretreatment, no difference in hematopoietic reconstitution was observed suggesting a minor role of VLA-5 in homing and engraftment. On the other hand, after VLA-5 blocking an inhibition of 59±7% in the homing of MPB progenitor cells in the bone marrow could be found, whereas homing in the spleen of the the recipients is only inhibited by 11±4%. To elucidate whether the observed enhanced reconstitution could be explained by a selective homing of VLA-5+ cells or a rapid upregulation of VLA-5 expression, cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. It could be demonstrated that at 3 hours post-transplant cells from MPB grafts showed a rapid increase from 38±3% up to 66±9% of VLA-5+ cells in the bone marrow of the recipient. In the spleen no significant increase in VLA-5+ cells was observed. When MPB cells were transplanted after pretreatment with SDF-1α an increase from 2±1% up to 33±5% of VLA-5+ cells in the bone marrow was detected. When calculating the number of cells recovered from bone marrow, a selective homing of VLA-5+ cells cannot be excluded. Therefore, we also assessed the number of VLA-5+ cells in the PKH+ fraction in peripheral blood from the recipient immediately (½-1 hour) after transplantation but found no increase during that time period. So far it can be concluded that MPB cells show low number of VLA-5+ cells but these cells possess an enhanced hematopoietic reconstitution potential. Homing of progenitor cells to the spleen seems to be less dependent on VLA-5 expression than homing to the bone marrow. A rapid upregulation of VLA-5 expression on engrafting MPB cells early after transplantation does not occur and hence our data are suggestive for the preferential homing of VLA-5+ cells in the bone marrow after transplantation.

Selective Migration of CD18+ Neutrophils into the Tissue Results in Reversal of the Phenotype in Canine Leukocyte Adhesion Deficiency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3084-3084
Author(s):  
Yuchen Gu ◽  
Robert A. Sokolic ◽  
Thomas R. Bauer ◽  
Laura M. Tuschong ◽  
Mehreen Hai ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Clinical Phenotype ◽  
Leukocyte Adhesion ◽  
Signal Molecules ◽  
Disease Phenotype ◽  
Leukocyte Adhesion Deficiency ◽  
Post Transplant ◽  
Mucosal Tissues

Abstract Leukocyte adhesion deficiency-1 (LAD-1) is a genetic immunodeficiency disease characterized by recurrent, life threatening bacterial infections. The phenotype of LAD-1 stems from the inability of leukocytes from affected children to adhere and migrate to sites of infection as a result of mutations in the leukocyte integrin CD18. Canine leukocyte adhesion deficiency (CLAD) represents the homologue of the severe phenotype of LAD-1 in humans. In previous studies we have demonstrated that less than 500 CD18+ neutrophils/μl following non-myeloablative stem cell transplantation from matched littermate donors resulted in reversal of the CLAD disease phenotype. In this report, we describe two CLAD dogs (D128 and D144) with donor microchimerism and improvement from a severe to a moderate clinical phenotype of CLAD following matched littermate hematopoietic stem cell transplant. Prior to transplant both dogs displayed the hallmarks of CLAD phenotype with fever, severe infections, leukocytosis and were following the natural history of CLAD, which invariably leads to death by 6 months of age. Both dogs are maintained on only prophylactic antibiotics and are alive more than two years post-transplant. The calculated level of CD18+ donor neutrophils was approximately 100 CD18+ neutrophils/μl in dog D128 and 50 CD18+ neutrophils/μl in dog D144, respectively. To determine how this persistently low level of CD18+ neutrophils/μl measured in the peripheral blood resulted in the moderation of the severe disease phenotype of CLAD, we assessed the level of CD18+ leukocytes in mucosal tissues. Mucosal tissues have a constant bacterial presence that is kept under control in part by a constant influx of neutrophils from surrounding periodontal tissues. The emigration of neutrophils from the circulation is a critical step during immune surveillance and inflammatory reactions, and is governed by a coordinated interaction involving a spectrum of adhesion and signal molecules. The oral rinse assay enables a non-invasive, in vivo measurement of neutrophil migration into tissues. Both dogs displayed evidence of selective extravasation of CD18+ donor cells into extravascular site as shown by FACS analysis with eleven-fold (D128) and five-fold (D144) more donor-derived CD18+ leukocytes present in saliva compared to the peripheral blood. The percentage of donor chimerism was confirmed using DNA microsatellite markers that distinguished donor from host. These results indicate that a level of less than 100 CD18+ neutrophils/μl in peripheral blood improves the CLAD phenotype from a severe to a moderate clinical phenotype. Moreover, measurement of neutrophils in peripheral blood alone fails to take into consideration of the total number of CD18+ donor derived cells in the recipient CLAD animals post-transplant. These results suggest that measurement of donor-derived CD18+ neutrophils in the tissue in this disease provides a more accurate assessment of the clinical effect than the measurement of CD18+ leukocytes in the peripheral blood and they support the treatment of CLAD by gene therapy, where a low number of gene corrected cells is anticipated with current gene therapy vectors and transduction conditions.

scholarly journals Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 939-948 ◽  
Author(s):  
Y Tomita ◽  
DH Sachs ◽  
M Sykes
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Whole Body ◽  
Mixed Chimerism ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Donor Type ◽  
Low Levels

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

scholarly journals Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7.

1995 ◽  
Vol 181 (1) ◽  
pp. 369-374 ◽  
Author(s):  
K J Grzegorzewski ◽  
K L Komschlies ◽  
S E Jacobsen ◽  
F W Ruscetti ◽  
J R Keller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Peripheral Blood Leukocytes ◽  
Gene Modification ◽  
Blood Leukocytes ◽  
Hematopoietic Stem ◽  
Interleukin 7 ◽  
Cell Repopulation

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

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