Expression and Function of Homing-Essential Molecules and Enhanced In Vivo Homing Ability of Human Peripheral Blood-Derived Hematopoietic Pr
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《干细胞学杂志》
a Department of Hematology and Oncology and
b Institute of Pathology, University of Regensburg, Regensburg, Germany
Key Words. CD34 ? Stem cell factor ? Integrin ? Hematopoietic progenitor cells
Correspondence: Burkhard Hennemann, M.D., Department of Hematology and Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Telephone: 0049-941-944-5531; Fax: 0049-941-944-5511; e-mail: burkhard.hennemann@klinik.uni-regensburg.de
ABSTRACT
The transplantation of peripheral blood-derived hematopoietic stem cells (HSCs) has become a routinely used procedure for the treatment of hematological diseases. In heavily pretreated patients, the harvest of sufficient numbers of CD34+ cells can be difficult even after mobilization with chemotherapy and granulocyte-colony stimulating factor (G-CSF) . In vitro studies revealed that the retention of hematopoietic progenitor cells (HPCs) in the bone marrow is primarily mediated via adhesion to extracellular matrix proteins by the ?1 integrins very-late antigen (VLA)-4 and VLA-5 . Bone marrow CD34+ cells express under steady-state conditions a variety of adhesion molecules, such as the 4, 5, ?1, CD11a/CD18, and CD11b/CD18 integrins, L-selectin, platelet and endothelial cell adhesion molecule (PECAM)-1, and CD44 . It has been shown that the expression or function of the ?1 integrins can be modulated by various cytokines; in HL60 cells, the expression of the 4 domain was reduced after incubation with interleukin (IL)-3, IL-6, IL-11, or stem cell factor (SCF) . The adhesive function of VLA-4 and VLA-5 on TF-1 and Mo7e cells to fibronectin increased after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, or SCF, although the expression of these integrins on the cell membrane was not altered . In vivo studies using HPCs treated with anti–VLA-4 antibodies before transplantation showed a significantly reduced engraftment ability of the transplanted cells . Phenotypic analysis and transplantation of human HPCs in nonobese diabetic-severe combined immune deficiency (scid)/scid (NOD/SCID) mice indicated that in addition to VLA-4, VLA-5 is also expressed on repopulating cells . In mice, it was shown that HPCs found in the blood after treatment with cyclophosphamide and G-CSF express significantly lower levels of 2, 4, and ?1 integrins, correlating with a 50% decrease in their ability to home to the bone marrow .
Based on the hypothesis that retention of HPC in hematopoietic organs is controlled by adhesive interactions, we reasoned that increasing the adhesiveness of peripheral blood-derived HPCs might enhance the proportion of the cells reaching and residing within the bone marrow. In the setting of HSC transplantation, this approach might improve the homing ability of the transplanted cells into the bone marrow of the recipient. The experiments described here were designed to test this hypothesis. First, we sought to identify progenitor cell populations that possibly were sensitive to stimulation with SCF by assessing the expression of c-kit. Then CD34+ cell–enriched suspensions of the peripheral blood of G-CSF–treated healthy volunteers or chemotherapy-treated cancer patients were cultured in the presence of SCF, and the expression and function of VLA-4 and VLA-5 were monitored. We used the long-term culture on murine stroma cells to detect and enumerate early human HPCs, referred to as long-term culture-initiating cells (LTC-ICs), to test the effect of SCF on the adhesive capabilities of these cells. Finally, the improvement of the in vivo homing abilities of SCF-stimulated HSCs was confirmed by transplantation into sublethally irradiated NOD/SCID mice.
MATERIALS AND METHODS
Isolation and Functional Characterization of CD34+/c-kit+ and CD34+/c-kit– Cells
The immunomagnetic enrichment of CD34+ cells from the leukapheresis products of chemotherapy and G-CSF–treated tumor patients resulted in a lineage-depleted cell fraction containing on average 80 ± 8% CD34+ cells with a 62% recovery of the CD34+ cells after the enrichment process (n = 10). A total of 36% of the CD34+ cells was additionally c-kit+ (n = 3). To assess the functional capacity of these cells, the CD34+/c-kit+ and the respective c-kit– fraction obtained from seven leukapheresis products were sorted and plated into CFC and LTC-IC assays. As shown in Table 1, no colony growth was detected in three specimens. In two of those samples, the LTC-IC frequency was too low (<1 in 7,800 and <1 in 28,800 cells) to give rise to any colonies within the c-kit+ cell fraction, but LTC-ICs were detectable within the c-kit– fraction. In four experiments, the CFC content of the c-kit+ and the c-kit– cells could be compared and showed a 2.2-fold higher number of CFCs within the c-kit+ fraction (p = .014). The LTC-IC frequency was calculated from the pooled data of six experiments and was 2.8-times higher within the c-kit– fraction compared with the c-kit+ fraction.
Table 1. Functional characterization of mobilized tumor patient (MTP)–derived CD34+/c-kit+ and CD34+/c-kit– cells
Flow Cytometric Analysis of c-kit, VLA-4, and VLA-5 on CD34+ Cells
Flow cytometry was performed with freshly isolated or cultured cells. Before culture, 14 ± 7% and 36 ± 4% of the CD34+ cells from healthy donors and chemotherapy-treated tumor patients were c-kit+. After culture, c-kit expression was reduced for both (Fig. 1). More than 50% of the CD34+ cells were spontaneously VLA-4+, and the proportion increased approximately 1.5-fold to 90% VLA-4+ cells after 24-hour SCF stimulation. The spontaneous expression of VLA-5 on CD34+ cells was remarkably lower and increased by a factor of 27 for cells from healthy donors and by a factor of 4 for patient-derived cells (Table 2) after SCF stimulation. The effect of SCF could be blocked by the addition of the tyrosine kinase inhibitor genistein (Fig. 2).
Figure 1. Representative flow cytometric dot blot diagram showing the expression of c-kit, VLA-4, and VLA-5 on CD34+ cells from a healthy donor before and after stimulation with SCF for 24 hours. The percentage of the cells is indicated in the corner of the respective quadrants. Abbreviations: SCF, stem cell factor; VLA, very-late antigen.
Table 2. Stimulation with stem cell factor (SCF) increases the surface expression very-late antigen (VLA)-4 and VLA-5 on CD34+ cells
Figure 2. CD34+ cells were stimulated with SCF with and without the addition of genistein for 24 hours and then analyzed by flow cytometry. Shown is the proportion of CD34+/CD117+ cells expressing VLA-4 and VLA-5. Mean values ± standard error of the mean from three experiments are documented. Abbreviations: SCF, stem cell factor; VLA, very-late antigen.
Effect of SCF on the Adhesiveness of Primary CD34+ Cells
The effect of stimulating sorted CD34+ cells for 30 minutes with SCF at a concentration of 100 ng/ml was evaluated. The proportion of cells adhering to Petri dishes coated with 4 μg/cm2 CH296 increased from 14.0% without the addition of SCF to 23.0% with SCF stimulation. Although the distribution of CFC of adherent and nonadherent cells did not differ, the frequency of LTC-ICs was approximately three times higher in the adherent cell population (39 versus 15 LTC-ICs per 105 cells plated without SCF stimulation). Stimulation with SCF did not alter the frequency of CFCs and LTC-ICs within the respective cell fractions. As shown in Table 3, subsequent limiting dilution analysis of the LTC-IC frequency within the adherent cell fractions revealed a net increase of adherent LTC-ICs of approximately 30% by stimulation with SCF.
Table 3. Stem cell factor (SCF) stimulation enhances the number of adherent long-term culture-initiating cells (LTC-ICs)
SCF Stimulation Increases the Homing Ability after Transplantation in NOD/SCID Mice
By transplanting human HPCs into irradiated NOD/SCID mice, we sought to confirm the improvement of the in vivo homing abilities of SCF-stimulated HSCs compared with untreated control. In two independent experiments, a total of 16 animals were injected with lin– cells with or without the addition of SCF. In both experiments, bone marrow cells from all animals could be analyzed by flow cytometry and PCR. Eight of eight mice in the SCF treatment group showed multilineage hematopoietic engraftment of the human cells as assessed by the detection of at least five human lymphocytes and five human granulocytes per 2 x 104 bone marrow cells analyzed by flow cytometry and the detection of human DNA by PCR. In the animals of the control group, no human cells could be detected by flow cytometry or by PCR (Fig. 3). The mean proportion of human CD45 and CD71+ cells as determined by flow cytometry was 8.5 ± 7.2% (mean ± standard deviation ; range, 1.2–17.2) and 3.0 ± 2.3% (mean ± SD; range, 1.2–6.4) after the transplantation of 3.0 x 105 and 3.6 x 105 lin– human cells, respectively (Table 4).
Figure 3. Bone marrow cells isolated from nonobese diabetic-scid/scid mice transplanted 6 weeks previously with lin– hematopoietic progenitor cells were analyzed by PCR for human 5' actin. (A): PCR analysis of mouse bone marrow cells mixed with a varying proportion of human cells. (B), (C): PCR analysis of two independent experiments in which two groups of mice were transplanted with (+SCF) or without (–SCF) the addition of SCF. As a negative control, bone marrow cells of a mouse that did not receive human cells were analyzed (C, mouse 0). Abbreviations: PCR, polymerase chain reaction; SCF, stem cell factor.
Table 4. Effect of stem cell factor (SCF) on the hematopoietic engraftment of enriched human CD34+ cells transplanted in irradiated nonobese diabetic-scid/scid (NOD/SCID) mice
DISCUSSION
The excellent technical assistance of Nadine Pi?ler and Marit Hoffmann is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (HE 3199/4-1; to B.H.).
REFERENCES
Stiff PJ. Management strategies for the hard-to-mobilize patient. Bone Marrow Transplant 1999;23(suppl 2):S29–S33.
Teixido J, Hemler ME, Greenberger JS et al. Role of ?1 and ?2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma. J Clin Invest 1992;90:358–367.
Mohle R, Murea S, Kirsch M et al. Differential expression of L-selectin, VLA-4, and LFA-1 on CD34+ progenitor cells from bone marrow and peripheral blood during G-CSF-enhanced recovery. Exp Hematol 1995;23:1535–1542.
Kinashi T, Springer TA. Steel factor and c-kit regulate cell-matrix adhesion. Blood 1994;83:1033–1038.
Dercksen MW, Gerritsen WR, Rodenhuis S et al. Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation. Blood 1995;85:3313–3319.
Watt SM, Williamson J, Genevier H et al. The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes. Blood 1993;82:2649–2663.
Laszik Z, Jansen PJ, Cummings RD et al. P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells. Blood 1996;88:3010–3021.
Becker PS, Nilsson SK, Li Z et al. Adhesion receptor expression by hematopoietic cell lines and murine progenitors: modulation by cytokines and cell cycle status. Exp Hematol 1999;27:533–541.
Levesque JP, Leavesley DI, Niutta S et al. Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J Exp Med 1995;181:1805–1815.
Papayannopoulou T, Craddock C, Nakamoto B et al. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A 1995;92:9647–9651.
van der Loo JC, Xiao X, McMillin D et al. VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin. J Clin Invest 1998;102:1051–1061.
Wagers AJ, Allsopp RC, Weissman IL. Changes in integrin expression are associated with altered homing properties of Lin(–/lo) Thy1.1 (lo) Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor. Exp Hematol 2002;30:176–185.
Hennemann B, Oh IH, Chuo JY et al. Efficient retro-virus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin. Blood 2000;96:2432–2439.
Hogge DE, Lansdorp PM, Reid D et al. Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor. Blood 1996;88:3765–3773.
Peled A, Kollet O, Ponomaryov T et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000;95:3289–3296.
Williams DA, Rios M, Stephens C et al. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991;352:438–441.
Papayannopoulou T, Nakamoto B. Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin. Proc Natl Acad Sci U S A 1993;90:9374–9378.
Yamaguchi M, Ikebuchi K, Hirayama F et al. Different adhesive characteristics and VLA-4 expression of CD34+ progenitors in G0/G1 versus S+G2/M phases of the cell cycle. Blood 1998;92:842–848.
Prosper F, Stroncek D, McCarthy JB et al. Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function. J Clin Invest 1998; 101:2456–2467.
Broudy VC. Stem cell factor and hematopoiesis. Blood 1997;90:1345–1364.
Kawashima I, Zanjani ED, Almaida-Porada G et al. CD34+ human marrow cells that express low levels of kit protein are enriched for long-term marrow-engrafting cells. Blood 1996;87:4136–4142.
Sakabe H, Yahata N, Kimura T et al. Human cord blood-derived primitive progenitors are enriched in CD34+ c-kit– cells: correlation between long-term culture-initiating cells and telomerase expression. Leukemia 1998;12:728–734.
Sakabe H, Ohmizono Y, Tanimukai S et al. Functional differences between subpopulations of mobilized peripheral blood-derived CD34+ cells expressing different levels of HLA-DR, CD33, CD38 and c-kit antigens. STEM CELLS 1997;15:73–81.
Zeller W, Kroger N, Berger J et al. Expression of the adhesion molecules CD49d and CD49e on G-CSF-mobilized CD34+ cells of patients with solid tumors or non-Hodgkin’s and Hodgkin’s lymphoma and of healthy donors is inversely correlated with the amount of mobilized CD34+ cells. J Hematother Stem Cell Res 1999;8:539–546.
Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nature Med 1996;2:876–882.
Denning-Kendall P, Singha S, Bradley B et al. Cytokine expansion culture of cord blood CD34+ cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions. STEM CELLS 2003;21:61–70.
Fuhlbrigge RC, Alon R, Puri KD et al. Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions. J Cell Biol 1996;135:837–848.
Hennemann B, Conneally E, Pawliuk R et al. Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables. Exp Hematol 1999;27:817–825.
Lynch DH, Jacobs C, DuPont D et al. Pharmacokinetic parameters of recombinant mast cell growth factor (rMGF). Lymphokine Cytokine Res 1992;11:233–243.
Martin FH, Suggs SV, Langley KE et al. Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 1990;63:203–211.
Zheng Y, Watanabe N, Nagamura-Inoue T et al. Ex vivo manipulation of umbilical cord blood-derived hematopoietic stem/progenitor cells with recombinant human stem cell factor can up-regulate levels of homing-essential molecules to increase their transmigratory potential. Exp Hematol 2003;31:1237–1246.
Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.(Christina Harta, Diana Dr)
b Institute of Pathology, University of Regensburg, Regensburg, Germany
Key Words. CD34 ? Stem cell factor ? Integrin ? Hematopoietic progenitor cells
Correspondence: Burkhard Hennemann, M.D., Department of Hematology and Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Telephone: 0049-941-944-5531; Fax: 0049-941-944-5511; e-mail: burkhard.hennemann@klinik.uni-regensburg.de
ABSTRACT
The transplantation of peripheral blood-derived hematopoietic stem cells (HSCs) has become a routinely used procedure for the treatment of hematological diseases. In heavily pretreated patients, the harvest of sufficient numbers of CD34+ cells can be difficult even after mobilization with chemotherapy and granulocyte-colony stimulating factor (G-CSF) . In vitro studies revealed that the retention of hematopoietic progenitor cells (HPCs) in the bone marrow is primarily mediated via adhesion to extracellular matrix proteins by the ?1 integrins very-late antigen (VLA)-4 and VLA-5 . Bone marrow CD34+ cells express under steady-state conditions a variety of adhesion molecules, such as the 4, 5, ?1, CD11a/CD18, and CD11b/CD18 integrins, L-selectin, platelet and endothelial cell adhesion molecule (PECAM)-1, and CD44 . It has been shown that the expression or function of the ?1 integrins can be modulated by various cytokines; in HL60 cells, the expression of the 4 domain was reduced after incubation with interleukin (IL)-3, IL-6, IL-11, or stem cell factor (SCF) . The adhesive function of VLA-4 and VLA-5 on TF-1 and Mo7e cells to fibronectin increased after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, or SCF, although the expression of these integrins on the cell membrane was not altered . In vivo studies using HPCs treated with anti–VLA-4 antibodies before transplantation showed a significantly reduced engraftment ability of the transplanted cells . Phenotypic analysis and transplantation of human HPCs in nonobese diabetic-severe combined immune deficiency (scid)/scid (NOD/SCID) mice indicated that in addition to VLA-4, VLA-5 is also expressed on repopulating cells . In mice, it was shown that HPCs found in the blood after treatment with cyclophosphamide and G-CSF express significantly lower levels of 2, 4, and ?1 integrins, correlating with a 50% decrease in their ability to home to the bone marrow .
Based on the hypothesis that retention of HPC in hematopoietic organs is controlled by adhesive interactions, we reasoned that increasing the adhesiveness of peripheral blood-derived HPCs might enhance the proportion of the cells reaching and residing within the bone marrow. In the setting of HSC transplantation, this approach might improve the homing ability of the transplanted cells into the bone marrow of the recipient. The experiments described here were designed to test this hypothesis. First, we sought to identify progenitor cell populations that possibly were sensitive to stimulation with SCF by assessing the expression of c-kit. Then CD34+ cell–enriched suspensions of the peripheral blood of G-CSF–treated healthy volunteers or chemotherapy-treated cancer patients were cultured in the presence of SCF, and the expression and function of VLA-4 and VLA-5 were monitored. We used the long-term culture on murine stroma cells to detect and enumerate early human HPCs, referred to as long-term culture-initiating cells (LTC-ICs), to test the effect of SCF on the adhesive capabilities of these cells. Finally, the improvement of the in vivo homing abilities of SCF-stimulated HSCs was confirmed by transplantation into sublethally irradiated NOD/SCID mice.
MATERIALS AND METHODS
Isolation and Functional Characterization of CD34+/c-kit+ and CD34+/c-kit– Cells
The immunomagnetic enrichment of CD34+ cells from the leukapheresis products of chemotherapy and G-CSF–treated tumor patients resulted in a lineage-depleted cell fraction containing on average 80 ± 8% CD34+ cells with a 62% recovery of the CD34+ cells after the enrichment process (n = 10). A total of 36% of the CD34+ cells was additionally c-kit+ (n = 3). To assess the functional capacity of these cells, the CD34+/c-kit+ and the respective c-kit– fraction obtained from seven leukapheresis products were sorted and plated into CFC and LTC-IC assays. As shown in Table 1, no colony growth was detected in three specimens. In two of those samples, the LTC-IC frequency was too low (<1 in 7,800 and <1 in 28,800 cells) to give rise to any colonies within the c-kit+ cell fraction, but LTC-ICs were detectable within the c-kit– fraction. In four experiments, the CFC content of the c-kit+ and the c-kit– cells could be compared and showed a 2.2-fold higher number of CFCs within the c-kit+ fraction (p = .014). The LTC-IC frequency was calculated from the pooled data of six experiments and was 2.8-times higher within the c-kit– fraction compared with the c-kit+ fraction.
Table 1. Functional characterization of mobilized tumor patient (MTP)–derived CD34+/c-kit+ and CD34+/c-kit– cells
Flow Cytometric Analysis of c-kit, VLA-4, and VLA-5 on CD34+ Cells
Flow cytometry was performed with freshly isolated or cultured cells. Before culture, 14 ± 7% and 36 ± 4% of the CD34+ cells from healthy donors and chemotherapy-treated tumor patients were c-kit+. After culture, c-kit expression was reduced for both (Fig. 1). More than 50% of the CD34+ cells were spontaneously VLA-4+, and the proportion increased approximately 1.5-fold to 90% VLA-4+ cells after 24-hour SCF stimulation. The spontaneous expression of VLA-5 on CD34+ cells was remarkably lower and increased by a factor of 27 for cells from healthy donors and by a factor of 4 for patient-derived cells (Table 2) after SCF stimulation. The effect of SCF could be blocked by the addition of the tyrosine kinase inhibitor genistein (Fig. 2).
Figure 1. Representative flow cytometric dot blot diagram showing the expression of c-kit, VLA-4, and VLA-5 on CD34+ cells from a healthy donor before and after stimulation with SCF for 24 hours. The percentage of the cells is indicated in the corner of the respective quadrants. Abbreviations: SCF, stem cell factor; VLA, very-late antigen.
Table 2. Stimulation with stem cell factor (SCF) increases the surface expression very-late antigen (VLA)-4 and VLA-5 on CD34+ cells
Figure 2. CD34+ cells were stimulated with SCF with and without the addition of genistein for 24 hours and then analyzed by flow cytometry. Shown is the proportion of CD34+/CD117+ cells expressing VLA-4 and VLA-5. Mean values ± standard error of the mean from three experiments are documented. Abbreviations: SCF, stem cell factor; VLA, very-late antigen.
Effect of SCF on the Adhesiveness of Primary CD34+ Cells
The effect of stimulating sorted CD34+ cells for 30 minutes with SCF at a concentration of 100 ng/ml was evaluated. The proportion of cells adhering to Petri dishes coated with 4 μg/cm2 CH296 increased from 14.0% without the addition of SCF to 23.0% with SCF stimulation. Although the distribution of CFC of adherent and nonadherent cells did not differ, the frequency of LTC-ICs was approximately three times higher in the adherent cell population (39 versus 15 LTC-ICs per 105 cells plated without SCF stimulation). Stimulation with SCF did not alter the frequency of CFCs and LTC-ICs within the respective cell fractions. As shown in Table 3, subsequent limiting dilution analysis of the LTC-IC frequency within the adherent cell fractions revealed a net increase of adherent LTC-ICs of approximately 30% by stimulation with SCF.
Table 3. Stem cell factor (SCF) stimulation enhances the number of adherent long-term culture-initiating cells (LTC-ICs)
SCF Stimulation Increases the Homing Ability after Transplantation in NOD/SCID Mice
By transplanting human HPCs into irradiated NOD/SCID mice, we sought to confirm the improvement of the in vivo homing abilities of SCF-stimulated HSCs compared with untreated control. In two independent experiments, a total of 16 animals were injected with lin– cells with or without the addition of SCF. In both experiments, bone marrow cells from all animals could be analyzed by flow cytometry and PCR. Eight of eight mice in the SCF treatment group showed multilineage hematopoietic engraftment of the human cells as assessed by the detection of at least five human lymphocytes and five human granulocytes per 2 x 104 bone marrow cells analyzed by flow cytometry and the detection of human DNA by PCR. In the animals of the control group, no human cells could be detected by flow cytometry or by PCR (Fig. 3). The mean proportion of human CD45 and CD71+ cells as determined by flow cytometry was 8.5 ± 7.2% (mean ± standard deviation ; range, 1.2–17.2) and 3.0 ± 2.3% (mean ± SD; range, 1.2–6.4) after the transplantation of 3.0 x 105 and 3.6 x 105 lin– human cells, respectively (Table 4).
Figure 3. Bone marrow cells isolated from nonobese diabetic-scid/scid mice transplanted 6 weeks previously with lin– hematopoietic progenitor cells were analyzed by PCR for human 5' actin. (A): PCR analysis of mouse bone marrow cells mixed with a varying proportion of human cells. (B), (C): PCR analysis of two independent experiments in which two groups of mice were transplanted with (+SCF) or without (–SCF) the addition of SCF. As a negative control, bone marrow cells of a mouse that did not receive human cells were analyzed (C, mouse 0). Abbreviations: PCR, polymerase chain reaction; SCF, stem cell factor.
Table 4. Effect of stem cell factor (SCF) on the hematopoietic engraftment of enriched human CD34+ cells transplanted in irradiated nonobese diabetic-scid/scid (NOD/SCID) mice
DISCUSSION
The excellent technical assistance of Nadine Pi?ler and Marit Hoffmann is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (HE 3199/4-1; to B.H.).
REFERENCES
Stiff PJ. Management strategies for the hard-to-mobilize patient. Bone Marrow Transplant 1999;23(suppl 2):S29–S33.
Teixido J, Hemler ME, Greenberger JS et al. Role of ?1 and ?2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma. J Clin Invest 1992;90:358–367.
Mohle R, Murea S, Kirsch M et al. Differential expression of L-selectin, VLA-4, and LFA-1 on CD34+ progenitor cells from bone marrow and peripheral blood during G-CSF-enhanced recovery. Exp Hematol 1995;23:1535–1542.
Kinashi T, Springer TA. Steel factor and c-kit regulate cell-matrix adhesion. Blood 1994;83:1033–1038.
Dercksen MW, Gerritsen WR, Rodenhuis S et al. Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation. Blood 1995;85:3313–3319.
Watt SM, Williamson J, Genevier H et al. The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes. Blood 1993;82:2649–2663.
Laszik Z, Jansen PJ, Cummings RD et al. P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells. Blood 1996;88:3010–3021.
Becker PS, Nilsson SK, Li Z et al. Adhesion receptor expression by hematopoietic cell lines and murine progenitors: modulation by cytokines and cell cycle status. Exp Hematol 1999;27:533–541.
Levesque JP, Leavesley DI, Niutta S et al. Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J Exp Med 1995;181:1805–1815.
Papayannopoulou T, Craddock C, Nakamoto B et al. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A 1995;92:9647–9651.
van der Loo JC, Xiao X, McMillin D et al. VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin. J Clin Invest 1998;102:1051–1061.
Wagers AJ, Allsopp RC, Weissman IL. Changes in integrin expression are associated with altered homing properties of Lin(–/lo) Thy1.1 (lo) Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor. Exp Hematol 2002;30:176–185.
Hennemann B, Oh IH, Chuo JY et al. Efficient retro-virus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin. Blood 2000;96:2432–2439.
Hogge DE, Lansdorp PM, Reid D et al. Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor. Blood 1996;88:3765–3773.
Peled A, Kollet O, Ponomaryov T et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000;95:3289–3296.
Williams DA, Rios M, Stephens C et al. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991;352:438–441.
Papayannopoulou T, Nakamoto B. Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin. Proc Natl Acad Sci U S A 1993;90:9374–9378.
Yamaguchi M, Ikebuchi K, Hirayama F et al. Different adhesive characteristics and VLA-4 expression of CD34+ progenitors in G0/G1 versus S+G2/M phases of the cell cycle. Blood 1998;92:842–848.
Prosper F, Stroncek D, McCarthy JB et al. Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function. J Clin Invest 1998; 101:2456–2467.
Broudy VC. Stem cell factor and hematopoiesis. Blood 1997;90:1345–1364.
Kawashima I, Zanjani ED, Almaida-Porada G et al. CD34+ human marrow cells that express low levels of kit protein are enriched for long-term marrow-engrafting cells. Blood 1996;87:4136–4142.
Sakabe H, Yahata N, Kimura T et al. Human cord blood-derived primitive progenitors are enriched in CD34+ c-kit– cells: correlation between long-term culture-initiating cells and telomerase expression. Leukemia 1998;12:728–734.
Sakabe H, Ohmizono Y, Tanimukai S et al. Functional differences between subpopulations of mobilized peripheral blood-derived CD34+ cells expressing different levels of HLA-DR, CD33, CD38 and c-kit antigens. STEM CELLS 1997;15:73–81.
Zeller W, Kroger N, Berger J et al. Expression of the adhesion molecules CD49d and CD49e on G-CSF-mobilized CD34+ cells of patients with solid tumors or non-Hodgkin’s and Hodgkin’s lymphoma and of healthy donors is inversely correlated with the amount of mobilized CD34+ cells. J Hematother Stem Cell Res 1999;8:539–546.
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