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发表于 2009-3-5 10:37 |只看该作者 |倒序浏览 |打印

a Department of Hematology and Oncology and
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b Institute of Pathology, University of Regensburg, Regensburg, Germany% `, p- Y( u$ X4 c: `, ]! F

Key Words. CD34 ? Stem cell factor ? Integrin ? Hematopoietic progenitor cells' L0 V4 t2 w' \8 T) O. k( n' a' b
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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, ~+ g8 ~3 ~# g4 [$ y5 ?0 k

ABSTRACT* U( P# q* x6 K9 ~% M4 X7 r

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 .% Y! }3 R/ V) }0 m# _& q$ n+ L

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.6 p1 g  S3 D0 b6 O( L

MATERIALS AND METHODS; p4 q2 D& Q0 o( A0 S

Isolation and Functional Characterization of CD34 /c-kit  and CD34 /c-kit– Cells# U# {4 x7 C0 }  N- b+ w0 g

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 (5 q  p+ A2 `$ t: g

Table 1. Functional characterization of mobilized tumor patient (MTP)–derived CD34 /c-kit  and CD34 /c-kit– cells
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Flow Cytometric Analysis of c-kit, VLA-4, and VLA-5 on CD34  Cells; [+ ?( O  N8 a+ A- @

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).2 B3 u9 ^2 O( Y& k9 M* p  u
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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/ a* T3 z+ C) _

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.- }) B: x) C2 `* {9 j! Q1 U7 L

Effect of SCF on the Adhesiveness of Primary CD34  Cells
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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.. A1 C/ c+ y, Y5 _" ]- t

Table 3. Stem cell factor (SCF) stimulation enhances the number of adherent long-term culture-initiating cells (LTC-ICs)  \. q( s* Z, G. p
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SCF Stimulation Increases the Homing Ability after Transplantation in NOD/SCID Mice( _+ s( E7 _! q+ ?% ]1 Q" g
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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).
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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." M8 k, v8 V6 p: l; \
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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/ w9 a8 M8 L5 k! i; o+ `

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.).
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