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First published online November 1, 2007
Stem Cells Vol. 26 No. 2 February 2008, pp. 543 -549
doi:10.1634/stemcells.2007-0476; www.StemCells.com
© 2008 AlphaMed Press

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THE STEM CELL NICHE

Ex Vivo Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Adversely Influences Their Distribution to Other Bone Marrow Compartments After Intra-Bone Marrow Transplantation

Kentaro Yamamuraa, Kohshi Ohishia, Masahiro Masuyab, Eri Miyataa, Yuka Sugimotoa, Shiho Nakamuraa, Atsushi Fujiedaa, Hiroto Arakia, Naoyuki Katayamaa

aDepartment of Hematology and Oncology, Mie University Graduate School of Medicine, Mie, Japan;
bBlood Transfusion Service, Mie University Hospital, Mie, Japan

Key Words. Cord blood • Intra-bone marrow • Transplantation • Migration • In vitro culture

Correspondence: Correspondence: Kohshi Ohishi, M.D., Ph.D., Department of Hematology and Oncology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. Telephone: 81-59-231-5016; Fax: 81-59-231-5216; e-mail: koishi{at}clin.medic.mie-u.ac.jp; or Naoyuki Katayama, M.D., Ph.D., Department of Hematology and Oncology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. Telephone: 81-59-231-5016; Fax: 81-59-231-5216; e-mail: n-kata{at}clin.medic.mie-u.ac.jp

Received on June 17, 2007; accepted for publication on October 22, 2007.

First published online in STEM CELLS EXPRESS  November 1, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Intra-bone marrow injection is a novel strategy for hematopoietic stem cell transplantation. Here, we investigated whether ex vivo culture of cord blood hematopoietic stem/progenitor cells influences their reconstitution in bone marrow after intra-bone marrow transplantation. Freshly isolated AC133+ cells or cells derived from AC133+ cells cultured with cytokines (stem cell factor, flt-3 ligand, and thrombopoietin) for 5 days were injected into the bone marrow of the left tibia in irradiated NOD/SCID mice. In the bone marrow of the injected left tibia, the engraftment levels of human CD45+ cells at 6 weeks after transplantation did not differ considerably between transplantation of noncultured and cytokine-cultured cells. However, the migration and distribution of transplanted cells to the bone marrow of other, noninjected bones were extremely reduced for cytokine-treated cells compared with noncultured cells. Similar findings were observed for engraftment of CD34+ cells. Administration of granulocyte colony-stimulating factor to mice after transplantation induced the migration of cytokine-cultured cells to the bone marrow of previously aspirated bone but not to other intact bones. These data suggest that ex vivo manipulation of hematopoietic progenitor/stem cells significantly affects their migration properties to other bone marrow compartments after intra-bone marrow transplantation. Our data raise a caution for future clinical applications of the intra-bone marrow transplantation method using ex vivo-manipulated hematopoietic stem cells.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Intra-bone marrow transplantation (IBMT) is a novel transplantation method that delivers cells directly into the bone marrow [1, 2]. During standard i.v. transplantation (IVT) of hematopoietic stem cells, significant numbers of the cells are trapped in various organs, such as the lung and liver, before they home to the bone marrow [3–5]. However, IBMT enables a variety of progenitor cells to be delivered into the bone marrow site, regardless of their homing ability. Because of this advantage, various clinical benefits have been proposed from studies in mice. First, IBMT of human cord blood hematopoietic stem cells in NOD/SCID mice was reported to enhance hematopoietic recovery and increase the engraftment rate compared with the IVT method [6, 7]. Second, cotransplantation of hematopoietic stem and stromal cells by IBMT was shown not only to enhance engraftment of hematopoietic cells in the bone marrow [8–10] but also to overcome human leukocyte antigen discordance between the donor and recipient to some extent, since the transferred stromal cells provided a niche for the hematopoietic stem cells [11]. In addition, the IBMT method has facilitated the identification of several new classes of human hematopoietic stem/progenitor cells, such as CD34+CD38lowCD36–lineage– cells with the potential to rapidly reconstitute bone marrow [7] and CD34–lineage– very primitive hematopoietic stem cells [12], neither of which could be identified by standard IVT, presumably because of their low homing capacities.

On the other hand, it has been revealed that following IBMT, cord blood hematopoietic stem cells not only reconstitute bone marrow in the injected bone but also migrate to and reconstitute bone marrow in other, noninjected bones [6–8, 10, 11, 13]. This property of hematopoietic stem/progenitor cells is considered to be important for efficient hematopoietic recovery after IBMT of cord blood in clinical settings. Here, we investigated whether ex vivo culture of cord blood hematopoietic stem/progenitor cells influences their reconstitution in bone marrow after IBMT. We observed that incubation of AC133+ cells with stem cell factor (SCF), flt3 ligand (Flt3L), and thrombopoietin (TPO) for 5 days did not considerably alter the engraftment levels in the bone marrow of the injected bone compared with noncultured AC133+ cells but did interfere with cell migration to the bone marrow of other, noninjected bones. Our data indicate that ex vivo culture of hematopoietic stem/progenitor cells significantly disturbs their migration properties to other bone marrow compartments after IBMT.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Isolation of AC133+ Cells
After obtaining informed consent, umbilical cord blood was obtained from full-term deliveries according to a protocol approved by the Ethics Committee of Mie University Hospital. AC133+ cells were isolated as described previously [14]. Briefly, mononuclear cells were separated by centrifugation on Ficoll-Hypaque, washed, and suspended in Ca2+-, Mg2+-free phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). AC133+ cells were positively isolated from the mononuclear cells using AC133 immunomagnetic beads (MACS; Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com), according to the manufacturer's instructions. The purity of the AC133+ cells routinely exceeded 90% after repeating the positive selection.

Recombinant Factors
Recombinant human TPO was a gift from Kirin Brewery Co. (Tokyo, http://www.kirin.co.jp/english). Recombinant human SCF and Flt3L were purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com). The cytokines were used at the following concentrations: SCF, 50 ng/ml; TPO, 20 ng/ml; Flt3L, 50 ng/ml. Human granulocyte colony-stimulating factor (G-CSF) was provided by Kirin Brewery.

Flow Cytometric Analysis
Immunofluorescence staining was performed as described previously [14]. The following murine monoclonal antibodies were used: anti-very late antigen 4 (VLA-4)-fluorescein isothiocyanate (FITC), anti-VLA-5-FITC (both from Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), anti-lymphocyte function associated antigen-1 (LFA-1)-FITC (DAKO, Golstrup, Denmark, http://www.dako.com) anti-CXCR4-phycoerythrin (PE) (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), anti-AC133-PE (Miltenyi Biotec), and anti-AC133-allophycocyanin (APC) (Miltenyi Biotec). Mouse IgG1-FITC, IgG2b-PE, IgG1-PE, IgG2a-PE (all from BD Pharmingen), and IgG1-APC (Miltenyi Biotec) served as isotype controls. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA, http://www.bdbiosciences.com). Dead cells were excluded by staining with propidium iodide (PI) (BD Pharmingen). The CellQuest software (Becton Dickinson Immunocytometry Systems) was used for data acquisition and analysis.

Culture
Suspension cultures were performed for 5 days in 24-well tissue culture plates (Nunc, Roskilde, Denmark, http://www.nuncbrand.com) containing serum-free medium (StemSpan; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 50 U/ml penicillin, 50 µg/ml streptomycin, SCF, Flt3L, and TPO as described previously [14]. On days 3–4, half the culture medium was replaced with fresh medium containing the same cytokines. Viable cell numbers were counted by the trypan blue dye exclusion method. Semisolid colony assays were performed as described previously [15].

Transplantation of Human Hematopoietic Cells into NOD/SCID Mice
Mice were bred and maintained at the Animal Research Facility of Mie University Graduate School of Medicine. The care of the mice was in accordance with institutional guidelines. NOD/SCID mice at 6–8 weeks of age were irradiated with 260 cGy at 4–6 hours prior to transplantation. Freshly isolated AC133+ cells and all cells derived from AC133+ cells cultured for 5 days with SCF, Flt3L, and TPO were injected via the tail vein or directly into the bone marrow cavity of the left tibia. Thereafter, the mice were given acidified water containing neomycin. At 6 weeks after the transplantation, the mice were sacrificed, and bone marrow cells were harvested from the left and right tibiae and the right femur. Bone marrow cells were sometimes aspirated from the left and right tibiae at earlier time points. Assessment of engraftment was performed as described previously [16]. Briefly, bone marrow cells were incubated with ammonium chloride-containing red blood cell lysis buffer; blocked with PBS containing 2% AB serum and an anti-mouse CD16/32 antibody (Fc{gamma}RII block, 2.4G2; BD Pharmingen); and incubated with anti-mouse CD45-FITC (BD Pharmingen), anti-human CD34-PE (Becton Dickinson, San Diego, http://www.bd.com), PI, and anti-human CD45-APC (BD Pharmingen). The cells were also stained with anti-mouse CD45-FITC, anti-human CD33-PE (Becton Dickinson), anti-human CD19-peridinin chlorophyll protein-conjugated anti-human CD19 (Becton Dickinson), and anti-human CD45-APC. The presence of human cells was assessed using a FACScan by analyzing cells stained with anti-human CD45, but not anti-mouse CD45, after excluding PI-stained dead cells.

Statistical Analysis
Student's t test was used to determine the statistical significance of differences in the obtained data.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Characteristics of Human Cell Engraftment After IBMT of Cord Blood AC133+ Cells
First, we examined the characteristics of human cell engraftment in the bone marrow after IBMT of freshly isolated cord blood hematopoietic stem/progenitor cells, compared with those after IVT. NOD/SCID-repopulating human hematopoietic cells were reported to be enriched in the CD34highAC133+ cell population [17]. Therefore, we isolated AC133 + cells from cord blood and injected these cells (5 x 104 cells per mouse) into irradiated NOD/SCID mice either directly into the bone marrow cavity of the left tibia or via the tail vein. At 6 weeks after transplantation, the engraftment levels of human CD45+ leukocytes in the bone marrow of the left tibia and other bones (right tibia and right femur) were assessed. As reported, IBMT resulted in a higher level of human CD45+ cell engraftment in the injected left tibia, compared with engraftment in the left tibia as well as the other bones after IVT (Fig. 1A). In addition, after IBMT, significant levels of human CD45+ cell engraftment were seen in the bone marrow of the other, noninjected bones (Fig. 1A).


Figure 1
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  		Figure 1. Characteristics of engraftment after IBMT of fresh AC133+ cells. (A) Human CD45+ cell engraftment in the Lt. tibia and other bones after IVT or IBMT of freshly isolated AC133+ cells. *, p < .05 versus engraftment by IVT. (B): Engraftment levels of CD45+CD34+ (Lt. panel) and CD45+CD34– (Rt. panel) cells in the injected Lt. tibia and noninjected Rt. tibia at 1, 2, and 3 weeks after IBMT of AC133+ cells. Data of two representative mice (mice 1 and 2) are shown. Abbreviations: IBMT, intra-bone marrow transplantation; IVT, i.v. transplantation; Lt., left; Rt., right.

 
To further examine the kinetics of engraftment in the bone marrow of injected and noninjected bones, AC133+ cells (5 x 104 cells per mouse) were similarly injected into the left tibia of NOD/SCID mice, and human CD45+ and CD34+ cell engraftment levels in the left and right tibiae were serially assessed by aspirating bone marrow at 1, 2, and 3 weeks after transplantation. At 1 week, small percentages of human CD45+ and CD34+ cells were detected not only in the injected left tibia but also in the noninjected right tibia. At this time, the majority of CD45+ cells engrafted in the noninjected tibia were positive for CD34, whereas the proportion of CD34–CD45+ relatively mature hematopoietic cells had already increased in the injected tibia (Fig. 1B). Thereafter, the percentage of CD45+CD34+ relatively immature hematopoietic cells, as well as CD45+ CD34– relatively mature cells, increased in both tibiae during the first 3 weeks, although the engraftment rate in the noninjected bone was relatively lower than that in the injected bone (Fig. 1B). These data suggest that hematopoietic stem/progenitor cells proliferate in the injected bone marrow and simultaneously migrate to and reconstitute other, noninjected bone marrow sites after IBMT.

Ex Vivo Culture of AC133+ Cells Disturbs Their Distribution to Other Bone Marrow Compartments After IBMT
In the next set of experiments, we attempted to investigate whether ex vivo culture of hematopoietic stem/progenitor cells influences their reconstitution in bone marrow after IVT and IBMT. To this end, AC133+ cells were cultured in serum-free medium containing SCF, Flt3L, and TPO for 5 days. In three independent experiments, 50%–70% of cultured cells remained positive for AC133, and the AC133+ cell population consisted of cells expressing relatively high levels of CD34 (Fig. 2A). The colony-forming efficiencies of AC133+ cells did not differ significantly before and after culture (39% ± 4% vs. 30% ± 7%). The number of AC133+ cells was increased by approximately 14-fold after the 5-day incubation. When freshly isolated AC133+ cells (5 x 104 cells per mouse) and all cells derived from AC133+ cells cultured in the same way were intravenously transplanted, the engraftment rates for cultured cells at 6 weeks after transplantation were lower than the rates for noncultured cells (Fig. 2B). Nevertheless, the engraftment of cultured cells was remarkably increased by IBMT compared with IVT (Fig. 2C). These data indicate that the engraftment of cultured as well as noncultured hematopoietic stem/progenitor cells after IVT is considerably influenced by their homing ability to bone marrow and that the IBMT method significantly increases the engraftment rates.


Figure 2
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  		Figure 2. Changes of phenotype and engraftment ability by ex vivo culture. (A): Expression levels of AC133 and CD34 before and after culture of AC133+ cells with stem cell factor, flt3 ligand, and thrombopoietin for 5 days. (B): Engraftment of human CD45+ cells at 6 weeks after IVT of freshly isolated AC133+ cells or AC133+ cell-derived cultured cells. (C): Engraftment of CD45+ cells after IVT or IBMT of AC133+ cell-derived cultured cells. Abbreviations: IBMT, intra-bone marrow transplantation; IVT, i.v. transplantation.

 
Next, we compared the engraftment rates of noncultured and cultured hematopoietic stem/progenitor cells after IBMT. Fresh AC133+ cells (5 x 104 cells per mouse) and all AC133+ cell-derived cytokine-cultured cells were injected to the bone marrow of the left tibia of irradiated NOD/SCID mice, and the engraftment levels of human CD45+ and CD34+ cells in the left tibia and other bones (right tibia and right femur) were analyzed at 6 weeks after transplantation. Assessment of the injected left tibia revealed that the mean percentages of CD45+ cells did not differ remarkably between transplantation of noncultured and cytokine-cultured cells, although they differed to some extent depending on the experiments. The engraftment levels of CD45+ cells in other, noninjected bones were almost half the levels in the injected left tibia after transplantation of noncultured cells. However, after IBMT of cytokine-cultured cells, the engraftment rate of CD45+ cells in other, noninjected bones was extremely lower than the rate in the injected left tibia (Table 1). Similar findings were observed for CD34+ cell engraftment. When we examined the engraftment of human CD45+ cells in each mouse following IBMT of cytokine-treated cells, the engraftment in other, noninjected bones was consistently low, even in mice exhibiting a high percentage of human CD45+ cells in the injected left tibia (Fig. 3A). We also analyzed the percentages of CD34+ immature, CD33+ myeloid, and CD19+ B cells in CD45+ leukocytes engrafted in bone marrow of the injected left tibia and other, noninjected bones. Significant percentages of the CD45+ cells were composed of CD33+ and CD19+ cells in addition to CD34+ cells in the injected and noninjected bone marrow after IBMT of noncultured cells and in the injected bone marrow after IBMT of cytokine-cultured cells (Fig. 3B–3D). The number of engrafted cells was too low to analyze in the noninjected bone marrow following IBMT of cytokine-cultured cells. These data suggest that although the migrated cells after IBMT of noncultured AC133+ cells and transplanted AC133+ cell-derived cultured cells included hematopoietic stem/progenitor cells that possess in vivo differentiation potential to myeloid and/or lymphoid cells, similar to the injected noncultured AC133+ cells, in vitro culture disturbed the migration of these cells to other bone marrow compartments. We further tested whether AC133+ cell-derived cultured cells could migrate to other organs, such as the thymus, spleen and peripheral blood after IBMT. When 53.9% ± 15.8% of human CD45+ cells were detected in the injected left tibia at 6 weeks after IBMT of 5 x 104 AC133+ cell-derived cultured cells (n = 9), engraftment rates in the other, noninjected bones, spleen, and peripheral blood were 1.8% ± 1.4%, 0.4% ± 0.3%, and 0.1% ± 0.2%, respectively. No engraftment was seen in thymus. Thus, although human CD45+ cells were detected in the spleen and peripheral blood, the engraftment levels were even lower than the levels of noninjected bones.


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  		Table 1. Ex vivo culture decreased the engraftment in other, noninjected bones after IBMT. Engraftment levels of human CD45+ and CD34+ cells in the bone marrow of the injected left tibia and other, noninjected bones after intra-bone marrow transplantation of noncultured AC133+ (5 x 104 cells per mouse) or AC133+ cell-derived cultured cells into NOD/SCID mice (n = 5 or 6)

 


Figure 3
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  		Figure 3. Engraftment in each mouse after IBMT of noncultured and cultured AC133+ cells. Engraftment levels of human CD45+ cells (A) and percentages of CD34+ (B), CD33+ (C), and CD19+ (D) cells among the engrafted CD45+ cells in the injected Lt. tibia and other, noninjected bones of each mouse after intra-bone marrow transplantation of noncultured AC133+ cells or AC133+ cell-derived cultured cells. Abbreviation: Lt., left.

 
Major integrins, such as VLA-4, VLA-5, and LFA-1, and a chemokine, stromal cell-derived factor-1 (SDF-1), have been shown to be coordinately involved in the engraftment and migration of human cord blood hematopoietic stem/progenitor cells in NOD/SCID mice [18–21]. We examined whether our in vitro culture condition alters the expression patterns of these integrins and the receptor for SDF-1, CXCR-4, on AC133+ cells isolated before and after culture, as reported by others [22]. In three independent experiments, the expression levels of LFA-1 on AC133+ cells isolated after culture were significantly higher than those on freshly isolated AC133+ cells, whereas the expressions of VLA-5 and VLA-4 on AC133+ cells remained unchanged (Fig. 4). In addition, the expression levels of CXCR-4 were significantly lower on AC133+ cells isolated after culture than on noncultured AC133+ cells.


Figure 4
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  		Figure 4. Expression levels of VLA-4, VLA-5, LFA-1, and CXCR-4 on AC133+ cells before and after culture with stem cell factor, flt3 ligand, and thrombopoietin. The results represent the mean ± SD of the median fluorescence intensity of three independent experiments. *, p < .05 versus noncultured AC133+ cells.

 
G-CSF is known to enhance the mobilization of hematopoietic progenitors from bone marrow [20, 21]. Therefore, we tested whether administration of G-CSF to mice after transplantation could stimulate the migration of transplanted cytokine-cultured cells to bone marrow in other, noninjected bones. We observed that cord blood AC133+ cells were engrafted in the bone marrow of NOD/SCID mice at 7 days after irradiation (data not shown). Based on this observation, we injected cells derived from AC133+ cells cultured with growth factors into the bone marrow of the left tibia, assessed the engraftment in the injected and noninjected tibiae at 7 days after transplantation, and then subcutaneously administered human G-CSF (250 µg/kg/day) for 5 days as described previously [23, 24]. Consistent with our previous observations, at 7 days after IBMT of cytokine-cultured cells, human CD45+ cells were detected in the injected left tibia, whereas few or no CD45+ cells were seen in the noninjected right tibia (Fig. 5). At 3 weeks after G-CSF treatment, significant percentages of human CD45+ cells were detected not only in the injected left tibia but also in the noninjected right tibia. Since the right tibia had been injured for aspiration of bone marrow before G-CSF administration, we further analyzed the CD45+ cell engraftment in the bone marrow of the intact right femur but detected few or no CD45+ cells in the intact bone marrow site even after G-CSF administration (Fig. 5). Little or no migration was also seen in the intact bone marrow site of the left femur (data not shown). Without G-CSF treatment, few or no CD45+ cells migrated to the right tibia, which had been similarly aspirated at 7 days after IBMT of cytokine-cultured cells into the left tibia (data not shown). These data indicate that migration of ex vivo-cultured hematopoietic stem/progenitor cells to the bone marrow in other, noninjected intact bones is not solely induced by G-CSF treatment but can also be induced by additional stimuli.


Figure 5
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  		Figure 5. Effect of G-CSF on the migration of transplanted AC133+ cell-derived cytokine-cultured cells. Following intra-bone marrow transplantation into the Lt. tibia, the engraftment levels of CD45+ cells in the Lt. and Rt. tibiae at 1 week after transplantation and those in Lt. and Rt. tibiae and Rt. femur at 3 weeks after G-CSF treatment were assessed. Similar data were observed in two other experiments. Abbreviations: G-CSF, granulocyte colony-stimulating factor; Lt., left; Rt., right.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
It has been reported that hematopoietic stem/progenitor cells directly transplanted into the bone marrow repopulate not only the injected bone marrow but also other, noninjected bone marrow sites. In the current study, we found that this distribution property of hematopoietic stem/progenitor cells to other bone marrow compartments after IBMT is impaired by preincubation of the hematopoietic stem/progenitor cells with growth factors. Our data raise a caution that ex vivo manipulation of hematopoietic stem/progenitor cells has a significant influence on their migration property, which would be important for efficient bone marrow reconstitution after IBMT.

Consistent with previous reports [8, 13], CD45+ cells are detected in the bone marrow of other, nonimplanted bones as early as 7 days after IBMT. At this time, the majority of CD45+ cells engrafted at noninjected bone marrow sites were CD34+ cells. Thereafter, the percentages of not only CD45+CD34– relatively mature cells but also CD45+CD34+ relatively immature cells increased over time for 3 weeks in noninjected bones as well as in the injected bone. These data support the notion that primitive hematopoietic progenitors proliferate in the injected bone marrow and simultaneously migrate to and reconstitute other bone marrow compartments after IBMT [7]. It has not been well studied how this migration takes place after IBMT and whether such migration also occurs after IVT. Nevertheless, since irradiated bone marrow is induced to produce higher levels of chemokines and cytokines, which would enhance the migration and homing of hematopoietic progenitors [21, 25], it is plausible to speculate that similar migration may occur even after IVT and that use of the IBMT method merely reveals the dynamic reconstitution process by primitive hematopoietic progenitors during the early recovery period after transplantation.

On the other hand, the migration to other, noninjected bone marrow sites was found to be extremely low when AC133+ cells were preincubated with growth factors before transplantation. It has been reported that cytokine activation of hematopoietic progenitors leads to a reduction in homing to the bone marrow after their IVT [26–29]. In fact, we observed that the engraftment rates after IVT of cytokine-cultured AC133+ cells were lower than those after IVT of noncultured AC133+ cells, whereas the engraftment rates in the injected site of the bone marrow after IBMT did not differ significantly between cultured and noncultured AC133+ cells in two of three experiments. Accordingly, our data demonstrate that in vitro culture reduces the migration as well as the homing ability of hematopoietic stem/progenitor cells. Migration and homing of hematopoietic progenitors have been considered to be a coordinated and multistep process involving adhesion to vascular endothelial cells, transendothelial migration, and anchoring to the bone marrow niche [18–21, 30–34]. We observed that in vitro culture increased the expression levels of a β2 integrin, LFA-1, on AC133+ cells but decreased their expression of CXCR-4, the receptor for the chemokine SDF-1. Since SDF-1 plays a central role in the homing and migration of hematopoietic stem cells and SDF-1-mediated activation of LFA-1 has been shown to be important for transendothelial migration [19], our data imply that reduced expression of CXCR-4 may be dominantly involved in the impaired migration of hematopoietic stem/progenitor cells to other bone marrow compartments.

Since administration of G-CSF enhances the migration of hematopoietic progenitors [20, 21, 23, 24], we attempted to stimulate the migration and distribution of transplanted cytokine-cultured hematopoietic cells, using G-CSF. Treatment of mice with G-CSF was not able to induce cell migration to intact bone marrow sites but did induce their distribution to bone that had been injured by aspiration. Collapse of the bone marrow cavity is supposed to increase the production of various kinds of chemotactic factors and cytokines [8]. Our data indicate that although the migration capacity of hematopoietic stem/progenitor cells is significantly deteriorated after ex vivo culture, their migration can be induced by the combined effects of G-CSF and additional stimuli.

Ex vivo culture is beneficial and required for the manipulation of hematopoietic stem cells for a broad range of clinical settings. For example, gene transfer into human hematopoietic stem cells is a promising tool for the correction of a wide variety of hematopoietic and genetic disorders [35]. Since ex vivo-transduced hematopoietic stem cells are reported to exhibit reduced homing ability [36], IBMT is an ideal transplantation method for transferring engineered hematopoietic stem cells to bone marrow compartments. Nonetheless, our data reveal that consideration and manipulation of the migration property are important for IBMT of ex vivo-treated hematopoietic stem cells in future clinical settings.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
IBMT is a recently established novel transplantation method that transfers various types of cells directly into the bone marrow, regardless of their homing ability. Nonetheless, our data reveal inhibitory effects of ex vivo culture of hematopoietic stem/progenitor cells on their migration property to other bone marrow compartments after IBMT. These findings will be important for future clinical applications of this transplantation strategy.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank the staff of Department of Radiology, Mie University Hospital. K.Y. and K.O. contributed equally to this study.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

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