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Stem Cells 2005;23:561-574 www.StemCells.com
© 2005 AlphaMed Press

Cycling G1 CD34+/CD38+ Cells Potentiate the Motility and Engraftment of Quiescent G0 CD34+/CD38–/low Severe Combined Immunodeficiency Repopulating Cells

Tamara Byka, Joy Kahna, Orit Kolleta, Isabelle Petita, Sarit Samiraa, Shoham Shivtiela, Herzl Ben-Hurb, Amnon Peledc, Wanda Piacibellod, Tsvee Lapidota

a Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel;
b Department of Obstetrics and Gynecology, Assaf-Harofeh Medical Center, Zerifin, Israel;
c Gene Therapy Institute, Hadassah University Hospital, Jerusalem, Israel;
d Oncological Sciences Department, Division of Clinical Oncology, IRCC Cancer Institute, Candiolo, Italy

Correspondence: Tsvee Lapidot, Ph.D., The Weizmann Institute of Science, Department of Immunology, P.O. Box 26, Rehovot, 76100, Israel. Telephone: 972-8-9342481; Fax: 972-8-9344141; e-mail: Tsvee.Lapidot{at}weizmann.ac.il


   ABSTRACT
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2- to 3-day in vitro cytokine stimulation of human cord blood CD34+-enriched cells induces the production of short-term repopulating, cycling G1 CD34+/CD38+ cells with increased matrix metalloproteinase (MMP)-9 secretion as well as increased migration capacity to the chemokine stromal cell–derived factor-1 (SDF-1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice. These cycling G1 cells enhance SDF-1–mediated in vitro migration and in vivo homing of quiescent G0 CD34+ cells, which is partially abrogated after inhibition of MMP-2/-9 activity. Moreover, the engraftment potential of quiescent G0 SCID repopulating cells (SRCs) is also increased by the cycling G1 CD34+/CD38+ cells. This effect is significantly abrogated after incubation of cycling G1 cells with a neutralizing anti-CXCR4 antibody. Our data suggest synergistic interactions between accessory cycling G1 CD34+/CD38+ committed progenitor cells and quiescent, primitive G0 CD34+/CD38–/low SRC/stem cells, the former increasing the motility and engraftment potential of the latter, partly via secretion of MMP-9.


   INTRODUCTION
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Hematopoietic stem cells (HSCs) are a small subfraction of the bone marrow (BM) cells that are able to either self-renew or differentiate into all the cellular lineages of the blood throughout life [1]. Stem cells are defined functionally by their ability to migrate to the BM of the recipient after transplantation, to durably repopulate it, and to produce all the blood lineages [2–4]. To identify and quantify human stem cells, functional in vivo assays using immune-deficient mice as recipients were developed [5–7].

Based on such an assay, human severe combined immunodeficiency (SCID) repopulating cells (SRCs), which efficiently produce high levels of myeloid and lymphoid cells in the BM of transplanted nonobese diabetic (NOD)/SCID or B2mnull NOD/SCID mice, were characterized [8–10]. We have demonstrated that CD34+/CD38–/lowCXCR4+ SRCs are true stem cells capable of high-level multilineage engraftment in primary and secondary transplanted immune-deficient mice. Homing and engraftment of human progenitor cells are dependent on interactions between their membrane-bound receptor CXCR4 and its ligand, the chemokine stromal cell–derived factor-1 (SDF-1) produced by the murine BM endothelium, and different stromal cell types[11–13]. Moreover, we have recently reported that overexpression of this receptor on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation [14]. Other key regulators of these processes are adhesion molecules, cytokines, and proteolytic enzymes [15]. In particular, matrix metalloproteinase (MMP)-9 was shown to be secreted by HSCs, facilitating their in vitro migration [16, 17]. We have further demonstrated a role for these proteinases in stem cell motility and also in the upregulation of cell-surface CXCR4 expression [18]. Recent studies have revealed the involvement of MMP-9 in in vivo HSC mobilization by shedding membrane-bound stem cell factor (SCF) [19].

Long-term maintenance of human stem cells in ex vivo cultures is a major challenge for clinical and experimental transplantation protocols [20]. Present results are limited because of the induction of differentiation and the concomitant loss of repopulation and self-renewal capacities [21]. Human cells capable of repopulating NOD/SCID mice have been maintained or expanded in vitro for a limited time using different combinations of cytokines [6, 22–24]. Other protocols used coculture with stromal cells to maintain NOD/SCID-repopulating cells in vitro [25] and overexpression of HOXB4 homeoprotein in stroma used for coculture [26].

In the murine system, actively cycling progenitor cells can repopulate transplanted recipients; however, long-term repopulation by HSCs is mostly restricted to the quiescent population [27]. Most primitive human CD34+-enriched progenitors are quiescent, and cytokine stimulation leads to their entrance into cell cycle [28]. Human adult mobilized peripheral blood (MPB) G1 CD34+ cells home to the BM of conditioned NOD/SCID mice [29]; however, Gothot et al. [30] have shown that long-term repopulation was achieved predominantly by quiescent human adult BM or MPB CD34+ cells within the G0 phase of the cell cycle. Moreover, human G0 CD34+ cells recovered after short-term ex vivo cytokine stimulation have reduced engraftment potential compared with freshly isolated G0 cells [30]. Studies with total CD34+ cells demonstrate a different effect of short-term in vitro stimulation, in which human cytokines induced an increase in the engraftment potential of NOD/SCID-repopulating cells [6, 22, 23]. Because SRCs are characterized as CD34+/CD38–/low/CXCR4+ cells [11], we aimed to determine the contribution of cytokine-induced, cycling G1 CD34+/CD38+ cells on their repopulating potential. In the present study, using stringent conditions for defining G0/G1 populations in which all the primitive CD34+/CD38–/low SRCs are within the quiescent G0 cell fraction, we show that the apparent loss of engraftment potential by cytokine-treated G0 CD34+/CD38–/low cells is attributable to the absence of accessory, non–short-term repopulating, cycling G1 CD34+/CD38+ cells.


   METHODS
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Human Cord Blood CD34+ Cell Isolation
Human cord blood (CB) samples were obtained from full-term deliveries after informed consent and were used in accordance with the procedures approved by the human experimentation and ethics committees of the Weizmann Institute. The blood samples were diluted 1:1 in phosphate-buffered saline (PBS) without Mg2+/Ca2+. Low-density mononuclear cells (MNCs) were collected after standard separation on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) and washed in PBS. CD34+ cells were purified using the MACS cell isolation kit and the autoMACS magnetic cell sorter (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purity of the enriched CD34+ cells was greater than 85%.

Enrichment of G0 and G1 Cells by Cell Sorting
Human CB CD34+ cells, freshly isolated or after in vitro cytokine stimulation (see below), were labeled with Hoechst and Pyronin Y as previously described [30]. Briefly, cells were resuspended at 3 x 106 cells per ml in cell cycle labeling buffer: Hanks’ balanced salt solution containing 20 mM HEPES (Biological Industries, Beit Haemek, Israel), 1 g/l glucose, and 10% fetal calf serum (FCS) (GIBCO BRL, Karlsruhe, Germany). Hoechst 33342 (Molecular Probes, Eugene, OR) was added to a final concentration of 1 µg/ml, and after an incubation of 45 minutes at 37°C, Pyronin Y (Sigma, St. Louis) was added to a final concentration of 1 µg /ml followed by incubation for another 45 minutes. After washing and resuspension in cell cycle labeling buffer, cells were sorted on a FAC Star+ (Becton, Dickinson, SanJose, CA) equipped with two lasers providing the 488-nm excitation for Pyronin Y and the 350-nm excitation for Hoechst. Sorting windows were fixed as shown in Figure 1AGo.



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  		Figure 1. Effect of cytokines on cell cycle status of CD34+ cells. Cord blood CD34+ cells, freshly isolated (time = 0), after 40 hours of pre-incubation with SCF (40 hours SCF) or after 3 days of preincubation with SCF, Flt-3 ligand, thrombopoietin, and interleukin-6 (3 days 4F), were labeled with Hoechst and Pyronin Y. (A): G0 and G1 fractions were separated using sorting windows as shown in a representative experiment. Numbers indicate percent of total CD34+ cells. (B): Bars indicate percent cells in G1 in the sorted (G0 + G1) cell populations. Values represent mean ± standard error of eight experiments. (C): Sorted cell fractions were fixed, permeabilized, and labeled with an anti-Ki-67 antibody and 7-AAD. Isotype control labeling was used to define negative labeling for Ki-67. The percentages of cells in each quadrant (G0, lower left; G1, upper left; S/G2/M, upper right) are indicated. Abbreviations: 7-AAD, 7-aminoactinomycin-D; FITC, fluorescein isothiocyanate; SCF, stem cell factor.

 
Cell Cycle Analysis of Sorted Cells
After sorting, 5 x 104 cells were washed, resuspended in PBS containing 0.4% formaldehyde, and incubated for 30 minutes at 4°C. An equal volume of PBS containing 0.2% Triton X-100 was then added, and the cells were stored overnight at 4°C. The cells were washed twice in PBS containing 1% bovine serum albumin (BSA) (Sigma) and incubated with a fluorescein isothiocyanate (FITC)–labeled anti-Ki-67 monoclonal antibody (MoAb) (Immunotech [bought by Beckman Coulter, Fullerton, CA]) for 1 hour at 4°C. Cells were finally washed and resuspended in PBS containing 1% BSA and 5 µg /ml 7-aminoactinomycin-D (7-AAD) (Sigma). After 3 hours on ice, cells were analyzed on a FACSort (Becton, Dickinson). Analysis was performed using CELLquest software (Becton, Dickinson).

Ex Vivo Cultures
Human CD34+-enriched CB cells were cultured in 24-well plates (106/ml) containing 10% FCS and SCF for 40 hours (50 ng/ml, R&D Systems, Minneapolis) or for 3 days with the following cytokines: SCF, Flt-3 ligand (both at 50 ng/ml, R&D Systems), thrombopoietin (TPO) (10 U/ml, Amgen, Thousand Oaks, CA), and interleukin (IL)-6 (50 ng/ml, InterPharm Laboratories, the Ares-Serono Group, Ness Ziona, Israel). The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2.

Transwell Migration Assay
RPMI (600 µl) plus 10% FCS containing 125 ng/ml SDF-1{alpha} (R&D Systems) or the concentration indicated was put into the lower chamber of a Costar 24-well transwell (Corning, NY). Cells (1 to 2 x105) in 100-µl medium were put into the upper chamber (poresize, 5 µm), and cells were collected from both chambers after 4 hours of migration at 37°C and counted by flow cytometry (FACSsort, Becton, Dickinson). Control migrations were performed without SDF-1{alpha} in the lower chamber. To distinguish G0/freshly isolated cells from G1/40-hour SCF-treated cells in comigration experiments, G0/freshly isolated cells were labeled with anti-human CD34-FITC/CD38-phycoerythrin (PE) MoAbs and washed twice before the migration assay. Labeled G0/freshly isolated cells (1 x 105) were put alone or together with 1 x 105 unlabeled G1/40-hour SCF-treated cells into the upper chamber to test the migration of the G0 cell population toward different SDF-1{alpha} concentrations. When indicated, G1/40-hour SCF-treated cells or their conditioned medium were incubated for 30 minutes at 37°C with 100 µM of a specific MMP-2/-9 inhibitor (MMP-2/MMP-9 Inhibitor III, Cal-biochem-Novabiochem Corp., San Diego) before migration. The number of labeled cells in the migrating and nonmigrating fractions was counted by fluorescence-activated cell sorting (FACS).

CFU Assay
Semisolid cultures were performed as previously described [31]. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 and scored 14 days later for myeloid, erythroid, and mixed colonies by morphologic criteria.

Zymography
After overnight incubation in RPMI only or in RPMI with SCF (50 ng/ml, R&D Systems) for 40 hours, human CB CD34+-enriched cells (106/ml) were centrifuged. Supernatants were collected and centrifuged again at 6,000 rpm, 4°C, collected, and kept on ice. Supernatants (3 µl of each) were loaded on 10% SDS-PAGE containing 1 mg/ml gelatin (Sigma). Gels were rinsed for 30 minutes in 2.5% Triton X-100, washed with ddH2O, and incubated at 37°C for 16 hours with developing buffer (50 mM Tris, pH 8, 5 mM CaCl2, 200 mM NaCl, 0.02% Brij [Sigma]). Gels were stained with Coomassie blue (0.25%) for 3 hours and destained with 5% acetic acid, 10% MetOH.

SCID Repopulating Cell Assay
NOD/LtSz-Prkdcscid/Prkdcscid (NOD/SCID) mice were bred and maintained under defined flora conditions at the Weizmann Institute in sterile intraventilated cages (Techniplast, Bugugiatte, Italy). All experiments were approved by the animal care committee of the Weizmann Institute. Eight-week-old mice were sub-lethally irradiated (4 hours or, in one series of experiments, 24 hours before injection at 375cGy at 67cGy/min) and transplanted with human cells. Human G0 or G1 cells were injected into the tail vein in 0.5 ml of RPMI supplemented with 10% FCS. When indicated, cells were incubated with 10 µg /ml of a CXCR4-neutralizing MoAb (clone 12G5, R&D Systems) or with 100 µM of a specific MMP-2/-9 inhibitor before injection. Mice were killed 5 weeks after transplantation, and BM cells were flushed from six bones (femur, tibia, and pelvis).

Homing Assay
In assays in which freshly isolated cells were coinjected with 40-hour SCF-treated cells, the freshly isolated cells were labeled with the intracellular amine-binding dye carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) in 1 ml PBS to a final concentration of 5 µM and incubated for 15 minutes in the dark at room temperature. Cells were washed with PBS, resuspended in RPMI with 10% FCS, and incubated for an additional 30 minutes at 37°C. After washing, cells were resuspended in RPMI for transplantation (0.5 x 106 cells/mouse). In other homing assays, transplanted cells remained unlabeled. When indicated, cells were pretreated with 100 µM of a specific MMP-2/-9 inhibitor before injection. Mice were irradiated 24 hours before injection, and the presence of human cells in the BM and spleen was assayed after 16 hours as previously reported [12].

Flow Cytometry Analysis
The purity of enriched subpopulations after magnetic bead separation was analyzed by staining with anti-human CD34 FITC MoAb (Becton, Dickinson). After G0/G1 cell sorting, cells were two color–stained with a peridinin-chlorophyll-protein (PerCP)–labeled anti-human CD34 MoAb (Pharmingen, San Jose, CA) and an FITC-labeled anti-human CD38 MoAb (Immunotech) or PE-labeled anti-CD34 and FITC-labeled anti-CD38 antibodies in the homing assay. The levels of human cells in the marrow of engrafted mice were detected by staining with anti-human CD45 FITC MoAb (Immuno Quality Products [IQP], Groningen, Netherlands). Human pre-B cells were detected by double staining with anti-human CD45-FITC/CD19-PE MoAbs (Coulter, Miami) and myeloid progenitors with CD45-FITC/CD33-PE MoAb (IQP). Human Fc receptors were blocked using human plasma (1:100) and murine Fc receptors by mouse immunoglobulin G. In homing assays, BM cells were labeled with anti-human CD34-FITC/CD38-PE MoAb (Coulter), and 1 x 106 cells were acquired for FACS analysis. In experiments in which cells were labeled with CFSE before transplantation, analysis by FACS of the FL-1 channel for CFSE-stained cells was performed. Isotype control antibodies were used to exclude false-positive cells (Coulter). Dead cells were gated out by staining with propidium iodide (Sigma). Cells were washed with PBS supplemented with 1% FCS and 0.02% azide, suspended to a volume of 1 to 5 x 105 cells per ml, stained with directly labeled MoAb, and incubated for 25 minutes on ice. After staining, cells were washed once in the same buffer and analyzed.


   RESULTS
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Enriched CD34+ Cells Enter the Cell Cycle After Short Cytokine Exposure
To investigate the effect of cytokine stimulation on the cell cycle status of CB CD34+ cells, purified CD34+ cells were incubated for 40 hours with SCF or for 3 days with a combination of four cytokines, SCF, Flt-3 ligand, TPO, and IL-6. Cells were then labeled with Hoechst/Pyronin Y and sorted for G0 and G1 fractions using gating regions as delineated in Figure 1AGo.

Before cytokine stimulation, 64% of the cells were in G0 (89% of the sorted cells) and only 3% of the cells in G1 (11 ± 1.6% of the sorted cells; Figs. 1A, 1BGo; time = 0). These stringent conditions for separating the G1 cell population were used to prevent contamination of the G1 population with quiescent G0 cells. Upon stimulation with SCF, 30% of the cells were in G1 (53 ± 4.0% of the sorted cells) and only 25% (47% of sorted cells) in G0. Preincubation of the cells with a combination of four growth factors for 3 days (SCF, Flt-3 ligand, TPO, and IL-6, shown to support SRC expansion in long-term cultures [23]) increased the G1 fraction further to 71± 5.0% of the sorted cells (Fig. 1BGo, 3 days 4F). It is noteworthy that there was no expansion of the total CD34+ cell number after 40-hour SCF treatment but the CD34+ cell number increased by 50%–100% after four factor treatment for 3 days.

To test the purity of the sorted G0 and G1 fractions, FACS analysis was performed by plotting the DNA marker 7-AAD against expression of the nuclear envelope protein Ki-67, which is expressed by cycling but not quiescent G0 cells. As shown in Figure 1CGo, the purity of the sorted fractions was high: 98.7% for G0-untreated cells (time = 0), 97.2% for G0, and 83.5% for G1 after 40 hours of exposure to SCF. In the latter cell fraction, most of the contaminating cells were in G2/M (9%). The G1 fraction of the four factor–treated cells was 85.1% pure.

SCF-Treated CD34+ Cells Express CD38 and Migrate Toward SDF-1
Stimulation with cytokines was shown to induce differentiation of HSCs. Therefore, the different cell fractions were analyzed for expression of the differentiation marker CD38 (Fig. 2Go). We found that freshly isolated untreated CD34+ cells in G0 showed the highest percentage of primitive cells, with approximately 33% of CD34+/CD38–/low cells. SCF-treated cells in G0 retained 24% of primitive CD34+/CD38–/low cells, whereas in the G1 fraction, primitive cells were almost absent (3.5%). Moreover, in the four cytokine–treated cells, the percentages of primitive CD38–/low cells in the G0 and G1 cell fractions were dramatically reduced to 3.1% and 0.2%, respectively. These results suggest that the cells stimulated by SCF or the combination of cytokines can also enter the cell cycle and differentiate into maturing CD34+/CD38+ cells.



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  		Figure 2. CD38 expression levels on sorted G0 and G1 cells. After sorting, cells were washed and labeled with anti-CD38-FITC and anti-CD34-PerCP monoclonal antibodies. Isotype control labeling was used to define negative or low labeling for CD38 (not shown). Numbers indicate percent of CD34+/CD38–/low cells. Figure shows a representative experiment. Abbreviations: FITC, fluorescein isothiocyanate; SCF, stem cell factor.

 
As we have shown previously, SCF induces increased motility and surface expression of CXCR4 on CD34+ cells and consequently a higher migration rate toward a gradient of SDF-1 [11, 12, 32]. To test the effect of SCF stimulation on the migration potential, cells from different sorted fractions were assayed for migration toward SDF-1 in a transwell migration assay. As shown in Figure 3AGo, 27 ± 4.7% of the fresh cells in G0 (time = 0) migrated toward SDF-1, whereas only 7.5 ± 2.1% of the cells in G1 did so. Taking into consideration that there was 4.5 ± 1.0% of SDF-1–independent random migration (control), almost none of the freshly isolated CD34+ cells in G1 migrate toward SDF-1. After SCF treatment, the G0 fraction demonstrated a slight increase in migration (36 ± 5.3%, p = .07), whereas the cells in G1 exhibited the most enhanced migration (40 ± 11.2%, p < .001 compared with G1, time = 0).



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  		Figure 3. Migration of CD34+ cells in G0 and G1 toward SDF-1 and clonogenic progenitor content of migrating and nonmigrating cell fractions. (A): Sorted CD34+ cells in G0 and G1 were tested for migration toward SDF-1 in a transwell assay. In control (cont.), no SDF-1 was added to the lower chamber. Bars represent mean ± standard error of seven independent experiments. *p = .02 compared with G1 at time = 0. (B): After the transwell migration assay, cells were collected, washed, and assayed for clonogenic progenitors in semisolid cultures. Bars represent mean ± standard error of four independent experiments performed in duplicate. **p < .01, *p < .05 compared with corresponding cell fractions at time = 0. BFU-E, burst-forming unit–erythroid (black bars); CFU-GEMM, colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte (white bars); CFU-GM, colony forming unit-granulocyte, macrophage (gray bars). Abbreviations: M, migrating cells; NM, nonmigrating cells; SCF, stem cell factor; SDF-1, stromal cell–derived factor-1.

 
The G1 Fraction of SCF-Treated CD34+ Cells Is Enriched for Progenitor Cells
To additionally characterize G0 and G1 subpopulations, cells were collected from the lower and upper chambers, and the clonogenic progenitors in the migrating and nonmigrating cell fractions were quantified in semisolid cultures. In freshly isolated CD34+ cells, almost all of the progenitors were present in the G0 fraction (Fig. 3BGo; time = 0), including primitive progenitors that gave rise to mixed colonies (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte [CFU-GEMM]). Cells in G1 gave rise to only very low numbers of colonies, indicating that these cells are mostly differentiated cells. SCF-treated cells in G0 showed higher numbers of progenitors than their fresh counterparts, but the difference was statistically significant only for the migrating cells (98 versus 138, p < .05). SCF-treated cells in G1 gave rise to the most colonies of all of the cell populations tested. Interestingly, these cells also produced the highest number of primitive CFU-GEMM colonies (15 per 1,000 plated CD34+ cells in the migrating and nonmigrating population), indicating that they are still in a relatively undifferentiated state (Fig. 3BGo, 40-hour SCF). As we have previously shown, in the entire CD34+ cell population there were very similar numbers of progenitors within the migrating and nonmigrating fractions [11]. The significant difference in both progenitor content and migration toward SDF-1 of G1 cells at time = 0 compared with SCF-induced G1 cells clearly indicates that the two G1 fractions are fundamentally different from each other.

Only the G0 Cell Fractions of CD34+ Cells Efficiently Engraft NOD/SCID Mice
We next tested the repopulating potential of the G0 and G1 fractions of SCF-treated and untreated cells by transplanting NOD/SCID mice. Only cells in G0 efficiently engrafted the BM of NOD/SCID mice. Equal numbers of SCF-treated G0 cells engrafted NOD/SCID at levels lower than (Fig. 4AGo) or close to (Fig. 5AGo) untreated, freshly isolated G0 cells but never at significantly higher levels. We have shown previously that SCF-treated cells engrafted better than freshly isolated cells [11]. It was therefore surprising that neither the G0 nor the G1 fraction alone of the SCF-treated cells showed significantly enhanced engraftment potential. The engraftment was strictly CXCR4 dependent, because preincubation of the cells with neutralizing anti-CXCR4 antibodies before injection (without washing) completely blocked the engraftment (Fig. 4AGo, +{alpha}-CXCR4). As we have shown previously, cells migrating toward SDF-1 in the transwell assay better engraft NOD/SCID mice than nonmigrating cells [11]. Similar results were observed for the purified G0 cells (Fig. 4BGo). Migrating G0 cells engrafted two to five times better than nonmigrating cells. Although the SCF-treated G1 cells were the best migrating cell fraction (Fig. 3AGo), they did not durably engraft. This was consistent with the fact that the G1 cells were almost exclusively non-engrafting, differentiating CD34+/CD38+ cells (Fig. 2Go). Similarly to what was shown in Figure 4AGo, freshly isolated G0 cells also engrafted better than G0 cells after SCF treatment when respective migrating or nonmigrating fractions were compared (Fig. 4BGo). Taken together, it seems that the beneficial influence of the SCF treatment was lost when G0 and G1 cells were separated.



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  		Figure 4. Engraftment and homing of G0 and G1 cells in NOD/SCID mice. Irradiated NOD/SCID mice were injected with 6 to 8 x 104 G0 or G1 cells. Where indicated, cells were incubated with 10 µg /ml of a neutralizing anti-CXCR4 antibody and washed before injection (+{alpha}-CXCR4). Mice were killed 5 weeks after transplantation, and BM cells were labeled for human CD45. Bars represent mean ± standard error of the engraftment levels resulting from at least eight independent experiments. (B): Cells (6 to 8 x 104) from the upper (nonmigrating, NM) and the lower (migrating, M) transwell chambers were collected and injected to NOD/SCID mice, and engraftment was analyzed as described in (A). *p = .002 and **p < .01 compared with the corresponding fraction in G0 after 40 hours of SCF treatment. (C): G1 cells (5 x 105) after 40 hours of SCF treatment were injected into irradiated NOD/SCID mice, and the BM wastested after 16 hours for the presence of human cells with antibodies to CD34 and CD38. R1: CD34+/CD38+ cells. In noninjected control mice, no CD34+ cells could be detected. A representative experiment is shown. Abbreviations: BM, bone marrow; FITC, fluorescein isothiocyanate; NOD/SCID, nonobese diabetic severe/combined immunodeficiency; PE, phycoerythrin.

 


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  		Figure 5. Accessory effect of G1 cells on the engraftment of G0 cells in NOD/SCID mice. (A): 8 x 104 fresh (time = 0) or 40-hour SCF-treated G0 cells were injected 4 hours after irradiation to NOD/SCID mice alone or in combination with 1.2 x 105 SCF-treated (white circles) or four factor-treated (dark circles) G1 cells. Because both G1 fractions gave similar results, the data were pooled. Data represent results of eight independent experiments. Numbers and bars indicate mean values. *p < .01 compared with G0, time = 0 alone; **p < .001 compared with G0, 40-hour SCF alone; Student’s paired t-test. (B): Mice were injected with G0 cells alone or with G0 cells in combination with G1 cells as described in (A). BM cells were stained with human-specific anti-CD45 and anti-CD19 MoAbs (upper panel) or with anti-CD34 and anti-CD38 MoAbs (lower panel). Data show a representative experiment. (C): Experiment as in (A), but mice were irradiated 24 hours after injection. Data represent results of at least six independent experiments. Numbers and bars indicate mean values. (D): Indicated numbers of G0 cells (white circles, fresh cells; dark circles, SCF-treated cells) alone or in combination with G1 cells (right column) after SCF treatment (circles) or four factor treatment (triangles) were injected into NOD/SCID mice 4 hours after irradiation. Data represent results of five independent experiments. *p < .001 compared with 4 x 104 G0 cells alone. (E): Experiment as in (A), but where indicated, 40-hour SCF and four cytokine-treated G1 cells were incubated with 10 µg /ml of a neutralizing anti-CXCR4 antibody and washed before injection (+{alpha}-CXCR4). Percent engraftment compares engraftment of G0 cells when cotransplanted with G1 cells to engraftment of G0 cells transplanted alone. Data represent mean ± standard error of five independent experiments. *p < .01, **p = .01, ***p = .03 compared with control. (F): Freshly isolated cord blood CD34+ cells (0.5 x 106) were injected either alone or together with 0.5 x 106 40-hour SCF-treated cells into irradiated NOD/SCID mice. The freshly isolated cells were labeled with CFSE before injection to distinguish them from the SCF-treated cells. The BM was tested after 16 hours for the presence of CFSE-stained human cells by fluorescene-activated cell sorter analysis. Data represent results of two independent experiments performed in triplicate. *p = .04 compared with freshly isolated cells alone. Abbreviations: BM, bone marrow; CFSE, carrboxyfluorescein diacetate succinimidyl ester; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; MoAb, monoclonal antibody; NOD/SCID, nonobese diabetic severe/combined immunodeficiency; PE, phycoerythrin; SCF, stem cell factor.

 
To elucidate a possible accessory role for the cycling G1 cells in the repopulation of NOD/SCID mice, we first tested their homing capacity to the BM. Taking into consideration that this cell population best migrates toward SDF-1 in vitro out of all populations assayed, it was most probable that it can also home to the BM of irradiated mice, because preconditioning with irradiation increases the production of SDF-1 in the murine BM and spleen [13]. To test this, 5 x 105 cycling G1 cells, sorted from CD34+ cells after 40-hour treatment with SCF, were injected into NOD/SCID mice preconditioned with total body irradiation (TBI). The BM was tested 16 hours after injection for the presence of homing human cells. In the analysis of a representative experiment (Fig. 4CGo, injected), the presence of 160 CD34+/CD38+ cells per 106 BM cells analyzed can clearly be seen. In the control mice that were not injected, no CD34+ cells could be detected (Fig. 4CGo, noninjected). This shows that although cycling CB G1 cells do not durably engraft NOD/SCID mice, they do home to the BM.

Cycling G1 Cells Enhance the Migration Ability of G0 Cells Toward SDF-1
The finding that cytokine-induced CD34+/CD38+ G1 cells efficiently home to the BM suggests that these nonrepopulating, cycling CB G1 cells may support the homing or motility of the repopulating CD34+/CD38–/low G0 cell fraction. To test this hypothesis, we first examined how the presence of cycling G1 cells affects in vitro migration of G0 cells toward different concentrations of SDF-1. Quiescent G0 cells (1 x 105) were assayed alone or together with 1x105 cytokine-stimulated cycling G1 cells for their migration capacity toward 10, 50, and 125 ng/ml SDF-1. To be able to distinguish sorted G0 from G1 cells, the G0 cells were labeled with antibodies to CD34 and CD38 before migration, whereas the G1 cells stayed unlabeled. Table 1Go shows that freshly isolated, untreated G0 cells alone only poorly migrated toward the lower (10 and 50 ng/ml) SDF-1 concentrations. However, when SCF-stimulated or four cytokine–stimulated G1 cells were loaded together with the G0 cells, the migration capacity of the G0 cells toward 10 and 50 ng/ml increased significantly and reached efficiencies resembling those of G0 cells alone toward 125 ng/ml of SDF-1. These results suggest that the presence of G1 cells enhances the motility of noncycling CD34+/CD38–/low cells and their sensitivity toward low SDF-1 concentrations.


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  		Table 1. Accessory effect of G1 cells on G0 cell migration in vitro
 
Cycling G1 CD34+/CD38+ Cells Enhance the Engraftment Potential of Quiescent G0 CD34+/CD38–/low Cells
The observations that cycling G1 cells are able to home to the BM as well as enhance the migration of quiescent G0 cells toward a gradient of SDF-1 in vitro led us to test the accessory effect of G1 cells on the engraftment potential of the G0 subset. When mice were irradiated 4 hours before transplantation, the combination of freshly isolated or SCF-treated G0 cells with G1 cells after 40-hour SCF or four factor treatment enhanced the engraftment level greater than twofold compared with the injection of the same number of G0 cells alone (Fig. 5AGo). Like in the experiments shown in Figures 4A and 4BGo, injection of the G1 cell fraction alone, either after SCF or after four factor treatment, never led to efficient engraftment (data not shown). Mice cotransplanted with freshly isolated G0 and cytokine-stimulated G1 cells were not only quantitatively but also qualitatively better engrafted than mice injected with G0 cells alone. Addition of G1 cells enhanced the CD45+/CD19+ pre-B cell level in the recipient’s BM from 17%–45% for G0 cells after SCF treatment (Fig. 5BGo). Furthermore, highly engrafted mice that were injected with cytokine-treated G0 cells combined with cycling G1 cells showed a more than threefold increase in the primitive CD34+/CD38–/low cell content in the recipient BM than mice injected with cytokine-treated G0 cells alone (Fig. 5BGo).

The accessory effect of cycling G1 cells on G0 cells was lost when the mice were irradiated 24 hours before transplantation (Fig. 5CGo). There was no significant increase in the engraftment level of G0 cells when they were coinjected with G1 cells, but the engraftment level after injection with G0 cells alone was significantly higher than in mice irradiated 4 hours before injection (Fig. 5AGo). This situation is mimicked by the in vitro migration assay in Table 1Go: The migratory accessory effect of G1 cells is reduced when the migration is performed toward high concentrations of SDF-1. In vivo, SDF-1 levels in the BM are lower 4 hours after irradiation compared with 24 hours after TBI, because ionizing irradiation and other DNA-damaging agents increase the production of SDF-1 (including the BM and spleen) 24–48 hours after TBI [13].

To get a quantitative insight into the in vivo G1 accessory effect, 8 x 104 or 4 x 104 freshly isolated G0 cells were injected into NOD/SCID mice, leading to a mean of 25% and 8%, respectively, of human CD45+ cells engrafted in the mouse BM (Fig. 5DGo). The combination of 1.2 x 105 nonrepopulating, cytokine-stimulated G1 cells with 4 x 104 freshly isolated G0 cells led to a mean engraftment level comparable with that of 8 x 104 G0 cells alone (30.5% versus 25%).

The accessory effect of G1 cells on G0 cell engraftment was significantly abrogated by 95% for fresh G0 cells (p < .01), 87% for 40-hour SCF-treated G0 cells (p = .01), and 75% for four factor–treated G0 cells (p = .03) when 40-hour SCF–treated or four factor–treated G1 cells were preincubated with a neutralizing {alpha}-CXCR4 antibody (with washing) before injection (Fig. 5EGo). Interestingly, enriched CD34+ cells (freshly isolated or incubated overnight with SCF) showed no increase in engraftment when cotransplanted with a high cell dose of 20 million (CD34-depleted) CB MNCs (data not shown), revealing that not the cell dose, but the function, is the dominant factor in the accessory effect of the cycling cells.

We then examined whether cycling 40-hour SCF-treated CB CD34+ cells could enhance the in vivo homing of their freshly isolated counterparts. The fresh cells were distinguished from the SCF-treated cells by staining with the intracellular amine-binding dye, CFSE, before transplantation. We observed a significant 1.7-fold (p = .04) increase in homing (16 hours after transplantation) of CFSE-stained freshly isolated CB CD34+ cells to the murine spleen when they were cotransplanted with SCF-treated cells compared with transplantation of these cells alone (Fig. 5FGo). We did not detect any differences in their homing capacity to the BM (data not shown). These results suggest that the cycling SCF-treated cells may first facilitate increased short-term homing of the freshly isolated CB CD34+ cells to the spleen before their long-term repopulation (5 weeks after transplantation) of the BM, as previously suggested [12, 14, 33].

All together, these results implicate that the observed cytokine-induced in vitro expansion of NOD/SCID-repopulating cells was at least partially mediated by differentiation of cycling, nonengrafting G1 accessory cells with increased migration and homing potential.

MMP-9 Is Involved in the Accessory Effect of Cycling SCF-Treated CD34+ Cells on Quiescent Freshly Isolated CD34+ Cell Motility
Stimulation of BM HSCs with various cytokines results in upregulation of MMP-2/-9 expression, thereby facilitating their transmatrigel migration [16]. We therefore compared MMP-9 secretion of freshly isolated mainly quiescent CB CD34+ cells with 40-hour SCF-treated CD34+ cells. Zymographic analysis of conditioned media revealed very little secretion by the freshly isolated cells, whereas a greater than 3.5-fold increase in MMP-9 was found in the 40-hour SCF-treated cells (Fig. 6AGo; p = .04).



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  		Figure 6. The role of MMP-9 in the accessory effect of G1 cells on the motility of G0 cells. (A): Conditioned media from freshly isolated or 40 hours SCF-treated CB CD34+ cells were assayed by zymography on 10% acrylamide gel containing 1 mg/ml gelatin. Media conditioned by HT1080 cells, known to secrete MMP-9 and MMP-2, as well as purified MMP-9 were used as the standards. Gel is one representative experiment. Histogram represents densitometry analysis of five independent experiments. *p = .04 compared with freshly isolated cells. (B): 1 x 105 freshly isolated CB CD34+ cells were assayed in a transwell with 40-hour SCF-treated cells or their conditioned medium in the absence (control) or presence of MMP-2/-9 inhibitor. To be able to distinguish fresh cells from SCF-treated cells, the former were labeled with antibodies to CD34 and CD38 before migration, whereas the latter stayed unlabeled. Percent migration compares stromal cell–derived factor-1 (10 ng/ml)-mediated migration of fresh cells in the presence of SCF-treated cells or their conditioned medium with migration of fresh cells alone. Data represent mean ± standard error of three independent experiments. *p < .05 compared with control. (C): CB CD34+ cells were pretreated with an MMP-2/-9 inhibitor and injected into sublethally irradiated NOD/SCID mice (0.5 x 105 cells/mouse). Mice were killed 16 hours later and analyzed for the presence of human cells per 1.5 x 106 acquired cells with antibodies to human CD34 and CD38. *p = .02 compared with control. (D): CB CD34+ cells were pretreated with an MMP-2/-9 inhibitor and injected into sublethally irradiated NOD/SCID mice (1 to 2 x 105 cells/mouse). Mice were killed 5 weeks later, and murine BM was labeled for the human panleukocyte marker CD45 and the pre-B cell marker CD19 and assayed by FACS. A representative FACS analysis is shown (left panel). Histogram (right panel) represents results of mean ± standard error of three independent experiments performed in duplicate. *p = .03 compared with control. Abbreviations: BM, bone marrow; CB, cord blood; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; MMP, matrix metalloproteinase; NOD/SCID, nonobese diabetic severe/combined immunodeficiency; SCF; stem cell factor; PE, phycoerythrin; SDF, stromal cell–derived factor.

 
Secretion of MMP-9 by the SCF-treated cells suggests that this metalloproteinase may be involved in the accessory effect of these cells on their freshly isolated counterparts. Indeed, we found that inhibition of MMPs by a specific MMP-2/-9 inhibitor abrogated the accessory effect of SCF-treated cells or their conditioned media by 75% and 48%, respectively, on migration of freshly isolated CD34+ cells to a low SDF-1 concentration (10 ng/ml) (Fig. 6BGo p, < .05).

Because MMP-2 and -9 play a role in the in vitro SDF-1–mediated motility of human CD34+-enriched CB cells [18], we hypothesized that these MMPs also play a role in the in vivo motility of HSCs. We therefore examined the role of MMP-2/-9 in homing of freshly isolated human CB CD34+ cells to the BM and spleen and their repopulation of sublethally irradiated NOD/SCID mice. We observed that inhibition of MMP-2/-9 partially reduced homing of CB CD34+ by 45% (p = .03) to the murine spleen, whereas only a minor effect was noted in their homing to the BM (Fig. 6CGo). Furthermore, 5 weeks after transplantation, we observed a 62% (p = .03) reduction in engraftment of human cells within the BM (Fig. 6DGo).

These results implicate a role for MMP-9 in the auxiliary effect of cycling SCF-treated CD34+ cells on freshly isolated CD34+ cell motility to a low SDF-1 gradient in vitro as well as the importance of this metalloproteinase for in vivo cell motility and long-term BM repopulation.


   DISCUSSION
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
HSC self-renewal and expansion are measured by functional repopulating assays. Donor stem cell engraftment requires efficient homing from the blood circulation into the recipient BM [20, 34, 35]. In the murine NOD/SCID model for human SRCs, we showed that homing and engraftment of these cells depends on CXCR4/SDF-1 signaling [11, 12]. We show here that ex vivo cytokine-stimulated, cycling CB G1 CD34+/CD38+ cells efficiently enhance the migration of quiescent CD34+/CD38–/low cells toward SDF-1 in vitro and their engraftment in transplanted NOD/SCID mice.

Several laboratories have developed short- and long-term in vitro human expansion protocols for NOD/SCID-repopulating cells using different combinations of cytokines, which led to increased frequencies of engraftment by limiting dilution analysis [6, 22, 23]. The increased repopulation potential was explained by a net expansion of the primitive repopulating cells. Using a fluorescent staining method, it was also shown that almost all human long-term culture-initiating cells and NOD/SCID competitive repopulating cells can be induced to undergo multiple cell divisions within a few days of cytokine stimulation [36, 37].

In freshly isolated BM and MPB CD34+ cells, the repopulating cells were found predominantly in the quiescent G0 phase [30]. Upon short-term in vitro cytokine stimulation, only G0 cells retained repopulation potential. Moreover, freshly isolated G0 cells exhibited a higher repopulation potential than G0 cells isolated after cytokine stimulation [30]. Similarly, we show here that cytokine-stimulated human CB G0 CD34+ cells exhibit in most experiments reduced or similar repopulating ability compared with freshly isolated G0 CD34+ cells. The reduced, or in the best case equal, repopulating capacity of short-term cytokine-stimulated G0 is in contrast to the ability of these cytokines to stimulate expansion of repopulating cells. Indeed, we found that coinjection of cytokine-stimulated, nonrepopulating, cycling G1 cells 4 hours after TBI enhanced the engraftment potential of both freshly isolated and cytokine-treated G0 cells. We further characterized the cytokine-induced G1 accessory cells as maturing CD34+/CD38+ cells, whereas the G0 fractions were highly enriched for undifferentiated, primitive CD34+/CD38–/low cells with repopulation potential.

The G1 cells may exert their accessory effect by enhancing the homing of repopulating cells to the BM. Indeed, we found that 40-hour SCF-treated cells, which are mainly cycling, increased the homing of freshly isolated quiescent CD34+ cells to the murine spleen; however, this effect was not observed in the BM. It has been previously suggested that intravenously administered cells are initially distributed in other organs such as the spleen and may at a later stage home specifically to the BM [33]. Furthermore, we have previously shown that more CB CD34+ cells reach the spleen shortly after infusion [12] and that human CD34+ cells that over-express CXCR4 also show increased homing only to this organ and yet demonstrate increased repopulation of the BM [14]. This suggests that SCF-treated cells may facilitate preferential homing of the freshly isolated progenitor cells to the spleen before they reside long-term in the BM. We further propose that other mechanisms such as increased proliferation in the BM may also facilitate the increased repopulation. This is supported by other studies suggesting that CD34+/CD38+ cells induce the proliferation of the primitive human CD34+/CD38–/low cell population in the murine BM [38, 39]. In this case, accessory cells themselves must home to the BM. Indeed, we found that cytokine-stimulated G1 cells migrate efficiently in vitro as well as in vivo (home) to the BM, although they failed to durably repopulate it. This is in agreement with a report showing very short and transient (2-week) repopulation of preimmune sheep [40] and NOD/SCID mice [41, 42] with differentiating human CD34+/CD38+ cells. Furthermore, Srour et al. [29] have shown that human MPB G1 CD34+ cells could home to the BM of NOD/SCID mice and that cycling CD34+/CD38+ cells can home short-term to the BM but do not engraft long-term because of preferential apoptosis of cycling cells [43]. From this it is evident that short-term assays such as in vitro migration and homing do not necessarily reflect the long-term in vivo outcome (which requires adhesion to extracellular matrix components and retention in the BM, leading to proliferation and differentiation), especially when comparing between cultured cells with reduced engraftment potential and freshly isolated progenitors.

We have demonstrated a significant increase in expression levels of SDF-1 mRNA in the BM and spleen of mice 24–48 hours after TBI or treatment with other DNA-damaging agents. This correlated well with an increase in homing and engraftment of primitive human SRCs [13]. Moreover, Weiss et al. [44] demonstrated, by limiting dilution analysis in a syngeneic murine transplantation model, increased survival of mice transplanted with BM cells 24 or 48 hours after lethal TBI compared with mice transplanted immediately after lethal TBI [44]. We demonstrate here that the accessory effect of cycling G1 cells on G0 cell engraftment was observed when mice were injected 4 hours, but not 24 hours, after irradiation. Irradiation-dependent SDF-1 modulation could explain the loss of this accessory effect when mice were irradiated 24 hours before injection and strongly suggested our hypothesis that the cycling G1 accessory effect increases migration of SRC to lower concentrations of SDF-1. Indeed, we found that cytokine-stimulated G1 cells, which efficiently migrate in vitro toward SDF-1, enhanced the migration of freshly isolated G0 cells, especially toward low concentrations of SDF-1 (10 and 50 ng/ml). Our results strongly suggest that the cycling G1 CD34+/CD38+ cells increase the motility of repopulating G0 CD34+/CD38–/low cells to low SDF-1 concentrations in the BM. It has been shown that SDF-1 production is restricted to cycling human peripheral blood CD34+ cells, with no SDF-1 production by the G0 population [45]. Therefore, in vivo local SDF-1 production by the G1 subset may stimulate the G0 cells and increase their sensitivity toward low SDF-1 BM concentrations. Additionally, once the cells home to the BM, SDF-1 produced by G1 cells might participate in the retention of the stem cells in the BM [46] or act as a survival factor [45, 47–51]. However, we did not reveal any SDF-1 production by cycling 40-hour SCF-treated CB CD34+ cells (data not shown), suggesting that other mechanisms such as direct cell–cell interactions or the secretion of other chemokines and cytokines may also be involved. It was recently shown that platelet-derived microparticles can bind human CD34+ cells and murine stem cells, thereby increasing their in vitro response to SDF-1–mediated adhesion to endothelial cells and in vivo engraftment [52]. Interestingly, Spooncer et al. [53] have recently shown that lysophospholipids can significantly enhance the low SDF-1–mediated in vitro motility of the most primitive Lin–Sca+c-Kit+ murine hematopoietic population [53]. We and others have demonstrated a role for MMP-2 and MMP-9 in HSC motility [17, 18]. Furthermore, stimulation of BM HSCs with various cytokines, including SCF, upregulates the secretion of these metalloproteinases [16]. Indeed, we found that cycling 40-hour SCF-treated cells have increased MMP-9 secretion and that inhibition of this enzyme partially (but significantly) abrogated the auxiliary effect of the cycling cells on the in vitro migration of quiescent CD34+ cells to a low SDF-1 concentration. These results, together with our findings that MMP-9 is also involved in the in vivo homing and repopulation of NOD/SCID mice by CB CD34+-enriched cells, strongly suggest a mechanism by which the cycling cells, via secretion of MMP-9, enhance the migration, homing, and repopulation of the quiescent, primitive cells.

We cannot rule out the possibility that it may also be the G0 cells that have an auxiliary effect on the defective G1 cell repopulation. However, our studies are supported by previous findings showing that the more differentiated cells (as is the case with our G1 cell population) are the ones that exert their auxiliary effect on the more primitive stem cell population (such as the G0 cells). For example, it was reported that NOD/SCID mice transplanted with very low cell numbers of human Lin–/CD34+/CD38– cells required the cotransplantation of CD34+/CD38+ accessory cells [39]. Additional data have also shown an accessory effect of human CD34+/CD38+ differentiating cells on the engraftment of CD34+/CD38–/low cells[38,39]. Further more, the primitive human CD34–Lin– cell population poorly migrates in vitro to SDF-1 [12] and also possesses very low in vivo engraftment abilities [54]. However, coculture of these cells together with CD34+Lin– cells and their subsequent cotransplantation into NOD/SCID ectopic murine virus-knockout (EMVnull) mice enhances the repopulating capacity of the CD34–Lin– population [55]. Moreover, we demonstrate herein increased MMP-9 secretion by the G1 population. This result together with our findings of enhanced in vitro migration and in vivo homing of the quiescent CD34+ cells after cotreatment with their cycling counterparts, as well as significant inhibition of the accessory effect on engraftment after pretreatment of G1 cells with an anti-CXCR4 antibody, strongly supports our hypothesis that it is indeed the G1 cells exerting their accessory effect on G0 cell repopulation. We further suggest that it is the function via MMP-9 secretion, and not the cell dose, that is the dominant factor in the accessory effect of the cultured cycling cells, because cotransplantation of a high cell dose of 20 million CD34-depleted CB MNCs together with enriched CD34+ cells (both freshly isolated and incubated overnight with SCF) did not improve the engraftment of the latter. This also suggests that the accessory effect of the cycling cells may be specific; however, we cannot rule out the possibility that other cell types, which secrete facilitating enzymes such as MMP-9, or are induced to do so, can also improve human SRC migration and repopulation.

Interestingly, Wilpshaar et al. [56] have demonstrated a similar NOD/SCID repopulation capacity of human CB CD34+ G0 compared with G1 cell subsets [56]. In our study, however, a very small, highly purified cell subset with the highest G1 profile failed to durably repopulate the BM of sublethally irradiated recipients. Therefore, there seems to be a hierarchy of cells also within the G1 population ranging from those able to repopulate to those who lack this repopulation capacity. Another possible explanation for the engraftment of G1 cells by this group may be an accessory effect contributed by the cotransplantation of BM CD34– irradiated cells, known to improve homing and engraftment, together with the G1 population. In addition, traces of G0 cells in the G1 population due to possible differences in settings used to sort the fractions may also contribute to repopulation.

In this study we found that repopulating, human CB CD34+/CD38–/low cells are mostly at the G0 stage and their repopulating capacity can be increased by nonrepopulating cytokine-stimulated, cycling CD34+/CD38+ G1 cells. We propose here that at least part of the observed ex vivo cytokine-induced expansion of NOD/SCID-repopulating cells is actually attributable to maturation of CD34+/CD38–/low G0 cells into CD34+/CD38+ G1 cells. These cells efficiently home to the BM and support the homing of quiescent repopulating G0 cells and may also increase their proliferation and retention within the BM.

In conclusion, our study adds insight into the process of human stem cell development and migration and can potentially be used for the development of improved clinical stem cell transplantation protocols.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T.B. and J.K. contributed equally to this study. This work was supported in part by grants from the Israel Science Foundation and MINERVA Foundation.


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 Introduction
 Methods
 Results
 Discussion
 References
 

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