Cycling G1 CD34+/CD38+ Cells Potentiate the Motility and Engraftment of Quiescent G0 CD34+/CD38–/low Severe Combined Immunodeficiency Repopu
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《干细胞学杂志》
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@weizmann.ac.il
ABSTRACT
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 . 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 . To identify and quantify human stem cells, functional in vivo assays using immune-deficient mice as recipients were developed .
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 . 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. Moreover, we have recently reported that overexpression of this receptor on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation . Other key regulators of these processes are adhesion molecules, cytokines, and proteolytic enzymes . In particular, matrix metalloproteinase (MMP)-9 was shown to be secreted by HSCs, facilitating their in vitro migration . We have further demonstrated a role for these proteinases in stem cell motility and also in the upregulation of cell-surface CXCR4 expression . Recent studies have revealed the involvement of MMP-9 in in vivo HSC mobilization by shedding membrane-bound stem cell factor (SCF) .
Long-term maintenance of human stem cells in ex vivo cultures is a major challenge for clinical and experimental transplantation protocols . Present results are limited because of the induction of differentiation and the concomitant loss of repopulation and self-renewal capacities . Human cells capable of repopulating NOD/SCID mice have been maintained or expanded in vitro for a limited time using different combinations of cytokines . Other protocols used coculture with stromal cells to maintain NOD/SCID-repopulating cells in vitro and overexpression of HOXB4 homeoprotein in stroma used for coculture .
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 . Most primitive human CD34+-enriched progenitors are quiescent, and cytokine stimulation leads to their entrance into cell cycle . Human adult mobilized peripheral blood (MPB) G1 CD34+ cells home to the BM of conditioned NOD/SCID mice ; however, Gothot et al. 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 . 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 . Because SRCs are characterized as CD34+/CD38–/low/CXCR4+ cells , 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
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 1A.
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, 1B; 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 ) increased the G1 fraction further to 71± 5.0% of the sorted cells (Fig. 1B, 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 1C, 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. 2). 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.
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 . 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 3A, 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).
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. 3B; time = 0), including primitive progenitors that gave rise to mixed colonies (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte ). 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. 3B, 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 . 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. 4A) or close to (Fig. 5A) 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 . 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. 4A, +-CXCR4). As we have shown previously, cells migrating toward SDF-1 in the transwell assay better engraft NOD/SCID mice than nonmigrating cells . Similar results were observed for the purified G0 cells (Fig. 4B). 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. 3A), 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. 2). Similarly to what was shown in Figure 4A, freshly isolated G0 cells also engrafted better than G0 cells after SCF treatment when respective migrating or nonmigrating fractions were compared (Fig. 4B). Taken together, it seems that the beneficial influence of the SCF treatment was lost when G0 and G1 cells were separated.
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 (+-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.
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 (+-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 . 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. 4C, 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. 4C, 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 1 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.
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. 5A). Like in the experiments shown in Figures 4A and 4B, 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. 5B). 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. 5B).
The accessory effect of cycling G1 cells on G0 cells was lost when the mice were irradiated 24 hours before transplantation (Fig. 5C). 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. 5A). This situation is mimicked by the in vitro migration assay in Table 1: 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 .
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. 5D). 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 -CXCR4 antibody (with washing) before injection (Fig. 5E). 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. 5F). 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 .
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 . 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. 6A; p = .04).
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. 6B p, < .05).
Because MMP-2 and -9 play a role in the in vitro SDF-1–mediated motility of human CD34+-enriched CB cells , 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. 6C). Furthermore, 5 weeks after transplantation, we observed a 62% (p = .03) reduction in engraftment of human cells within the BM (Fig. 6D).
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
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.
REFERENCES
Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100:157–168.
Morrison S, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol 1995;11:35–71.
To LB, Haylock DN, Simmons PJ et al. The biology and clinical uses of blood stem cells. Blood 1997;89:2233–2258.
Moore MA. "Turning brain into blood": clinical applications of stem-cell research in neurobiology and hematology. N Engl J Med 1999;341:605–607.
Lapidot T, Pelumio F, Doedens M et al. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 1992;255:1137–1141.
Conneally E, Cashman J, Petzer A et al. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lymphomyeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 1997;94:9836–9841.
Dao MA, Shah AJ, Crooks GM et al. Engraftment and retroviral marking of CD34+ and CD34+CD38– human hematopoietic progenitors assessed in immune-deficient mice. Blood 1998;91:124–1255.
Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mice using retroviral gene marking and cell purification: implications for gene therapy. Nat Med 1996;2:1329–1337.
Wang JC, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997;89:3919–3924.
Kollet O, Peled A, Byk T et al. ?2 microglobulin deficient (B2mnull) NOD/SCID mice are excellent recipients for studying human stem cell function. Blood 2000;95:3102–3105.
Peled A, Petit I, Kollet O etal. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.
Kollet O, Spiegel A, Peled A et al. Rapid and efficient homing of human CD34+CD38–/low CXCR4+ stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice. Blood 2001;97:3283–3291.
Ponomaryov T, Peled A, Petit I et al. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest 2000;106:1331–1339.
Kahn J, Byk T, Jansson-Sjostrand L et al. Overexpression of CXCR4 on CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation. Blood 2004;103:2942–2949.
Lapidot T, Kollet O. The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2mnull mice. Leukemia 2002;16:1992–2003.
Janowska-Wieczorek A, Marquez L, Nabholtz J-M. Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34+ cells and their transmigration through reconstituted basement membrane. Blood 1999;93:3379–3390.
Janowska-Wieczorek A, Marquez L, Dobrowsky A. Differential MMP and TIMP production by human marrow and peripheral blood CD34+ cells in response to chemokines. Exp Hematol 2000;28:1274–1285.
Kollet O, Shivtiel S, Chen YQ et al. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 2003;112:160–169.
Heissig B, Hattori K, Dias S et al. Recruitment of stem and progenitor cells from the bone marrow niche requires mmp-9 mediated release of kit-ligand. Cell 2002;109:625–637.
Moore MA. Umbilical cord blood: an expandable resource. J Clin Invest 2000;105:855–856.
Emerson SG. Ex vivo expansion of hematopoietic precursors, progenitors and stem cells: the next generation of cellular therapeutics. Blood 1996;87:3082–3088.
Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997;186:619–624.
Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34+ cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93:3736–3749.
Kollet O, Aviram R, Chebath J et al. The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34+CD38–/low cells capable of repopulating severe combined immunodeficiency mice. Blood 1999;94:923–931.
Gan OI, Murdoch B, Larochelle A et al. Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. Blood 1997;90:641–650.
Amsellem S, Pflumio F, Bardinet D et al. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nat Med 2003;9:1423–1427.
Orschell-Traycoff CM, Hiatt K, Dagher RN et al. Homing and engraftment potential of Sca-1+lin– cells fractioned on the basis of adhesion molecule expression and position in cell cycle. Blood 2000;96:1380–1387.
Ladd AC, Pyatt R, Gothot A et al. Orderly process of sequential cytokine stimulation is required for activation and maximal proliferation of primitive human bone marrow CD34+ hematopoietic progenitor cells residing in G0. Blood 1997;90:658–668.
Srour EF, Jetmore A, Wolber FM et al. Homing, cell cycle kinetics and fate of transplanted hematopoietic stem cells. Leukemia 2001;15:1681–1684.
Gothot A, van der Loo JC, Clapp DW et al. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641–2649.
Lapidot T, Pflumio F, Doedens M et al. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 1992;255:1137–1141.
Kollet O, Petit I, Kahn J et al. Human CD34+CXCR4- sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation. Blood 2002;100:2778–2886.
Papayannopoulou T. Bone marrow homing: the players, the playfield, and their evolving roles. Curr Opin Hematol 2003;10:214–219.
Muench MO, Firpo MT, Moore MAS. Bone marrow transplantation with interleukin-1 plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without the loss of stem cell lineage and proliferative potential. Blood 1993;81:3463–3473.
Hendrikx PJ, Martens ACM, Hagenbeek A et al. Homing of fluorescently labeled murine hematopoietic stem cells. Exp Hematol 1996;24:129–140.
Glimm H, Eaves CJ. Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture. Blood 1999;94:2161–2168.
Oostendorp RA, Audet J, Eaves CJ. High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo. Blood 2000;95:855–862.
Bierhuizen MF, Westerman Y, Visser TP et al. Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells. Blood 1997;90:3304–3315.
Bonnet D, Bhatia M, Wang JC et al. Cytokine treatment or accessory cells are required to initiate engraftment of purified primitive human hemato-poietic cells transplanted at limiting doses into NOD/SCID mice. Bone Marrow Transplant 1999;23:203–209.
Civin CI, Almeida-Porada G, Lee MJ et al. Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo. Blood 1996;88:4102–4109.
Kerre TC, De Smet G, De Smedt M et al. Both CD34+38+ and CD34+38– cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion. J Immunol 2001;167:3692–3698.
Hogan CJ, Shpall EJ, Keller G. Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD-SCID mice. Proc Natl Acad Sci U S A 2002;99:413–418.
Jetmore A, Plett P, Tong X et al. Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients. Blood 2002;99:158–1593.
Weiss L, Bullorsky E, Ashkenazi YJ et al. Optimal time interval between myeloblative whole body irradiation and reconstitution with syngeneic bone marrow graft. Bone Marrow Transplant 1988;3:20–210.
Lataillade JJ, Clay D, Bourin P et al. Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in Cd34+ cells: evidence for an autocrine/paracrine mechanism. Blood 2002;99:1117–1129.
Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 1999;10:463–471.
Broxmeyer HE, Hangoc G, Cooper S et al. Enhanced myelopoiesis in sdf-1-transgenic mice: sdf-1 modulates myelopoiesis by regulating progenitor cell survival and inhibitory effects of myelosuppressive chemokines . Blood 1999;94:650a.
Broxmeyer H, Kohli L, Kim C et al. Stromal cell-derived factor-1/CXCL12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and Gai proteins and enhances engraftment of competitive, repopulating stem cells. J. Leukoc Biol 2003;73:630–638.
Lataillade JJ, Clay D, Dupuy C et al. Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood 2000;95:756–768.
Lee Y, Gotoh A, Kwon H et al. Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines. Blood 2002;99:4307–4317.
Grafte-Faure S, Leveque C, Ketata E et al. Recruitment of primitive peripheral blood cells: synergism of interleukin 12 with interleukin 6 and stromal cell-derived factor-1. Cytokine 2000;12:1–7.
Janowska-Wieczorek A, Majka M, Kijowski J et al. Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment. Blood 2001;98:3143–3149.
Whetton AD, Lu Y, Pierce A et al. Lysophospholipids synergistically promote primitive hematopoietic cell chemotaxis via a mechanism involving Vav 1. Blood 2003;102:279–2802.
Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038–1045.
Hess DA, Karanu FN, Levac K et al. Coculture and transplant of purified CD34+Lin– and CD34–Lin– cells reveals functional interaction between repopulating hematopoietic stem cells. Leukemia 2003;17:1613–1625.
Wilpshaar J, Falkenburg JH, Tong X et al. Similar repopulating capacity of mititically active and resting umbilical cord blood CD34+ cells in NOD/SCID mice. Blood 2000;96:2100–2107.(Tamara Byka, Joy Kahna, O)
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@weizmann.ac.il
ABSTRACT
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 . 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 . To identify and quantify human stem cells, functional in vivo assays using immune-deficient mice as recipients were developed .
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 . 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. Moreover, we have recently reported that overexpression of this receptor on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation . Other key regulators of these processes are adhesion molecules, cytokines, and proteolytic enzymes . In particular, matrix metalloproteinase (MMP)-9 was shown to be secreted by HSCs, facilitating their in vitro migration . We have further demonstrated a role for these proteinases in stem cell motility and also in the upregulation of cell-surface CXCR4 expression . Recent studies have revealed the involvement of MMP-9 in in vivo HSC mobilization by shedding membrane-bound stem cell factor (SCF) .
Long-term maintenance of human stem cells in ex vivo cultures is a major challenge for clinical and experimental transplantation protocols . Present results are limited because of the induction of differentiation and the concomitant loss of repopulation and self-renewal capacities . Human cells capable of repopulating NOD/SCID mice have been maintained or expanded in vitro for a limited time using different combinations of cytokines . Other protocols used coculture with stromal cells to maintain NOD/SCID-repopulating cells in vitro and overexpression of HOXB4 homeoprotein in stroma used for coculture .
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 . Most primitive human CD34+-enriched progenitors are quiescent, and cytokine stimulation leads to their entrance into cell cycle . Human adult mobilized peripheral blood (MPB) G1 CD34+ cells home to the BM of conditioned NOD/SCID mice ; however, Gothot et al. 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 . 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 . Because SRCs are characterized as CD34+/CD38–/low/CXCR4+ cells , 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
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 1A.
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, 1B; 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 ) increased the G1 fraction further to 71± 5.0% of the sorted cells (Fig. 1B, 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 1C, 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. 2). 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.
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 . 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 3A, 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).
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. 3B; time = 0), including primitive progenitors that gave rise to mixed colonies (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte ). 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. 3B, 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 . 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. 4A) or close to (Fig. 5A) 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 . 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. 4A, +-CXCR4). As we have shown previously, cells migrating toward SDF-1 in the transwell assay better engraft NOD/SCID mice than nonmigrating cells . Similar results were observed for the purified G0 cells (Fig. 4B). 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. 3A), 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. 2). Similarly to what was shown in Figure 4A, freshly isolated G0 cells also engrafted better than G0 cells after SCF treatment when respective migrating or nonmigrating fractions were compared (Fig. 4B). Taken together, it seems that the beneficial influence of the SCF treatment was lost when G0 and G1 cells were separated.
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 (+-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.
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 (+-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 . 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. 4C, 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. 4C, 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 1 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.
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. 5A). Like in the experiments shown in Figures 4A and 4B, 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. 5B). 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. 5B).
The accessory effect of cycling G1 cells on G0 cells was lost when the mice were irradiated 24 hours before transplantation (Fig. 5C). 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. 5A). This situation is mimicked by the in vitro migration assay in Table 1: 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 .
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. 5D). 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 -CXCR4 antibody (with washing) before injection (Fig. 5E). 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. 5F). 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 .
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 . 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. 6A; p = .04).
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. 6B p, < .05).
Because MMP-2 and -9 play a role in the in vitro SDF-1–mediated motility of human CD34+-enriched CB cells , 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. 6C). Furthermore, 5 weeks after transplantation, we observed a 62% (p = .03) reduction in engraftment of human cells within the BM (Fig. 6D).
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
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.
REFERENCES
Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100:157–168.
Morrison S, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol 1995;11:35–71.
To LB, Haylock DN, Simmons PJ et al. The biology and clinical uses of blood stem cells. Blood 1997;89:2233–2258.
Moore MA. "Turning brain into blood": clinical applications of stem-cell research in neurobiology and hematology. N Engl J Med 1999;341:605–607.
Lapidot T, Pelumio F, Doedens M et al. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 1992;255:1137–1141.
Conneally E, Cashman J, Petzer A et al. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lymphomyeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 1997;94:9836–9841.
Dao MA, Shah AJ, Crooks GM et al. Engraftment and retroviral marking of CD34+ and CD34+CD38– human hematopoietic progenitors assessed in immune-deficient mice. Blood 1998;91:124–1255.
Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mice using retroviral gene marking and cell purification: implications for gene therapy. Nat Med 1996;2:1329–1337.
Wang JC, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997;89:3919–3924.
Kollet O, Peled A, Byk T et al. ?2 microglobulin deficient (B2mnull) NOD/SCID mice are excellent recipients for studying human stem cell function. Blood 2000;95:3102–3105.
Peled A, Petit I, Kollet O etal. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.
Kollet O, Spiegel A, Peled A et al. Rapid and efficient homing of human CD34+CD38–/low CXCR4+ stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice. Blood 2001;97:3283–3291.
Ponomaryov T, Peled A, Petit I et al. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest 2000;106:1331–1339.
Kahn J, Byk T, Jansson-Sjostrand L et al. Overexpression of CXCR4 on CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation. Blood 2004;103:2942–2949.
Lapidot T, Kollet O. The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2mnull mice. Leukemia 2002;16:1992–2003.
Janowska-Wieczorek A, Marquez L, Nabholtz J-M. Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34+ cells and their transmigration through reconstituted basement membrane. Blood 1999;93:3379–3390.
Janowska-Wieczorek A, Marquez L, Dobrowsky A. Differential MMP and TIMP production by human marrow and peripheral blood CD34+ cells in response to chemokines. Exp Hematol 2000;28:1274–1285.
Kollet O, Shivtiel S, Chen YQ et al. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 2003;112:160–169.
Heissig B, Hattori K, Dias S et al. Recruitment of stem and progenitor cells from the bone marrow niche requires mmp-9 mediated release of kit-ligand. Cell 2002;109:625–637.
Moore MA. Umbilical cord blood: an expandable resource. J Clin Invest 2000;105:855–856.
Emerson SG. Ex vivo expansion of hematopoietic precursors, progenitors and stem cells: the next generation of cellular therapeutics. Blood 1996;87:3082–3088.
Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997;186:619–624.
Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34+ cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93:3736–3749.
Kollet O, Aviram R, Chebath J et al. The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34+CD38–/low cells capable of repopulating severe combined immunodeficiency mice. Blood 1999;94:923–931.
Gan OI, Murdoch B, Larochelle A et al. Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. Blood 1997;90:641–650.
Amsellem S, Pflumio F, Bardinet D et al. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nat Med 2003;9:1423–1427.
Orschell-Traycoff CM, Hiatt K, Dagher RN et al. Homing and engraftment potential of Sca-1+lin– cells fractioned on the basis of adhesion molecule expression and position in cell cycle. Blood 2000;96:1380–1387.
Ladd AC, Pyatt R, Gothot A et al. Orderly process of sequential cytokine stimulation is required for activation and maximal proliferation of primitive human bone marrow CD34+ hematopoietic progenitor cells residing in G0. Blood 1997;90:658–668.
Srour EF, Jetmore A, Wolber FM et al. Homing, cell cycle kinetics and fate of transplanted hematopoietic stem cells. Leukemia 2001;15:1681–1684.
Gothot A, van der Loo JC, Clapp DW et al. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641–2649.
Lapidot T, Pflumio F, Doedens M et al. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 1992;255:1137–1141.
Kollet O, Petit I, Kahn J et al. Human CD34+CXCR4- sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation. Blood 2002;100:2778–2886.
Papayannopoulou T. Bone marrow homing: the players, the playfield, and their evolving roles. Curr Opin Hematol 2003;10:214–219.
Muench MO, Firpo MT, Moore MAS. Bone marrow transplantation with interleukin-1 plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without the loss of stem cell lineage and proliferative potential. Blood 1993;81:3463–3473.
Hendrikx PJ, Martens ACM, Hagenbeek A et al. Homing of fluorescently labeled murine hematopoietic stem cells. Exp Hematol 1996;24:129–140.
Glimm H, Eaves CJ. Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture. Blood 1999;94:2161–2168.
Oostendorp RA, Audet J, Eaves CJ. High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo. Blood 2000;95:855–862.
Bierhuizen MF, Westerman Y, Visser TP et al. Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells. Blood 1997;90:3304–3315.
Bonnet D, Bhatia M, Wang JC et al. Cytokine treatment or accessory cells are required to initiate engraftment of purified primitive human hemato-poietic cells transplanted at limiting doses into NOD/SCID mice. Bone Marrow Transplant 1999;23:203–209.
Civin CI, Almeida-Porada G, Lee MJ et al. Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo. Blood 1996;88:4102–4109.
Kerre TC, De Smet G, De Smedt M et al. Both CD34+38+ and CD34+38– cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion. J Immunol 2001;167:3692–3698.
Hogan CJ, Shpall EJ, Keller G. Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD-SCID mice. Proc Natl Acad Sci U S A 2002;99:413–418.
Jetmore A, Plett P, Tong X et al. Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients. Blood 2002;99:158–1593.
Weiss L, Bullorsky E, Ashkenazi YJ et al. Optimal time interval between myeloblative whole body irradiation and reconstitution with syngeneic bone marrow graft. Bone Marrow Transplant 1988;3:20–210.
Lataillade JJ, Clay D, Bourin P et al. Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in Cd34+ cells: evidence for an autocrine/paracrine mechanism. Blood 2002;99:1117–1129.
Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 1999;10:463–471.
Broxmeyer HE, Hangoc G, Cooper S et al. Enhanced myelopoiesis in sdf-1-transgenic mice: sdf-1 modulates myelopoiesis by regulating progenitor cell survival and inhibitory effects of myelosuppressive chemokines . Blood 1999;94:650a.
Broxmeyer H, Kohli L, Kim C et al. Stromal cell-derived factor-1/CXCL12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and Gai proteins and enhances engraftment of competitive, repopulating stem cells. J. Leukoc Biol 2003;73:630–638.
Lataillade JJ, Clay D, Dupuy C et al. Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood 2000;95:756–768.
Lee Y, Gotoh A, Kwon H et al. Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines. Blood 2002;99:4307–4317.
Grafte-Faure S, Leveque C, Ketata E et al. Recruitment of primitive peripheral blood cells: synergism of interleukin 12 with interleukin 6 and stromal cell-derived factor-1. Cytokine 2000;12:1–7.
Janowska-Wieczorek A, Majka M, Kijowski J et al. Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment. Blood 2001;98:3143–3149.
Whetton AD, Lu Y, Pierce A et al. Lysophospholipids synergistically promote primitive hematopoietic cell chemotaxis via a mechanism involving Vav 1. Blood 2003;102:279–2802.
Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038–1045.
Hess DA, Karanu FN, Levac K et al. Coculture and transplant of purified CD34+Lin– and CD34–Lin– cells reveals functional interaction between repopulating hematopoietic stem cells. Leukemia 2003;17:1613–1625.
Wilpshaar J, Falkenburg JH, Tong X et al. Similar repopulating capacity of mititically active and resting umbilical cord blood CD34+ cells in NOD/SCID mice. Blood 2000;96:2100–2107.(Tamara Byka, Joy Kahna, O)