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Dependence of Human Stem Cell Engraftment and Repopulation of NOD/SCID Mice on CXCR4

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Science  05 Feb 1999:
Vol. 283, Issue 5403, pp. 845-848
DOI: 10.1126/science.283.5403.845

Abstract

Stem cell homing and repopulation are not well understood. The chemokine stromal cell–derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38−/low cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.

Stem cells within the bone marrow microenvironment actively maintain continuous production of all mature blood cell lineages throughout life. These rare primitive cells are functionally defined by their ability to home to the bone marrow and to durably repopulate transplanted recipients with both myeloid and lymphoid cells (1, 2). Several groups have established in vivo models for engrafting human stem cells (3–8). We developed a functional in vivo assay for primitive human SCID repopulating cells (SRCs) based on their ability to repopulate the bone marrow of intravenously transplanted SCID or non- obese diabetic SCID (NOD/SCID) mice with high levels of both myeloid and lymphoid cells (5, 6, 8).

Chemokines are cytokines that are best known for their ability to selectively attract subsets of leukocytes to sites of inflammation (9). The role that chemokines and their receptors play in homing and repopulation of human stem cells is not fully understood. The chemokine SDF-1 (10) binds to its receptor CXCR4, which is expressed on many cell types, including some CD34+CD38cells (11, 12). In vitro SDF-1 attracts certain CD34+CXCR4+ cells, and in vivo it is produced by bone marrow stromal cells as well as by epithelial cells in many organs (11, 13, 14). Mice that lack SDF-1 or do not express CXCR4 exhibit many defects, including the absence of both lymphoid and myeloid hematopoiesis in the fetal bone marrow (10, 15). Overexpression of human CD4 and CXCR4 receptors on murine CD4+ T cells led to enhanced homing of these cells to the murine bone marrow (16).

To examine the in vivo role of SDF-1 and its receptor CXCR4 in migration and repopulation by human SRCs, we treated CD34+-enriched cord blood cells either with two different antibodies to CXCR4 or with control antibodies to CD34 (anti-CD34) before transplantation of NOD/SCID mice (Fig. 1A, panel a). Only anti-CXCR4 reduced engraftment. Similar treatment of human CD34+-enriched cells from mobilized peripheral blood or adult bone marrow also resulted in inhibition of engraftment (Fig. 1A, panel b). Antibodies to SDF-1 coinjected with human CD34+ cord blood cells and readministered after 24 hours significantly reduced the level of engraftment (Fig. 1A, panel a). The first 24 hours were critical to the engraftment process. Antibodies administered intraperitoneally 30 min after transplantation blocked engraftment (Fig. 1B). Antibodies administered 24 hours later reduced engraftment less effectively and when administered 4 days after transplantation were completely ineffective (Fig. 1B) (17).

Figure 1

Effect of antibodies to CXCR4 and SDF-1 on engraftment of NOD/SCID bone marrow (BM) by human CD34+cells. (A) (Panel a) Human cord blood CD34+cells (6) treated with two alternative antibodies to CXCR4 or with anti-CD34 as a control were transplanted into mice. Alternatively, anti–SDF-1 was coinjected with the cells and reinjected 24 hours later. After 2 weeks human progenitor cells were quantified in semi-solid media assays (5). The following cell types were counted: colony-forming unit–granulocyte/macrophage (CFU-GM) (white bars), blast-forming unit–erythroid (BFU-E) (dashed bars), and multilineage colony (CFU-GEMM) (striped bars). Data are average ± SE (*P < 0.01, as determined by paired Student's t test) of three experiments. (Panel b) Human bone marrow (black bars) or mobilized peripheral blood (stippled bars) CD34+ cells were treated with the indicated antibodies and transplanted into NOD/SCID mice, and total human progenitors were quantified after 1 month as for panel a. (B) Antibodies to CXCR4 were injected at the indicated times after transplantation of NOD/SCID mice with cord blood CD34+ cells. Control cells were incubated with anti-CD34. After 2 weeks, bone marrow was assayed by Southern blot for human DNA with a human-specific α satellite probe (5). (C) Cord blood CD34+ cells were either not treated (CT) or treated for 24 hours with SDF-1 or PMA. (Panel a) CXCR4 surface expression of CD34+ cells. (Panel b) Transwell migration assay (13) of untreated cells without SDF-1 (CT−) or with SDF-1 (CT+), and migration to SDF-1 of treated cells. (Panel c) The percent of human cells in NOD/SCID mice 1 month after transplantation was determined by FACS analysis with antibodies to human CD45. Data are average ± SE (*P < 0.01) of three experiments.

The effects of SDF-1 desensitization and CXCR4 down-regulation on the ability of human CD34+ cells to migrate and engraft NOD/SCID mice were further studied. SDF-1 and phorbol esters [phorbol 12-myristate 13-acetate (PMA)] cause internalization and down-regulation of CXCR4 surface expression on human CD4+ T cells (18). Cord blood CD34+ cells were incubated overnight with high doses of SDF-1. Cells were subsequently washed and tested for CXCR4 expression and migration to SDF-1 in a transwell assay. Treatment of CD34+ cells with SDF-1 or PMA reduced CXCR4 cell surface expression (Fig. 1C, panel a) and abolished the migration of CD34+ cells in response to SDF-1 (Fig. 1C, panel b) (19). Prolonged treatment of CD34+cells with SDF-1 significantly blocked the engraftment of transplanted NOD/SCID mice (Fig. 1C, panel c). Thus, SDF-1 probably affects SRC engraftment by mediating chemotaxis to the bone marrow, linking migration to SDF-1 in vitro to human stem cell function in vivo.

The migration potential of human CD34+ cells from cord blood, bone marrow, or mobilized peripheral blood was tested in vitro in a transwell assay. Consistent with previous studies (13), 20 to 25% of cord blood and bone marrow CD34+ cells migrated in response to a chemotactic gradient of SDF-1 in all donors tested (Fig. 2A, panel a). Migration of mobilized peripheral blood CD34+ cells from multiple donors in response to SDF-1 was variable (between 8 to 60%), suggesting the involvement of SDF-1 in the mobilization process (Fig. 2A, panel a). The migrating and nonmigrating CD34+ cell populations did not differ in the incidence of progenitor cells, as determined by in vitro colony assays (Fig. 2A, panel b); however, the engraftment potential of the migrating and nonmigrating CD34+ cells was different. Equal numbers of migrating (M) and nonmigrating (NM) CD34+ cells were washed and transplanted into NOD/SCID or NOD/SCID β2 microglobulin knockout mice (20). Whereas mice transplanted with nonmigrating cells were poorly engrafted, mice transplanted with migrating cells were significantly better engrafted (Fig. 2A, panel c). The low concentrations of SDF-1 and the limited exposure time caused only a transient decrease of CXCR4 expression that did not prevent engraftment. These results are further evidence for the link between in vitro motility to SDF-1 and in vivo stem cell function.

Figure 2

SDF-1 induces the migration of SRCs. (A) (Panel a) Transwell migration assay with CD34+ cord blood (C), bone marrow (B), or mobilized peripheral blood (MB) cells (13, 14). CT, migration without SDF-1. SDF-1 migrating (M) and nonmigrating (NM) cells were assayed for progenitors (panel b) or transplanted into NOD/SCID or β2-microglobulin knockout NOD/SCID mice (3 × 104cells per mouse) (panel c) (20,23). The percent of human cells was quantified as in Fig. 1C, panel c. Data are average ± SD of 11 (panel a) or 3 experiments (panel b), or average ± SE of three experiments (panel c) (*P < 0.01). (Panels CB and BM) SDF-1 preferentially induces migration of CD34+CD38-/lowCXCR4+ cells. Surface expression of CD38 on cord blood (panel CB) and bone marrow (panel BM) CD34+ cells was analyzed by flow cytometry on SDF-1 migrating (M) or nonmigrating (NM) cells. R gates CD34+/CD38 cells. (B) Sorted cord blood CD34+CD38-/low cells, (Panel a) SDF-1 migrating (M) or nonmigrating (NM) cells were transplanted into NOD/SCID mice (3 × 104cells per mouse). After 6 weeks, percent of engraftment was quantified as in Fig. 1C, panel c. Data are average ± SE (*P < 0.01) of three experiments. Phenotype analysis of engrafted M and NM cells. Numbers indicate percent of human cells. (Panels Ma and Mb) The presence of human lymphoid CD45+CD19+ pre-B cells (panel Ma) and progenitors for human CD45+CD56+natural killer cells (panel Mb) is shown.

Although only 20 to 25% of cord blood CD34+ cells migrated toward SDF-1, this population contained a significantly higher percentage of primitive CD34+CD38 cells than did nonmigrating cells (Fig. 2A, panel CB). In CD34+cells from bone marrow, the proportion of immature CD34+CD38−/low cells migrating to SDF-1 was larger than in cord blood (Fig. 2A, panel BM). Nevertheless, most cord blood CD34+CD38 cells (60%) did not migrate to SDF-1, demonstrating that CD34+CD38 cells are a heterogeneous population composed mostly of nonmigrating cells. Sorted CD34+CD38−/low cord blood cells from different donors were evaluated for their ability to migrate toward a chemotactic gradient of SDF-1 in vitro on the basis of surface CXCR4 expression and for their content of SRCs in vivo. Only 26% (±7%) of the CD34+CD38−/low cells migrated to a gradient of SDF-1 in the transwell assay. Transplantation of migrating CXCR4+ cells into NOD/SCID mice resulted in high levels of multilineage engraftment (Fig. 2B). This was reflected in the engraftment of primitive CD34+CD38 cells (Fig. 2B, panel M) and lymphoid (Fig. 2B, panels Ma and Mb), and myeloid colony-forming cells. In contrast, little engraftment was observed with nonmigrating CXCR4−/low cells (Fig. 2B, panel NM). Thus, the CD34+CD38−/lowCXCR4+migrating cell population representing less than one-third of all CD34+CD38−/low cells engrafts the murine bone marrow with SRCs.

Kim and Broxmeyer have demonstrated that stem cell factor (SCF) attracts CD34+ cells, increases their motility, and synergizes with SDF-1, increasing migration to both cytokines in vitro (21). Unexpectedly, prolonged (24- to 48-hour) stimulation of mobilized peripheral blood CD34+ cells with SCF resulted in increased CXCR4 expression (Fig. 3A), enhanced migration toward SDF-1 (Fig. 3B), and enhanced engraftment potential dependent on the exposure time to SCF (Fig. 3C). Engraftment potential was similarly increased when only half the cell number was injected after 40 hours of SCF treatment, compared with 16 hours of exposure or untreated cells transplanted at time 0 (Fig. 3D). Thus, enhanced CXCR4-dependent migration to SDF-1 was accompanied by an increase in the SRC fraction. Incubation of SCF-stimulated, mobilized peripheral blood CD34+ cells with anti-CXCR4 prevented engraftment (Fig. 3C). Sorted CD34+-CD38−/lowCXCR4−/lowcord blood cells that did not migrate toward SDF-1 were either transplanted or treated with SCF for 48 hours. Whereas nontreated cells had low engraftment efficiency (Fig. 4A), SCF treatment resulted in increased migration toward SDF-1 and efficient engraftment by converted CD34+CD38−/lowCXCR4+ cells, properties that were similar to those of the original migrating fraction (M) (Fig. 4A).

Figure 3

SCF potentiates CXCR4 expression, cell migration, and SRC engraftment. (A) Mobilized peripheral blood CD34+ cells stained with control antibody (curve a) or with anti-CXCR4 before (curve b) or after (curve c) 40 hours of treatment with SCF. (B) SDF-1 transwell migration of untreated (0), SCF-treated (16 and 40 hours), or control cells cultured for 40 hours without SCF (CT). Data are average ± SE of three experiments. (C) Percent of engraftment in NOD/SCID mice transplanted with 2 × 105 cells before (0) or after 16 or 40 hours of exposure to SCF and 40 hours of exposure to SCF followed by incubation with anti-CXCR4 (+ anti CXCR4). Control cells (CT) as in (B). Percent of engraftment was quantified as inFig. 1C, panel c. Data are average ± SE (*P < 0.01, SCF 40 hours versus 0 hours, SCF+anti-CXCR4, and CT 40 hours) of three mice per treatment, in a representative experiment. (D) Exposure times of mobilized peripheral blood CD34+ to SCF as in (C). At time 0 and after 16 hours 1 × 105 cells per mouse were transplanted, and after 40 hours 0.5 × 105 cells per mouse were transplanted. Human engraftment was quantified after 1 month by Southern blot analysis.

Figure 4

Increase in SRCs and of stem cell self-renewal by up-regulation of CXCR4 expression. (A) Sorted CD34+CD38−/low cord blood cells migrating toward SDF-1 were transplanted into NOD/SCID mice (3 × 104 cells per mouse) (M). Nonmigrating cells were either injected directly (NM) or treated with SCF for 48 hours and then injected (+SCF). After 6 weeks engraftment levels were quantified as inFig. 1C, panel c. Data in the left panel are average ± SE (*P < 0.01) of four experiments. (B) Bone marrow cells from mice transplanted 4 to 6 weeks before with human cord blood CD34+ cells in panels a and b were retransplanted untreated (2nd in panel a) or after SCF and IL-6 treatment for 48 hours (panel b) into secondary β2-microglobulin knockout NOD/SCID mice. Data in panels a and b are the average ± SE of four experiments (panel a, *P < 0.01, 1st versus 2nd; **P < 0.05, 2nd in panel b versus 2nd in panel a). (Panel c) Human CXCR4 expression on cord blood cells from transplanted mice immediately labeled (solid) or after 48 hours treatment with SCF and IL-6 (open). (Panel d) SDF-1 migration of cord blood cells from the marrow of transplanted mice before and after treatment with SCF and IL-6 for 48 hours. Data in panel d are the average of triplicates in a representative experiment. (C) Cord blood CD34+cells were stained with control antibody (curve a) or antibody to CXCR4 after a 48-hour exposure to SCF (curve b) or SCF and IL-6 (curve c). Percent of engraftment in (A) and (B) was quantified as inFig. 1C, panel c.

Self-renewal of stem cells can only be determined by their ability to also repopulate secondary transplanted recipients with high numbers of both myeloid and lymphoid cells. Consistent with previous studies, secondary transplanted mice that received untreated human cells showed little engraftment (Fig. 4B, panel a) (22). Human interleukin-6 (IL-6) synergizing with SCF induced high levels of CXCR4 expression on CD34+ cord blood cells (Fig. 4C). Incubation of bone marrow cells from primary transplanted mice with SCF and IL-6 for 48 hours resulted in up-regulation of surface CXCR4 expression (Fig. 4B, panel c) and increased migration of human progenitor cells to SDF-1 in vitro (Fig. 4B, panel d). Transplantation of similar numbers of human cells from the bone marrow of primary transplanted mice after treatment with these cytokines resulted in higher engraftment levels in secondary transplanted mice compared with mice transplanted with untreated cells (Fig. 4B, panel b versus panel a). Thus, by up-regulating surface CXCR4 expression on primitive cells, the population of self-renewing CD34+CD38−/low SRC stem cells could be increased.

Our data provide evidence that CXCR4-dependent migration to SDF-1 is essential for human stem cell function in NOD/SCID mice. We characterized SRCs further as CD34+CD38−/lowCXCR4+ stem cells and showed that CD34+CD38−/lowCXCR4−/low cells can be converted into functional CXCR4+ stem cells by cytokine treatment. This suggests that migration to SDF-1 is associated with localization of stem cells in the bone marrow, permitting differentiating cells with reduced migration levels to exit into the blood circulation. In conclusion, our findings define human CD38−/lowCXCR4+ cells as stem cells endowed with migration and repopulation potential and provide insights into human stem cell biology.

  • * To whom correspondence should be addressed. E-mail: litsvee{at}weizmann.weizmann.ac.il

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