Chemokine-mobilized adult stem cells; defining a better hematopoietic graft

Abstract

Stem cell research is currently focused on totipotent stem cells and their therapeutic potential, however adult stem cells, while restricted to differentiation within their tissue or origin, also have therapeutic utility. Transplantation with bone marrow hematopoietic stem cells (HSC) has been used for curative therapy for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow or mobilize to peripheral blood by G-CSF, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. The chemokine/chemokine receptor axis also mobilizes HSC that occurs more rapidly than with G-CSF. In mice, the HSC and progenitor cells (HPC) mobilized by the CXCR2 receptor agonist GRObeta can be harvested within minutes of administration and show significantly lower levels of apoptosis, enhanced homing to marrow, expression of more activated integrin receptors and superior repopulation kinetics and more competitive engraftment than the equivalent cells mobilized by G-CSF. These characteristics suggest that chemokine axis-mobilized HSC represent a population of adult stem cells distinct from those mobilized by G-CSF, with superior therapeutic potential. It remains to be determined if the chemokine mobilization axis can be harnessed to mobilize other populations of unique adult stem cells with clinical utility.

Figure 1

Competitive transplantation. In primary transplants, one half million marrow cells from BoyJ mice (CD45.1) were mixed with one million PBMC from C57Bl/6 mice (CD45.2) mobilized by G-CSF, GROβ, AMD3100 or the combination of GROβ or AMD3100 with G-CSF and transplanted into lethally irradiated C57Bl/6 mice. Chimerism in recipient mice was analyzed after 6 months. In secondary transplants, individual mice that had received transplants containing marrow and mobilized peripheral blood stem cells were killed and one million marrow cells transplanted in noncompetitive fashion to lethally irradiated C57Bl/6 mice. Chimerism was analyzed at 6 months post-transplant. *P<0.05 compared to G-CSF. ^†P<0.05 compared to G-CSF, GROβ or AMD3100 alone, analyzed by ANOVA with Bonferroni correction. ^‡P<0.05 compared to GROβ or AMD3100 plus G-CSF primary transplants, analyzed by ANOVA with Bonferroni correction.

Figure 2

Immunophenotype and function comparison of GROβ- and G-CSF-mobilized SKL and KL cells. Data are presented as the average percent change for GROβ-mobilized cells relative to G-CSF-mobilized cells. KL=c-kit⁺, lineage ^neg cells; SKL=Sca-1⁺, c-kit⁺, lineage^neg cells; CFU-GM=colony forming unit granulocyte–macrophage. Annexin V (FITC) was determined on lin ^neg cells prestained with APC-c-kit and PE-Cy7-Streptavidin–biotin–Sca-1. Lineage ^neg cells were subjected to in vitro migration to 100 ng ml^−/1 rmSDF-1 for 4 h. Input and migrated cells were collected, washed and CFU-GM enumerated in 1% methylcellulose with 30% FBS, 1Uml^−/1 rhEpo, 10 ng ml^−/1 rmGM-CSF and 50 ng ml^−/1 rmSCF or stained with anti-c-kit and anti-Sca-1 and migrating cells analyzed by flow cytometry. PBMC mobilized by G-CSF or GROβ were stained with 5 μM 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4 × 10⁷ cells transplanted into lethally irradiated syngeneic recipients. Recipient marrow cells were harvested 16 h after transplantation. Total KL or SKL cells homed to marrow were calculated as: (% CFSE⁺ KL or SKL cells in recipient marrow) × 100^−/1 × (% CFSE⁺ cells in recipient marrow) × 100^−/1 × (total nucleated recipient marrow cells recovered) × (100/30), assuming marrow cells from two femurs, two tibias, two humerus and the pelvis represents 30% of total body marrow. Percent homing was determined as (KL or SKL cells homed to the marrow)÷(KL or SKL cells transplanted) × 100 in each recipient and the data were averaged. In some experiments (see inset) CFSE-stained lin ^neg cells were pretreated with 10 μM AMD3100 or PBS for 15 min at 37 °C, washed and transplanted, to determine the effect of CXCR4 antagonism on homing. Surface KL and SKL cell CXCR4 expression was evaluated on lin ^neg cells using anti Fc-γIII/II block and anti-CXCR4. Intracellular CXCR4 in mobilized KL and SKL cells was determined by staining lin ^neg cells with FITC-Sca-1, APC-c-kit and a saturating amount of nonconjugated anti-mouse CXCR4. Cells were fixed with 1% paraformaldehyde, permeabilized with 0.25% Triton X/0.5% BSA/PBS and stained with PE-CXCR4. Mobilized PBMC in 0.5% BSA/RPMI-1640 (4×10⁴ cells per well) were added to plates precoated with 5 μgml^−/1 human fibronectin or fibronectin plates seeded with C166 cells 48 h prior to adhesion assays. The plates were briefly centrifuged to facilitate adhesion. After 30 min at 37 °C, wells were washed and non-adherent cells collected. Input cells and non-adherent KL and SKL cells were quantitated and percent adhesion determined by subtracting % non-adherent cells from 100. Surface expression of CD62, CD49d, CD11a, CD49e and CD26 was analyzed on lin ^neg cells blocked with anti Fc-γIII/II block using specific antibodies (MEL14; 9C10; 2D7; 5H10-27; H194-112; all from BD Biosciences). CD26 expression on SKL cells was quantitated following staining of lin ^neg cells with PE-Sca-1, APC-c-kit and FITC CD26. *P<0.05.