Superior long-term repopulating capacity of G-CSF+plerixafor-mobilized blood: implications for stem cell gene therapy by studies in the Hbb(th-3) mouse model

Abstract

High numbers of genetically modified hematopoietic stem cells (HSCs) equipped with enhanced engrafting potential are required for successful stem cell gene therapy. By using thalassemia as a model, we investigated the functional properties of hematopoietic stem and progenitor cells (HSPCs) from Hbb(th3)/45.2(+) mice after mobilization with G-CSF, plerixafor, or G-CSF+plerixafor and the engraftment kinetics of primed cells after competitive primary and noncompetitive secondary transplantation. G-CSF+plerixafor yielded the highest numbers of HSPCs, while G-CSF+plerixafor-mobilized Hbb(th3)/45.2(+) cells, either unmanipulated or transduced with a reporter vector, achieved faster hematologic reconstitution and higher levels of donor chimerism over all other types of mobilized cells, after competitive transplantation to B6.BoyJ/45.1(+) recipients. The engraftment benefit observed in the G-CSF+plerixafor group was attributed to the more primitive stem cell phenotype of G-CSF+plerixafor-LSK cells, characterized by higher CD150(+)/CD48 expression. Moreover, secondary G-CSF+plerixafor recipients displayed stable or even higher chimerism levels as compared with primary engrafted mice, thus maintaining or further improving engraftment levels over G-CSF- or plerixafor-secondary recipients. Plerixafor-primed cells displayed the lowest competiveness over all other mobilized cells after primary or secondary transplantation, probably because of the higher frequency of more actively proliferating LK cells. Overall, the higher HSC yields, the faster hematological recovery, and the superiority in long-term engraftment indicate G-CSF+plerixafor-mobilized blood as an optimal graft source, not only for thalassemia gene therapy, but also for stem cell gene therapy applications in general.

FIG. 1.

Distribution of mobilized HSPCs among hematopoietic compartments and the mobilization effect on the spleen. (A) Absolute numbers of LSK cells and CFCs are presented per milliliter of blood. (B) Absolute numbers of SLAM LSKs are presented per milliliter of blood. (C) Absolute numbers of LSK cells and CFCs are presented per two femurs and spleen (× 10⁻¹). Data are expressed as mean±SEM from untreated (n=13), G-CSF-treated (n=13), plerixafor-treated (n=13), and G-CSF+plerixafor-treated (n=16) thalassemic mice. *p<0.05 vs. untreated, ^‡p<0.05 vs. G-CSF, ^#p<0.05 vs. plerixafor, and ^†p<0.05 vs. all other. (D) Quantitation and representative images of SDF-1 expression by immunohistochemistry in spleen sections of untreated (a), G-CSF-mobilized (b), plerixafor-mobilized (c), and G-CSF+plerixafor-mobilized (d) mice. Original magnification×200. (E) Absolute numbers of LSK cells and CFU-GM are presented per milliliter of blood of untreated, G-CSF-treated, plerixafor-treated, and G-CSF+plerixafor-treated, nonsplenectomized, and splenectomized mice. Data are expressed as mean±SEM (n=3–6 animals/group per experiment×3 independent experiments). *p<0.05. (F) Splenic infarcts were detected in all mobilization groups. Original magnification×20 (left) and ×100 (right). CFCs, colony forming cells; HSPCs, hematopoietic stem and progenitor cells; SLAM, signaling lymphocyte activation marker.

FIG. 2.

In vitro properties of differently mobilized LK cells. (A) Migration of Lin⁻/c-kit⁺ (LK) mobilized and steady-state blood cells in response to 100 ng/ml SDF-1a. Data are expressed as percentage±SEM of migrated cells (n=3 mice per group assayed in duplicate). Expression of CXCR4 and CD26 on LK cells shown as frequency (B) and as MFI±SEM (C) (n=9 mice per group). (D) Cell cycle status expressed as the average S&G2&M and G0&G1 frequency±SEM of LK cells (n=4 mice per group), *p<0.05 vs. untreated, ^‡p<0.05 vs. G-CSF, and ^†p<0.05 vs. all other.

FIG. 3.

Competitive repopulation in mice after 3:1 transplantation. Whole blood from mobilized thalassemic 45.2⁺ mice was infused into lethally irradiated B6.BoyJ (45.1⁺) mice at a 3:1 (donor:recipient) ratio, along with a steady number of competitor 45.1⁺ cells (0.5×10⁶). (A) Hematologic recovery in terms of platelet recovery (%) is depicted as a percentage to average normal platelet levels and neutrophil recovery as absolute neutrophil cell count (ANC) at different time points after transplantation. Data are expressed as mean±SEM of recipients receiving unmobilized and G-CSF-, plerixafor-, and G-CSF+plerixafor-mobilized grafts. (B) Engraftment based on the % donor's (45.2⁺) chimera of 45.1⁺ recipients. (C) Representative dot plots depictive of multilineage reconstitution capacity of differently mobilized CD45.2⁺ (donor) cells. Each experiment was performed 3 times; n=5 animals per group per experiment. *p<0.05 vs. all other groups. Content of infused grafts in LSK and CFCs, expressed as mean±SEM; *p<0.05 vs. all other groups.

FIG. 4.

Competitive repopulation after transplantation of equal number of LSKs and secondary transplantation from primary engrafted recipients. (A and B) Mobilized peripheral blood containing equal numbers of immunophenotypically characterized G-CSF-, plerixafor-, G-CSF+plerixafor-mobilized 45.2⁺ LSKs (1×10⁴) was transplanted in a competitive fashion either unmanipulated (A) or transduced with a reporter lentiviral vector (B), to lethally irradiated recipients, along with 0.25×10⁶ competitor 45.1⁺ BM cells. Donor's chimera is shown as mean±SEM (n=5 recipients per group). (C) Composition in progenitor cells of infused grafts containing equal number of LSK cells. Data from three grafts per group are expressed as mean±SEM. (D) Frequency of CD150⁺/CD48⁻ cells (SLAM) within differently mobilized LSK populations. Data from 9 to 11 animals per group, represented as mean±SEM. (E) An amount of 2×10⁶ BM cells from primary recipients of each group were injected in a noncompetitive fashion into lethally irradiated secondary recipients. Primary donor's chimera in secondary recipients is shown as 45.2⁺ mean frequency±SEM; *p<0.05 and **p<0.0001 vs. all other groups.