A novel competitive repopulation strategy to quantitate engraftment of ex vivo manipulated murine marrow cells in submyeloablated hosts

Abstract

Objective-Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts.Methods-Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation conditioned hosts using a "three-way" marking system, so that test, competitor and host cell chimerism could be reliably determined post-transplant.Results-We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced ~10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel "three-way" transplant assay.Conclusions-Murine marrow cells transduced using a clinically-applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.

Enhanced homing and engraftment of fresh but not ex vivo cultured murine marrow cells in submyeloablated hosts following CD26 inhibition by Diprotin A

“…We previously showed that ex vivo cultur for gamma-retroviral transduction results in decreased HSC engraftment in submyeloablated hosts [1, 18], and developed a novel competitive repopulation assay in submyeloablated hosts to quantify the engraftment defect [2]. Engraftment impairment, however, is not limited to submyeloablated hosts; the long-term repopulating ability of lin - marrow cultured for gamma-retroviral transduction, with or without exposure to gamma-retroviral supernatant, is also ~10-fold lower than that of fresh lin - marrow in ablated hosts (Figure 1).…”

“…Gamma-retroviral transduction was performed as previously described [2] as adapted from Li and colleagues [17]. Briefly, lineage depleted marrow was prepared using a mouse Lineage Cell Depletion kit and VarioMACS apparatus (Miltenyi Biotec, Auburn, CA, USA).…”

“…Competitive repopulation assays in 1100 cGy-conditioned hosts were performed as described [2]. Peripheral blood total donor chimerism was determined monthly for 4-6 months post-transplant using antibodies to CD45.1 and CD45.2 [2]. The fraction of donor-derived cells expressing the transgene was determined as described above.…”

“…We previously reported that gamma-retrovirus-transduced murine marrow cells acquire an engraftment defect leading to ~three-fold lower engraftment in 160 cGy-conditioned hosts than fresh marrow cells, despite the transduced cell population being enriched for primitive-phenotype marrow cells [1]. More recently, we developed a quantitative submyeloablative competitive repopulation assay and showed that murine marrow cells transduced using two gene-transfer protocols (a standard research protocol and a clinically-applicable approach) engraft ~10-fold less well than freshly-isolated marrow cells in submyeloablated hosts [2]. However, the mechanism(s) responsible for this engraftment defect remains unclear.…”

T Cell and B Cell Immunity can be Reconstituted with Mismatched Hematopoietic Stem Cell Transplantation Without Alkylator Therapy in Artemis-Deficient Mice Using Anti-Natural Killer Cell Antibody and Photochemically Treated Sensitized Donor T Cells

“…Competitive repopulation experiments were performed by co-injecting into the dorsal tail vein of the irradiated recipients equal numbers of unmanupulated mArt -/- B6 HSC or mArt -/- B6 HSC from animals which had been previously injected with 4×10 5 BALB/c PCT-4 STC 24 hours earlier, combined with naïve WT BALB/c bone marrow cells [25]. At 2 and 4 months post transplant, peripheral blood was obtained from the lateral saphenous vein of the recipients and stained by using fluorescently conjugated antibodies to BALB/c (anti-H-2 d ) and B6 (anti-H-2 b )[16].…”