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

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. MATERIALS AND 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 posttransplantation. RESULTS: We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced approximately 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.

Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model.

Dental pulp stem cells (DPSCs) have drawn much interest for the regeneration of mineralized tissues, and several studies have compared DPSCs to bone marrow-derived mesenchymal stem cells (BMMSCs). However, conflicting results, possibly due to donor-associated variability, have been published and the regenerative potential of DPSCs is currently unclear. In the present study we have sought to address this problem using a donor-matched experimental design to robustly compare the biological properties of DPSCs and BMMSCs. All experiments were performed using cells isolated from a single adult Sprague-Dawley rat. Our results show that DPSCs and BMMSCs had similar morphologies and flow cytometry profiles, were capable of forming colonies in vitro and were capable of osteogenic, chondrogenic and adipogenic differentiation. However, quantitative comparisons revealed that DPSCs had a faster population doubling time and a higher percentage of stem/progenitor cells in the population, as determined by clonogenic assays. Furthermore, while both cell populations formed mineral in vitro, DPSCs had significantly higher alkaline phosphatase activity than BMMSCs after 3 weeks in osteogenic medium. These data show several key differences between DPSCs and BMMSCs and support the possibility of using DPSCs for mineralized tissue regeneration.

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

OBJECTIVE: We recently reported that murine marrow cultured ex vivo for gamma-retrovirus transduction engrafts approximately 10-fold less well than fresh marrow upon transplantation into submyeloablated hosts. Here, we evaluated homing efficiency as a potential mechanism for this engraftment disparity, and whether CD26 inhibition with the tripeptide Diprotin A (DipA) would enhance engraftment of ex vivo cultured cells in submyeloablated hosts. MATERIALS AND METHODS: Homing and engraftment of fresh and ex vivo cultured lineage-negative (lin(-)) marrow cells in submyeloablated congenic hosts with and without DipA treatment was evaluated. Expression of CXCR4 and CD26 on fresh and cultured lin(-) marrow cells was compared. RESULTS: Homing of lin(-) cells cultured for gamma-retrovirus transduction was at least threefold less than that of fresh lin(-) cells 20 hours after transplantation into submyeloablated hosts. DipA treatment of fresh lin(-) cells resulted in at least twofold increased homing and engraftment in submyeloablated hosts. DipA treatment, however, did not significantly improve homing or engraftment of cells undergoing a 3-day culture protocol for gamma-retrovirus transduction in submyeloablated hosts. CXCR4 expression on lin(-) cells was significantly decreased following 3 days of culture; CXCR4 expression was not significantly altered following overnight culture. CONCLUSIONS: Ex vivo culture of lin(-) cells for gamma-retroviral transduction downregulates CXCR4 expression and markedly impairs homing and engraftment of murine lin(-) marrow in submyeloablated hosts. While inhibition of CD26 activity with DipA increases homing and engraftment of fresh lin(-) cells, DipA treatment does not improve homing and engraftment of cultured lin(-) marrow cells in submyeloablated congenic hosts.