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.


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.
Wyss
Brandon K
BK
Herman B Wells Center for Pediatric Research.
Meyers
Justin L
JL
Sinn
Anthony L
AL
Cai
Shanbao
S
Pollok
Karen E
KE
Goebel
W Scott
WS
D000818:Animals; D001681:Biological Assay / Q000379:methods; D001854:Bone Marrow Cells / Q000166:cytology*; D016026:Bone Marrow Transplantation; D002452:Cell Count; D018929:Cell Culture Techniques; D019070:Cell Lineage; D049109:Cell Proliferation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006085:Graft Survival; D006412:Hematopoietic Stem Cells / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D011828:Radiation Chimera; D011829:Radiation Dosage; D019172:Transplantation Conditioning