SRC within the Lin-CD34+CD38+/Lo Population Possess Heterogeneous Migration, Repopulation, and Self-Renewal Potential.

Abstract The SCID repopulating cell (SRC) xenotransplant assay is a powerful tool for characterizing human hematopoietic stem cells. Injection of hematopoietic cells directly into the intrafemoral (IF) cavity along with injection of a neutralizing antibody against residual murine NK cells and macrophages provides additonal improvements to the method (ASH 2004). IF injection permitted identification of a novel rapid-SRC (R-SRC) within the Lin-CD34+CD38+/Lo population that generated an erythromyeloid graft within 2 weeks post-transplant (Nat Med 2003). We found that this population also provides multi-lineage engraftment at 6 weeks post-transplant raising the question of whether the R-SRC had self-renewal potential. Lentivector-mediated clonal tracking was used to determine the self-renewal capacity of the individual cells within the Lin-CD34+CD38+/Lo population. Clonal analysis in primary recipients injected by IF with 5 x 10e4 Lin-CD34+CD38+/Lo and analyzed at 6 weeks showed that a subset of clones present in the injected femur were found in other bones, indicating that some individual SRC had self-renewed in the injected femur and migrated to other hematopoietic tissues. To directly test for self-renewal of migrating and non-migrating SRC, the original injected femur and the other bones (non-injected femur, two tibias and the pelvis) from each primary mouse was injected by IF into two individual secondary mice, respectively. 1) At 6 weeks post-transplant, each cell source produced substantial secondary grafts establishing that the Lin-CD34+CD38+/Lo population contains SRC with self-renewal potential. 2) Clonal analysis revealed heterogeneous self-renewal properties of individual SRC found in the primary mice; some made major contributions to all hematopoietic territories of secondary mice while others did not engraft secondary mice. 3) Interestingly, in some cases clones were detected in secondary mice that had been below detection in the primary mouse, suggesting that upon transplant into primary mice the SRC either did not divide or if they divided they returned to quiescence. Secondary transplantation was a stimulus for their activation to produce a graft of differentiated progeny. 4) Cases were observed where an active clone was found in a secondary mouse (transplanted from the primary injected femur) that had been below detection within the primary injected femur. However, the non-injected bones from this primary mouse as well as secondary mice derived from these bones all contained that same clone. We conclude that upon IF injection this SRC underwent self-renewal divisions and some of these progeny migrated to other bones and established a graft and also self-renewed, while in the injected bone the SRC likely returned to quiescence, only to be reactivated by secondary transplant. 5) Evaluation of two secondary recipients derived from one primary injected femur at 3 weeks when the graft is mainly myeloerythroid and at 6 weeks when it is mainly B cell and myeloid demonstrated that different lineage compositions were initiated by the same stem cell. The combination of clonal marking and IF injection provides an unprecedented insight into the earliest steps of stem cell function following transplantation. Although our clonal analysis is ongoing, it appears that rapid self-renewal and migration following IF injection represents a hallmark of a primitive subclass of SRC. It is essential to gain insight into the complex composition of the human stem cell compartment to develop effective stem cell-based therapies.

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