Clonal Evidence of Human Hematopoietic Stem Cell Hierarchy: Distinct SCID-Repopulating Cell Subsets Contribute to Various Phases of Hematopoiesis.

The SCID-repopulating cell (SRC) pool is shown to be heterogeneous and is composed of at least two distinct subsets; short-term and long-term repopulating cells (STRCs and LTRCs), which appear in different time points following transplantation. However, the precise characteristics and their relationships regarding the stem cell function remain elusive. To clarify the specific stem cell activity of each SRC clones that contribute to various stages of hematopoietic reconstitution, we examined the functional aspects of individual SRCs. To determine the repopulating dynamics of individual SRC clones in vivo, we traced the kinetics of individual SRC clones by LAM-PCR based virus integration site analysis. Individual SRC clones which repopulate in each NOG mouse that received EGFP-transduced fractionated CD34+ populations were analyzed at two time points. At 3 weeks after transplantation, BM cells were aspirated from tibia of each recipient, and at 18 weeks recipients were sacrificed and BM cells were recovered from 4 long bones. At each time point, EGFP-expressing human hematopoietic lineage cells were sorted for integration site analysis by LAM-PCR, and the fate of individual SRC clones in the same recipient was examined by clone-tracking analysis. Using primers that were designated based on the genomic sequence information of the CD33+ myeloid cell integration site, we clonally traced distribution of each clone in lineage cells; CD34+ stem/progenitor, T-, and B-lymphoid cells. We found that the early phase of hematopoietic reconstitution was attributed to transient myeloid-restricted clones which rapidly exhausted from the CD34+ stem cell pool. Interestingly, the multilineage cell-producing clones that were responsible for the later phase of hematopoiesis were distinct from the transient myeloid-restricted clones, and these clones continuously self-replicated in the CD34+ stem cell pool. Next, CD34+ cells from the primary recipients were divided into two secondary recipients, and the fate of individual SRC clones in different phases was traced using the paired secondary mice. One recipient was sacrificed at 3 weeks, and the other recipient was sacrificed at 18 weeks after secondary transplantation. First, clones that were detected at the early phase in one recipient were also detected at the later phase in the other recipient (80%). This is clonal evidence that LTRC in the primary recipient produces STRC as well as self-replicating secondary transplantable LTRC. Second, all clones in the secondary recipients were also detected in the primary donor; however, most of clones (68.3%) found in the primary recipients did not contribute to the secondary recipient. In addition, LTRC clones detected in the CD34+ stem cell pool of secondary recipient demonstrated much larger clone size compared to primary recipient. These indicated that the quiescent LTRC clones in the primary recipients were stimulated by transplantation, there by expanded clonally in the secondary recipient and contributed to the later phase of hematopoiesis. Our clonal tracking study clearly demonstrated that the hierarchical structure of the human HSC pool composed of distinct clonal subsets which were heterogeneous in the self-renewal capacity and differentiation ability.