The multilineage differentiation and self-renewal of HSC represented by a single SRC are yet to be proven. To analyze the multilineage differentiation capacity of individually transduced SRCs, we performed in vivo virus integration site analysis by LAM-PCR. Based on the genomic sequence information of the LAM-PCR products of CD4/CD8 double positive (DP)-thymocytes, we designed primers corresponding to individual integration sites. Using these primers that were unique to each clone, we were able to track the individual clones and their progenies, CD34+ stem/progenitor, myeloid and B-lymphoid cells. The majority of SRC clones found in the recipient mice were p-MTB multilineage type, in which insertion sites originally detected in DP cells were also detected in highly purified myeloid and B-lymphoid cell population. All p-MTB clones were found to contain CD34+ cell population, which suggested that those SRC clones replicated within the stem cell pool without loosing their ability during long-term hematopoiesis. On the other hand, as the differentiation ability of clone became limited to bipotent (p-TB) or unipotent (p-T), the proportion of clones that was common to CD34+ cells decreased, which indicated that some SRC clones had exhausted from the stem cell pool during lineage commitment. To demonstrate the self-renewal ability of SRCs, we injected BM samples from each primary mouse into two secondary mice. PCR tracking analysis was then performed to examine the fate of individual SRC clones in paired secondary mice using insertion sites found in DP cells of each secondary recipient. In most secondary recipient pairs, at least one of the two clones inherited p-MTB differentiation potential from its parent cell. The other daughter clone either remained as p-MTB clone or became committed to specific lineages. Since clones detected in paired secondary recipients were also detected in the primary donor, these observations confirmed that the multilineage repopulating SRC clone underwent self-renew. Existence of common p-MTB clones in both of paired secondary recipients indicates expansion of multipotent SRC clones. We found that although the same multipotent SRC clone was detected in paired secondary recipients, less than half of them retained stem cell phenotype, determined by the presence of common integration site in CD34+ cell populations. In approximately half of p-MTB clone pairs found in secondary paired recipients, the clones divided asymmetrically, leaving only one of the pair to have stem cell phenotype. Moreover, stem cell phenotype was not retained in 11.1% of p-MTB clone pairs. Considering that 100% of p-MTB clones originally found in primary recipient possessed stem cell phenotype, these results indicate that SRCs with stem cell phenotype progressively decrease during serial transplantation process, leading to exhaustion of SRCs. Our data indicated that even though the number of total SRC population appears to expand, the ability of individual SRCs might be restricted during long-term hematopoiesis.

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