Ex vivo expansion of hematopoietic stem cells (HSC) is a major challenge for clinical and experimental transplantation protocols. However, no significant clinical benefit has been demonstrated to date. Clonal kinetics of ex vivo-expanded HSCs is one of the basic transplantation biology questions to be addressed before we can optimize ex vivo expansion approaches. To characterize human HSC, xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSC, LDA is a statistical method and does not directly establish that a single HSC has self renewed in vitro. By using lentiviral gene-marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro.
To detect multiplied clones, 5x104 gene-marked CD34+ cells were cultured for 4 days in our ex vivo expansion culture system (
Exp Hematol, 27:904–915, 1999
), and then divided into 10 lots, each of which was transplanted directly into the bone marrow of a NOD/SCID mouse. We used linear amplification-mediated (LAM)-PCR to detect unique genomic-proviral junctions as clonal markers. Detection of the same clones in different mice would provide direct evidence of ex vivo multiplication of a SRC clone. We identified 20 clone-specific genomic-proviral junction sequences by LAM-PCR on 10 mice. Although 14 clones were detected in only one mouse, six clones were detected in more than 2 mice. In the next experiment, purified CD19+EGFP+ and CD33+EGFP+ cells from each mouse were analyzed for each clone to detect multi-lineage differentiation of amplified SRCs. We identified 15 clonal markers from 6 mice. While 12 clones were present in only one mouse, 3 clones were present in 2 independent mice and reconstituted both CD19+and CD33+cells. Finally, we designed a secondary transplantation experiment to confirm the self-renewal ability of each clone. We identified 39 clonal markers from 10 primary and 10 secondary transplanted mice, 11 of which were detected in multiple mice with secondary transplantable ability. Together, of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than two mice after serum-free and stroma-dependent culture. Some of them were secondary transplantable. Furthermore, we identified new class of stem cells based not on repopulation, or cell surface markers, but on response to cytokine stimulation in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.
