Using the intra-bone marrow injection (IBMI) method, we have identified human cord blood (CB)-derived CD34-negative (CD34) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with multi-lineage repopulating ability (

Blood 101:2924,2003
). Functional studies revealed that these CD34 SRCs have different hematopoietic stem cell (HSC) characteristics from CD34+ SRCs. In order to further clarify the HSC characteristics of CD34 SRCs, here we investigate the proliferative potential and redistribution kinetics of human CB-derived CD34 SRCs, and compare them with those of CD34+CD38+/− SRCs using IBMI. First, we performed limiting dilution analyses and revealed that the incidence of CD34+CD38 SRCs in CB-derived LinCD34+CD38 cells was 1 out of 41 cells by IBMI. In contrast, the incidence of CD34 SRCs in LinCD34 cells was 1 out of 24,100, as we previously reported. Based on these data, we transplanted 200 to 5,000 LinCD34+CD38 cells (containing 5 to 120 SRCs), 15,000 to 50,000 LinCD34+CD38+ cells (containing 10 to 30 SRCs), or 60,000 to 70,000 LinCD34 cells (containing 3 SRCs) into primary recipient NOD/Shi-scid mice. After 5 weeks, all mice that received transplants of LinCD34+CD38+/− cells showed the human CD45+ cell repopulation in the other bones as well as the injected left tibiae. However, the human CD45+ cells were only detected in the injected left tibiae in mice that received transplants of LinCD34 cells 5 weeks after the transplantation. In the mice that received transplants of 200 LinCD34+CD38 cells (containing 5 SRCs), the CD45+CD34+ as well as CD45+CD34 cells were detected in both sites. In contrast, only CD45+CD34 cells were detected in the mice that received transplants of 70,000 LinCD34 cells (3 SRCs). These results suggested that CD34 SRCs might remain or slowly proliferate as CD34 cells at the site of injection for at least 5 weeks. Next, we serially investigated the human CD45+ cell repopulation in the injected site and the other bones, separately. Very interestingly, CD34+CD38+/− SRCs began to migrate 2 weeks after the transplantation. The human cell repopulation in these mice was observed in other bones by 3 weeks after transplantation. Moreover, these CD34+ SRCs actively proliferated at both sites and produced CD34+ progenies. In contrast, CD34 SRCs began to migrate 5 weeks after the transplantaion. Furthermore, these CD34 SRCs showed significantly higher proliferative potential 8 weeks after transplantation than CD34+ SRCs and produced more CD34+ progenies not only at the site of injection, but also in the other bones. These results indicated that CD34 SRC as well as CD34+CD38+/− SRCs could actively migrate from the injected site to the other bones. However, the time of initiation of migration was different between CD34+/− SRCs. All these findings indicate that CD34 SRCs show different proliferative potential and redistribution kinetics, and suggest that our identified CD34 SRCs are distinct class of primitive HSCs in comparison with CD34+CD38+/− SRCs. We are now in the progress of clarifying whether the CD34 SRCs migrate to other bones with the CD34 immunophenotype or after their conversion (differentiation) to the CD34+ cells.

Disclosure: No relevant conflicts of interest to declare.

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