Different Proliferative Potential and Redistribution Kinetics of Human Cord Blood-Derived CD34 - SCID-Repopulating Cells (SRCs) in Comarison with CD34 + CD38 +/− SRCs Using Intra-Bone Marrow Injection.

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 (Blood101:2924,2003). 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 Lin − CD34 + CD38 − cells was 1 out of 41 cells by IBMI. In contrast, the incidence of CD34 − SRCs in Lin − CD34 − cells was 1 out of 24,100, as we previously reported. Based on these data, we transplanted 200 to 5,000 Lin − CD34 + CD38 − cells (containing 5 to 120 SRCs), 15,000 to 50,000 Lin − CD34 + CD38 + cells (containing 10 to 30 SRCs), or 60,000 to 70,000 Lin − CD34 − cells (containing 3 SRCs) into primary recipient NOD/Shi-scid mice. After 5 weeks, all mice that received transplants of Lin − CD34 + 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 Lin − CD34 − cells 5 weeks after the transplantation. In the mice that received transplants of 200 Lin − CD34 + 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 Lin − CD34 − 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.