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.
Authors: Sonoda Yoshiaki , Kimura Takafumi , Asada Rumiko , Kimura Takashi , Morioka Miho , Sasaki Yutaka , Ikehara Susumu 
Published: October 2019

Abstract
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.