Identification and Long-Term Repopulating Potential of Human Cord Blood-Derived CD34−Flt3− Cells with Very Immature Phenotype Using Intra-Bone Marrow Injection.
Authors: Sonoda Yoshiaki , Kimura Takafumi , Asada Rumiko , Wang Jianfeng , Kimura Takashi , Ishihara Hiroyasu , Kouda Katsuyasu , Sasaki Yutaka , Tsuji Takashi , Ikehara Susumu 
Published: April 2020

Abstract
Recently, we have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood101:2924,2003). These CD34− SRCs could home into the BM niche only by IBMI, because they expressed lower levels of homing receptors including CXCR4. These CD34− SRCs did not express CD38 as well as c-kit. It is well documented that the tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. In murine model, it was reported that Lin−CD34−Sca−1+c-kit+flt3− cells supported long-term multilineage hematopoietic reconstitution. In contrast, Lin−CD34−Sca-1+c-kit+flt3+ cells are progenitors for the common lymphoid progenitor. More recently, these Lin−CD34−Sca-1+c-kit+flt3+ hematopoietic stem cells (HSCs) have been revealed to lack erythro-megakaryocytic potential. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34− SRCs. First, we studied the SRC activity of CB-derived Lin-CD34+Flt3+/− or CD34−Flt3+/− cells using IBMI. Both CD34+FLT3+/− cells repopulated all 13 recipient mice. The level of human CD45+ cells in murine BMs received transplants of CD34+Flt3+ cells (29.3~90.8%, median 62.8%) was higher than those received transplants of CD34+Flt3− cells (9.8~45.1%, median 17.7%). On the other hand, only CD34−Flt3− cells repopulated all 7 recipient mice and the level of human CD45+ cells in murine BMs was 11.7~63.3% (median 37.9%). To further evaluate the long-term repopulating potential of these three populations, including CD34+Flt3+/− and CD34−Flt3− cells, BM cells obtained from each primary recipient mice were accessed for their SRC activities by secondary transplantation by IBMI. While CD34+Flt3+ cells did not show secondary repopulating activity, CD34+Flt3− cells could repopulate 83% (5/6) of secondary recipients. Moreover, CD34−Flt3− cells did repopulate all 5 secondary recipient mice with higher repopulating rate. Next, we cocultured CD34−Flt3−cells with the murine stromal cell line, HESS-5 in the presence of SCF, TPO, IL-3, IL-6, and G-CSF. After one week, significant numbers of CD34+Flt3− and CD34+Flt3+ cells were generated. Then we sorted these two populations, CD34+Flt3+/− cells, and tested their SRC activities by IBMI. Seven out of 10 and 5 out of 10 mice received CD34+Flt3+/− cells were repopulated with human cells, respectively. These results indicated that human CB-derived Lin−CD34−Flt3− cells produced CD34+Flt3− as well as CD34+Flt3+ SRCs in vitro. Our present study has demonstrated that human CB-drived CD34− SRCs do not express Flt3 tyrosine kinase receptor as did murine CD34− KSL cells. Based on these data, we propose that the immunophenotype of very primitive long-term repopulating human HSC is Lin−CD34−CD38−c-kit-Flt3−.