Prospective Isolation and Functional Characterization of Human Bone Marrow-Derived Hematopoietic Stem Cell-Supportive Mesenchymal Stromal Cells.

The identification of human CD34-negative (CD34⁻) SCID-repopulating cells (SRCs) provide a new concept for the hierarchy in the human HSC compartment (Blood 101:2924, 2003). Recently, we succeeded to highly purify these CD34-SRCs using 18 lineage specific antibodies (Blood 114:336, 2009). It has been suggested that human hematopoietic stem cell (HSC)-supportive microenvironment exist in the bone marrow (BM), which play a pivotal role in the maintenance of self-renewal capacity and dormancy of primitive HSCs. It was reported that osteoblasts and vascular endothelial cells played an important role to organize HSC niches. However, whether mesenchymal stromal cells (MSCs) contribute to organize HSC niches or not is not clearly understood, because MSCs are heterogeneous population. Therefore, it is important to clarify their origin and functional characteristics.

The aim of this study was to prospectively isolate/identify human BM-derived MSCs and investigate their functional characteristics including HSC-supportive abilities.

First, human BM-derived Lin⁻CD45⁻ cells were subdivided into 4 fractions according to their expression levels of CD271 and SSEA-4 by FACS. We succeeded to isolate 3 MSC lines from these 4 fractions, including CD271^+/−SSEA-4^+/− cells. Approximately 1 out of 6 CD271⁺SSEA-4⁺ (DP) cells could form MSC-derived colony. These DP cells-derived MSCs could differentiate into osteoblasts and chondrocytes, but could not differentiate into adipocytes. In contrast, CD271⁺SSEA-4⁻ cells and CD271⁻SSEA-4⁻ cells-derived MSCs could differentiate into three lineages. Then, we assessed CD34⁻ SRC-supportive activity of these 3 MSC lines. First, certain numbers of 18Lin⁻CD34⁻ cells were cocultured with 3 MSC lines for 1 week, respectively. Next recovered cells were transplanted into NOD/SCID mice by intra-bone marrow injection (IBMI) to investigate SCID-repopulating cell (SRC) activity. After 8 weeks, the highest CD45⁺ human cell engraftments (0.1 % to 32.4 %, median 8.6 %) were observed in mice received 18Lin⁻CD34⁻ cells cocultued with DP cells-derived MSCs. As recently reported (Cell Stem Cell 1:635,2007), Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺ cells contained most primitive human CD34⁺CD38⁻ SRCs. Very interestingly, these Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺ cells were generated from the above mentioned cocultures. In order to evaluate SRC activity of these Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺ cells generated from 18Lin⁻CD34⁻ cells in vitro, Lin⁻CD34⁺CD38⁻CD45RA⁻CD90^+/− cells were sorted by FACS and then transplanted into NOD/SCID mice by IBMI. Eight weeks after transplantation, 8 out of 16 mice received Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺ cells (400 to 3000 cells/mouse) were repopulated with human cells. In contrast, only 2 out of 16 mice received Lin⁻CD34⁺CD38⁻ CD45RA⁻CD90⁻ cells (1500 to 7000 cells/mouse) were repopulated. These results demonstrated that human CB-derived 18Lin⁻CD34⁻ cells could generate very primitive CD34⁺CD38⁻ SRCs in vitro.

These findings elucidate that human BM-derived DP cell-derived MSCs can support very primitive human CB-derived CD34⁻ SRCs in vitro and suggest that these CD34⁻ SRCs seem to be more immature than CD34⁺CD38⁻ SRCs. These results provide a new concept of hierarchy in the human primitive HSC compartment.

No relevant conflicts of interest to declare.