GPI-80 Defines Primitive Human Cord Blood-Derived CD34-Positive and Negative Hematopoietic Stem Cells

Background. We identified very primitive CD34-negative (CD34^-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) in human cord blood (CB) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). These CD34^- SRCs possess myeloid-biased differentiation potential, which suggests that they are a distinct class of primitive hematopoietic stem cell (HSC) and that they reside at the apex of the human HSC hierarchy (Blood Cancer J 2015:5;e290). We recently developed high-resolution purification methods for CD34^- SRCs using 18 Lineage (18Lin)-specific antibodies, which can enrich CD34^- SRC at the 1/1,000 level in 18Lin^-CD34^- fractions (Exp Hematol 2011:39:203). In addition, we previously identified CD133 as a positive marker for CD34^- SRCs as well as for CD34⁺ SRCs (Leukemia 2014:28;1308). The results showed that CD34^+/- SRCs were enriched to approximately 1/100 and 1/140 in 18Lin^-CD34^+/-CD133⁺fractions, respectively.

Aim. In order to further elucidate the details of the characteristics of human CD34^+/- HSCs, we aimed to identify additional positive markers for the high-level purification of CB-derived CD34^+/-SRCs.

Materials and Methods. First, we extensively analyzed the candidate positive markers, including cell adhesion molecules and homing receptors that are expressed on 18Lin^-CD34⁺CD38^- and 18Lin^-CD34^- cells by multicolor FACS. Finally, we discovered that glycosylphosphatidylinositol-anchored protein GPI-80, which has recently been reported as a marker for human fetal liver hematopoietic stem/progenitor cells (HSPCs) (Cell Stem Cell 2015:16;80), was also expressed on human full-term CB-derived 18Lin^-CD34⁺CD38^- and 18Lin^-CD34^- cells. Next, we sorted 18Lin^-CD34⁺CD38^-GPI-80^+/- and 18Lin^-CD34^-GPI-80^+/- cells from human CB. The HSPC characteristics of the 18Lin^-CD34⁺CD38^-GPI-80^+/- and 18Lin^-CD34^-GPI-80^+/- cells were assessed as follows: (1) the in vitro maintenance/production capacities of CD34⁺ and CD34⁺CD38^- cells were examined in co-cultures with mesenchymal stroma cells (MSCs) established from human bone marrow-derived CD45^-Lin^-CD271⁺SSEA-4⁺ cells (Stem Cells 2015:33;1554); (2) an SRC assay was performed using NOD/Shi-scid/IL-2Rγ_c^null (NOG) mice; (3) limiting dilution analyses (LDAs) were performed to determine the SRC frequencies in these four fractions of cells.

Results. In the CB-derived 18Lin^-CD34⁺CD38^- and 18Lin^-CD34^- fractions, 10.1% and 14.4% of cells expressed GPI-80, respectively. The 18Lin^-CD34⁺CD38^-GPI-80^+/- and 18Lin^-CD34^-GPI-80^+/- cells were then co-cultured with human MSCs for 7 days in the presence of SCF and TPO. As a  result, the 18Lin^-CD34⁺CD38^-GPI-80⁺ cells maintained significantly higher percentages of CD34⁺ (86.4%) and CD34⁺CD38^- cells (24.8%) in comparison to 18Lin^-CD34⁺CD38^-GPI-80^- cells (78.7% and 14.3%, respectively). However, 18Lin^-CD34^-GPI-80^+/- cells produced comparable levels of CD34⁺ (50.3% and 50.8%) and CD34⁺CD38^- cells (4.4% and 5.3%). These four fractions of cells were then transplanted into NOG mice using the IBMI method. All of these four fractions of cells showed long-term repopulating SRC activities with multi-lineage differentiation potential in mouse bone marrow. However, LDAs demonstrated that the frequencies of SRC in the 18Lin^-CD34⁺CD38^-GPI-80^+/- and 18Lin^-CD34^-GPI-80^+/- fractions were 1/21, 1/35 and 1/28, 1/874, respectively. These data clearly demonstrate that both CD34^+/- SRCs are enriched in GPI-80⁺ fractions. Surprisingly, a number of mice received a limited number of 18Lin^-CD34⁺CD38^-GPI-80⁺ (2 cells) and 18Lin^-CD34^-GPI-80⁺cells (10 cells), and thus also showed multi-lineage long-term human hematopoietic cell repopulation.

Conclusion. These observations clearly demonstrated that GPI-80 defines CB-derived human primitive HSCs. Furthermore, these results indicate that GPI-80 is a useful marker for the high-level purification of human CB-derived CD34^+/- SRCs (HSCs).