GPI-80 expression highly purifies human cord blood–derived primitive CD34-negative hematopoietic stem cells

To the editor:

We identified very primitive CD34-negative (CD34⁻) severe combined immunodeficiency–repopulating cells (SRCs) in human cord blood (CB) using the intrabone marrow injection method¹  and provided a new concept for the hierarchy in the human hematopoietic stem cell (HSC) compartment.^2,3  However, the incidence of CD34⁻ SRC in 13 lineage-negative (Lin⁻) CD34⁻ cells (1/25 000) was very low.¹  We then developed a high-resolution purification method for CD34⁻ SRCs using 18Lin-specific antibodies, which can enrich CD34⁻ SRC at the 1/1000 level in 18Lin⁻CD34⁻ fraction.⁴  In addition, we recently identified CD133 as a positive marker for CD34⁻ as well as CD34⁺ SRCs.⁵  A limiting dilution analysis (LDA) demonstrated that CD34^+/− SRCs were enriched to ∼1/100 and 1/140 in 18Lin⁻CD34^+/−CD133⁺ fractions, respectively.^3,5  Our goal is to purify CD34⁻ HSCs to the single-cell level, in order to further elucidate the details of the characteristics of human CB-derived CD34⁻ HSCs. It was therefore necessary to identify additional specific positive/enrichment markers for CD34⁻ HSCs. Until recently, several investigators have reported the usefulness of CD49f, CD93, and CD82 in the enrichment of human CB- and bone marrow (BM)-derived CD34^+/− HSCs.^6-8 

Using the above-mentioned 18Lin⁻CD34⁻ cell population, we extensively analyzed the expression of candidate markers by flow cytometry (FCM). Finally, we demonstrated that a glycosylphosphatidylinositol-anchored protein GPI-80, which had originally been reported to regulate neutrophil adherence and migration,^9,10  was also expressed on human full-term CB-derived 18Lin⁻CD34⁺CD38⁻ and 18Lin⁻CD34⁻ cells. Interestingly, human CB-derived CD34⁻ SRCs were highly enriched in the 18Lin⁻CD34⁻GPI-80⁺ cell fraction.

Details of the experimental methods can be found in the supplemental Data, available on the Blood Web site. First, we extensively explored candidate positive/enrichment markers, which could be useful for the efficient purification of rare CB-derived CD34⁻ SRCs (HSCs), including known HSC markers and various adhesion molecules, using highly purified CB-derived 18Lin⁻CD34^+/− cells⁴  by multicolor FCM. Out of the 25 molecules that were analyzed, GPI-80, c-kit, CD90, CXCR4, CD49f, and CD93 were expressed on both types of cells. The 18Lin⁻CD34^+/− cells could be subdivided into positive and negative fractions (supplemental Figure 1A). In order to select an appropriate marker from these candidate molecules, we back-gated the positive fractions of these markers in 18Lin⁻CD34⁻ cells into FSC/SSC scattergrams. As clearly seen in supplemental Figure 1B, the distribution of c-kit⁺, CD90⁺, CXCR4⁺, CD49f⁺, and CD93⁺ cells in the 18Lin⁻CD34⁻ cells was scattered inside and around the blast window. In contrast, most of the 18Lin⁻CD34⁻GPI-80⁺ cells were concentrated in the low FSC blast window. Based on these data, we selected GPI-80 as a candidate positive/enrichment marker for human CB-derived CD34⁻ SRCs (HSCs). We then purified 18Lin⁻CD34⁺CD38⁻GPI-80⁺ and 18Lin⁻CD34⁻GPI-80⁺ cells, as shown in Figure 1Av-vi. Approximately 5% to 30% of the 18Lin⁻CD34⁺CD38⁻ and 18Lin⁻CD34⁻ cells expressed GPI-80. These 18Lin⁻CD34⁺CD38⁻GPI-80^+/− and 18Lin⁻CD34⁻GPI-80^+/−cells were then sorted for the following in vivo as well as in vitro experiments.

Using the 4 above-mentioned cell fractions, we performed an LDA to demonstrate the frequency of the CD34^+/− SRCs. As shown in Figure 1Bi-iv and supplemental Table 2, the incidences of SRCs in these 4 cell fractions were as follows: 1/20.7 in 18Lin⁻CD34⁺CD38⁻GPI-80⁺ cells; 1/35.4 in 18Lin⁻CD34⁺CD38⁻GPI-80⁻ cells; 1/27.5 in 18Lin⁻CD34⁻GPI-80⁺ cells; and 1/874 in 18Lin⁻CD34⁻GPI-80⁻ cells. These results clearly demonstrated that GPI-80 expression highly purified the CD34⁻ SRCs and achieved a purification level of ∼1000 times higher than the initial incidence (1/25 000) of CD34⁻ HSCs.¹  This purification level (1/27.5) is the highest to date.

We next analyzed the long-term multilineage reconstitution abilities of CD34⁺CD38⁻GPI-80⁺ and CD34⁻GPI-80⁺ SRCs. In these experiments, all of the mice that received transplants of 200 18Lin⁻CD34⁺CD38⁻GPI-80⁺ cells containing 9.7 SRCs and 200 18Lin⁻CD34⁻GPI-80⁺ cells containing 7.3 SRCs showed distinct human cell repopulation at 20 weeks after transplantation (supplemental Table 3). The level of human cell repopulation ranged from 9.3% to 84.7% (median, 66.2%) for 18Lin⁻CD34⁺CD38⁻GPI-80⁺ cells and from 0.7% to 52.8% (median, 31.9%) for 18Lin⁻CD34⁻GPI-80⁺ cells.

To further evaluate the functional differences between the CD34⁺CD38⁻GPI-80⁺ and CD34⁻GPI-80⁺ SRCs, we studied their multilineage reconstitution abilities in the BM, peripheral blood, spleen, and thymus, in the above-mentioned NOG mice. The results of the repopulation patterns are precisely shown in supplemental Figure 2. An analysis of the BM of the 2 representative mice that received either (supplemental Figure 2A) CD34⁺CD38⁻GPI-80⁺ SRC or (supplemental Figure 2B) CD34⁻GPI-80⁺ SRC demonstrated that both of the SRCs have an in vivo differentiation capacity that was comparable to that of CD34⁺ stem/progenitor cells, CD19⁺ B-lymphoids, CD33⁺ myeloids, CD11b⁺ monocytes, CD235a⁺ erythroid, and CD41⁺ megakaryocytic lineages. The CD56⁺ NK cells were detected in the spleen in both NOG mice. The CD3⁺ T cells were also detected in the thymi in both NOG mice. These results confirmed that both the CD34⁺CD38⁻GPI-80⁺ and the CD34⁻GPI-80⁺ SRCs had multilineage differentiation potential.

Next, we cocultured CB-derived 18Lin⁻CD34⁺CD38⁻GPI-80^+/− and 18Lin⁻CD34⁻GPI-80^+/− cells with human BM-derived mesenchymal stromal cells (abbreviated as DP MSCs).¹¹  As shown in supplemental Figure 3, both 18Lin⁻CD34⁺CD38⁻GPI-80⁺ and 18Lin⁻CD34⁻GPI-80⁺ cells could generate 12Lin⁻CD45RA⁻CD34⁺CD38⁻GPI-80^+/− (abbreviated as 34⁺38⁻80^+/−) cells. However, 18Lin⁻CD34⁺CD38⁻GPI-80⁻ and 18Lin⁻CD34⁻GPI-80⁻ cells could only generate 34⁺38⁻80⁻ cells. Moreover, the former 34⁺38⁻80^+/− cells showed distinct SRC activities. All of the recipient mice (6/6) showed multilineage human cell repopulation (supplemental Table 4). In contrast, the latter 34⁺38⁻80⁻ cells showed weak SRC activities. In other words, some of the recipient mice (3/6 for the 18Lin⁻CD34⁺CD38⁻GPI-80⁻ cell-derived 34⁺38⁻80⁻ cells and 1/6 for the 18Lin⁻CD34⁻GPI-80⁻ cell-derived 34⁺38⁻80⁻ cells) were repopulated with human cells. However, the repopulation rates were low in comparison with their GPI-80⁺ counterparts. All 4 types of input cells generated 12Lin⁻CD45RA⁻CD34⁻GPI-80⁻ (abbreviated as 34⁻80⁻) cells (supplemental Figure S3). However, none of the mice that received these 34⁻80⁻ cells were repopulated with human cells (supplemental Figure 3 and supplemental Table 4). Overall, CD34^+/−GPI-80⁺ SRCs could generate/maintain CD34⁺GPI-80^+/− SRCs, but not CD34⁻GPI-80^+/− SRCs in the cocultures with DP MSCs. In contrast, CD34^+/−GPI-80⁻ SRCs could not generate/maintain CD34^+/−GPI-80⁺ SRCs in cocultures with DP MSCs. They could only generate/maintain CD34⁺GPI-80⁻ SRCs in cocultures with DP MSCs. These results suggest that CD34^+/−GPI-80⁺ SRCs seem to be more immature than CD34^+/−GPI-80⁻ SRCs in the human HSC hierarchy. In other words, GPI-80 defines human CB-derived primitive HSCs that can maintain SRC activity in cocultures with DP MSCs.

In conclusion, these observations clearly demonstrated that GPI-80 is a useful enrichment marker for the high-level purification of human CB-derived CD34^+/− SRCs (HSCs). GPI-80 has very recently been reported as a positive marker for human fetal liver hematopoietic stem/progenitor cells (HSPCs), and only CD34⁺CD38^lo/−CD90⁺GPI-80⁺ cells have shown SRC activity.¹²  Because human placenta/CB-derived HSPCs reflect fetal hematopoiesis,^13-15  it is conceivable that GPI-80 is expressed on CB-derived primitive HSCs. From another point of view, leukemia-initiating cells (LICs) were detected in the CD34⁻ cell fractions of leukemia patients.^16-18  In order to eradicate these chemotherapy-resistant CD34⁻ LICs, it is necessary to identify useful markers as targets for molecular-targeting therapy. Thus, it would be interesting to analyze whether these CD34⁻ LICs express GPI-80. It is also interesting to note that both GPI-80 and CD133 were correlated with the polarization and migration of leukocytes and HSPCs.^9,10,19,20  Because our goal is to purify CD34^+/− HSCs to the single-cell level in order to elucidate key molecules/signals for maintaining their stemness, the next step is to combine GPI-80 with CD133.⁵  Using these 2 markers, we are now working to develop a method that achieves the ultra-high-resolution purification of human CB-derived CD34^+/− SRCs (HSCs).²¹