Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Biology: Poster II
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-2Rγcnull (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).
Disclosures: No relevant conflicts of interest to declare.
See more of: Hematopoietic Stem and Progenitor Biology
See more of: Oral and Poster Abstracts
*signifies non-member of ASH