Th1+ and Thy1- stem cells expressing CD49f are capable of long-term, multilineage engraftment in mice.

Scientists claim to have identified markers that enable the segregation of rare, truly pluripotent human hematopoietic stem cells (HSCs) from multipotent progenitors (MPPS). They identified CD49f as a specific marker for pluripotent HSCs, enabling their isolation using flow cytometry.  

The team, at the Campbell Family Institute for Cancer Research/Ontario Cancer Institute, tracked the expression of a number of adhesion molecules in HSC-enriched subsets. They report their work in Science in a paper titled “Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment.”

Self-renewing human HSCs that are capable of giving rise to all the different cell types in the blood system are rare and hard to isolate from the mix of transient MPPS in stem cell populations, report John E. Dick, Ph.D., and colleagues. Although transplant and xenograft repopulation studies have suggested that the bulk of HSCs are CD34+, most CD34+ cells are actually lineage-restricted progenitors.

HSCs can be enriched further on the basis of CD45RA, Thy1, and CD38 expression. Previous results suggesting that Thy1 represents a marker for true HSCs is tenuous, however, the Ontario researchers claim.

To try and identify markers that more definitively segregate true HSCs from MPPs, the team optimized an HSC xenograft assay in experimental mice to functionally characterize  flow-sorted lineage-depleted cord blood HSCs (CD34+CD38CD45RAThy1+; designated as Thy1+)  and MPPs (CD34+CD38CD45RAThy1; designated as Thy1).  The main criterion for HSC function was defined by lymphomyeloid engraftment that persisted for at least 20 weeks after transplant, the authors note.

Interestingly, they found that Thy1 cells could be serially transplanted over long periods, albeit at lower efficiencies than Thy1+ cells but nevertheless suggesting that cells with extensive self-renewal potential exist in both Thy1+ and Thy1 subsets.

Limiting dilution analysis (LDA) studies showed that 1/20 Thy1+ cells clonally initiated long-term hematopoiesis in the mice, compared with 1/100 Thy1 cells. “Thus, although Thy1+ enriches for HSCs, long-term repopulating activity persists in the Thy1 fraction previously believed to represent MPPS,” the authors write.

Culturing both Thy1 and Thy+ cells with stromal cells threw up the unexpected finding that Thy1 cells consistently generated Thy1+ cells and that these Thy1+ cells demonstrated robust repopulating activity in mice 20 weeks after transplantation. In fact, engraftment and lineage potentials were identical for Thy1+ cells derived from either the originally Thy or the originally Thy1+ subfractions. Conversely, cells that remained Thy1 after being cultured on stroma did not sustain long-term grafts.

The researchers therefore extended their search for a different cell surface marker that could identify HSCs in both Thy1+ and Thy1 fractions. They compared the surface expression of several adhesion molecules in HSC-enriched (Thy1+) and -depleted (Thy1) fractions. Among the potential candidates, only ITGA6 (integrin α6, or CD49f ) was differentially expressed, with 50 -70% of Thy1+ cells, and 10-20% of Thy1 cells expressing CD49f.

The team then evaluated the capacity of CD49f and CD49+ Thy1+ cells for long-term multilineage chimerism in mouse recipients. This showed that the mean chimerism was 6.7-fold higher for Thy1+CD49f + cells than for Thy1+CD49f cells, and only Thy1+CD49f + cells could be serially transplanted. Importantly, LDA revealed that 9.5% of Thy1+CD49f + cells had long-term repopulating activity, compared with only 0.9% of the Thy1+CD49f cells.

Repeating the studies with CD49f and CD49f+ Thy1 cells, demonstrated that even some Thy1CF49+ cells were capable of reconstitution in mice, and 4.5% of cells in this fraction had long-term multilineage engraftment potential. No difference in lineage potential was observed between Thy1+CD49f + and Thy1CD49f + cells, although recipients of Thy1+CD49f+ cells tended to demonstrate higher levels of chimerism at similar cell doses.

By studying the peripheral blood of bone marrow and blood in mice transplanted with all four subsets of cells (Thy1+CD49+, Thy1CD49+, Thy1+CD49, and Thy1CD49), Dr. Dick’s team was further able to determine that Thy1CD49f cells can still give rise to all major hematopoietic lineages but fail to engraft longterm, indicating that these are bona fide MPPS, they write. More specifically, they suggest, “considering that Thy1CD49f cells differ from CD49f + HSCs solely in the ability to engraft durably, an alternate interpretation is that Thy1CD49f cells are short-term HSCs.”

Gene-expression analysis of CD49f+ and Thy1CD49f subsets found no major differences, although Thy1CD49f + HSCs displayed an intermediate pattern to Thy1+CD49f +HSCs and MPPs. In contrast, 70 differentially expressed genes segregated Thy1CD49f MPPs from the HSC subset. “These findings support our functional delineation of Thy1+CD49f + and Thy1CD49f + HSCs as distinct from Thy1CD49f MPPS and identify gene-expression changes associated with the earliest steps of human HSC differentiation,” the authors state. “The demarcation of human HSCs and MPPS will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell-based therapeutics.”

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