Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment
Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment
A variety of distinct progenitors arising from self-renewing hematopoietic stem cells (HSCs) allow for the production of mature blood cell lineages. Human HSCs are poorly understood due to their rarity and difficulty to segregate them from multipotent progenitors (MPPS) to obtain a pure population for analysis. This study investigates the determining factors of HSCs. It appears that the majority of HSCs are CD34+, as shown by transplantation and xenograft repopulation assays, however most of these cells are lineage-restricted progenitors and HSCs are therefore rare. Enrichment of HSCs seems reliant on CD45RA, Thy1and CD38 expression. Further study into the role of each of these factors in HSC differentiation is required. In this study a range of assays were carried out in an attempt to identify and separate HSCs from MPPs. Recently it was shown that a depletion in Thy1 expression in the CD34+CD38-CD45RA- compartment of lineage-depleted cord blood was sufficient to separate HSCs from MPPs. However, further studies gave rise to concern surrounding this theory. They then used an optimized HSC xenograft assay and flow-sorted cord blood HSCs and MPPs into functionally characterized fractions. Data obtained from this assay suggested that cells with extensive self-renewal potential exist in both Thy1+ and Thy1- subsets. However, more extensive research was required to investigate the disparity in secondary transfer efficiency between subsets. To distinguish the cause of such disparity the Thy1 subsets were sorted into Thy1+ and Thy- cells and cultured with stroma cells known to express HSC supportive ligands. The results demonstrated that the Thy1- compartment is heterogeneous and contains a small fraction with repopulating activity and a larger fraction with MMP-like activity and therefore may account for the differences in efficiency between subsets. To further
A variety of distinct progenitors arising from self-renewing hematopoietic stem cells (HSCs) allow for the production of mature blood cell lineages. Human HSCs are poorly understood due to their rarity and difficulty to segregate them from multipotent progenitors (MPPS) to obtain a pure population for analysis. This study investigates the determining factors of HSCs. It appears that the majority of HSCs are CD34+, as shown by transplantation and xenograft repopulation assays, however most of these cells are lineage-restricted progenitors and HSCs are therefore rare. Enrichment of HSCs seems reliant on CD45RA, Thy1and CD38 expression. Further study into the role of each of these factors in HSC differentiation is required. In this study a range of assays were carried out in an attempt to identify and separate HSCs from MPPs. Recently it was shown that a depletion in Thy1 expression in the CD34+CD38-CD45RA- compartment of lineage-depleted cord blood was sufficient to separate HSCs from MPPs. However, further studies gave rise to concern surrounding this theory. They then used an optimized HSC xenograft assay and flow-sorted cord blood HSCs and MPPs into functionally characterized fractions. Data obtained from this assay suggested that cells with extensive self-renewal potential exist in both Thy1+ and Thy1- subsets. However, more extensive research was required to investigate the disparity in secondary transfer efficiency between subsets. To distinguish the cause of such disparity the Thy1 subsets were sorted into Thy1+ and Thy- cells and cultured with stroma cells known to express HSC supportive ligands. The results demonstrated that the Thy1- compartment is heterogeneous and contains a small fraction with repopulating activity and a larger fraction with MMP-like activity and therefore may account for the differences in efficiency between subsets. To further