Isolation of Single Human Hematopoietic Stem Cells Capable

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 hown 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, Thyl and 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 Thyl expression in the CD34+CD38-CD45RA- compartment of lineage-depleted cord blood was ufficient 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 Thyl+ and Thyl- 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 Thyl subsets were sorted into

Thyl+ and Thy- cells and cultured with stroma cells known to express HSC supportive ligands. The results demonstrated that the Thyl- 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 distinguish HSCs in both Thy+ and Thy- subsets they analysed cell surface markers and found only CD49f to be differentially expressed between subsets.
Next they investigated whether HSCs could be identified using CD49f expression. Data recovered indicated that human HSCs are indeed marked by CD49f. Further tests were carried out on Thyl-CD49f+ and Thyl-CD49f- subsets. Results revealed Thyl-CD49f- cells are short term HSCs as they fail to engraft long-term, indicating that these are MPPs. Investigation into the role of mitochondrial dye rhodamine-123 (Rho) in HSC enrichment. Results indicated twofold enrichment for HSCs compared to Thyl+ alone as opposed to Thyl +Rholo.
The next aspect nvestigated was whether the addition of Rho to Thyl +CD49f+ would permit robust engraftment of single human HSCs. This investigation was carried out using single cell transplantation with two separate experiments focusing on engraftment and Thyl +RholoCD49f+ cells provides evidence that human HSCs express CD49f. These findings illustrated by Notta et al. will aid in identifying gene regulatory networks that govern human HSC function and therefore allow manipulation of human HSCs ex vivo with the aim of overcoming the barriers associated with transplantation.

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