Human hematopoietic stem cells (HSCs) and progenitors can be isolated by enriching for a rare cell population with a combination of monoclonal antibodies (MAbs). Such an isolation scheme involves multi-step procedures including ficoll-density fractionation and presort enrichment followed by cell sorting. Over the past decade, various cell-surface and metabolic markers have been identified and used to isolate human HSCs and progenitors as summarized in Table 1. Among them, CD34 has become the most critical cell-surface marker for positively selecting a rare cell population (1,2). Within the CD34+ cell population, the differential expression of Thy-1, CD38, and AC133 have been used to fractionate HSCs and progenitors. In order to subfractionate CD34+ cells by these markers, the cells can be further purified by flow cytometry. HSCs can be further enriched into a Thy-1+ (3–7), CD38-lo (8–10), Thy-1+ CD38-lo (11), or AC133+ (12,13) fraction of CD34+ cells.
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Table 1 |
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Commercially Available Cell-Surface and Metabolic Markers for Isolation of Human HSC and Progenitor Cells |
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Marker |
Expression/Remark |
Fluorochrome conjugate recommended |
Reference |
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Positive marker |
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CD34 |
Positive |
FITC, PE, APC, BIO |
1,2,33 |
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Thy-1 |
Positive |
PE, BIO |
3,4 |
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AC133 |
Positive |
PE |
12,34 |
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Negative/low marker |
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CD38 |
Negative /low |
FITC, PE, APC |
8,9 |
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HLA-DRa |
Negative to low |
FITC, PE |
35,36 |
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Mature lineage marker, Lin- |
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CD2 |
T-cell lineage |
FITC, PE, BIO |
3 |
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CD3 |
T-cell lineage |
FITC, PE, APC, BIO |
3 |
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CD19 |
B-cell lineage |
FITC, PE, APC, BIO |
3 |
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CD16 |
NK-cell lineage |
FITC, PE, APC, BIO |
3 |
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CD14 |
Myeloid lineage |
FITC, PE |
3 |
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CD15 |
Myeloid lineage |
FITC, PE |
3 |
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Glycophorin A |
Erythroid lineage |
FITC, PE |
3 |
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2nd Step reagent |
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Avidin/Streptavidin |
For BIO MAb |
FITC, PE, APC, TXRD, PharRed, Cy-chromed |
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Metabolic markerb |
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Rhodamine 123c |
Low |
Mitochondria-binding dye |
37,38 |
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Hoechst 33342c |
Low |
DNA-binding dye |
39,40 |
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Pyronin Y |
Low |
RNA-binding dye |
39,40 |
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Propidium iodide |
Negative to low |
Dead-cell exclusion |
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Abbreviations: FBM, fetal bone marrow; MPB, mobilized peripheral blood; ABM, adult bone marrow; HSC, hematopoietic stem cells; FITC, fluorescein; PE, phycoerythrin; APC, allophycocyanin; TXRD, Texas red; BIO, biotinylated.aFBM, MPB HSCs express HLA-DR(41,42).bTo isolate quiescent HSC.cSubstrates for p-glycoprotein, encoded by MDR-1. HSC possess high levels of p-glycoprotein efflux activity.dRecommended for single laser flow cytometry only, lineage marker positive and PI positive cells can be excluded simultaneously. |
