Method for Purification of Human Hematopoietic Stem Cells by Flow Cytometry
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.
Table 1 | |||
Commercially Available Cell-Surface and Metabolic Markers for Isolation of Human HSC and Progenitor Cells | |||
Marker | Expression/Remark | Fluorochrome conjugate recommended | Reference |
Positive marker | |||
CD34 | Positive | FITC, PE, APC, BIO | 1,2,33 |
Thy-1 | Positive | PE, BIO | 3,4 |
AC133 | Positive | PE | 12,34 |
Negative/low marker | |||
CD38 | Negative /low | FITC, PE, APC | 8,9 |
HLA-DR a | Negative to low | FITC, PE | 35,36 |
Mature lineage marker, Lin- | |||
CD2 | T-cell lineage | FITC, PE, BIO | 3 |
CD3 | T-cell lineage | FITC, PE, APC, BIO | 3 |
CD19 | B-cell lineage | FITC, PE, APC, BIO | 3 |
CD16 | NK-cell lineage | FITC, PE, APC, BIO | 3 |
CD14 | Myeloid lineage | FITC, PE | 3 |
CD15 | Myeloid lineage | FITC, PE | 3 |
Glycophorin A | Erythroid lineage | FITC, PE | 3 |
2nd Step reagent | |||
Avidin/Streptavidin | For BIO MAb | FITC, PE, APC, TXRD, PharRed, Cy-chrome d | |
Metabolic marker b | |||
Rhodamine 123 c | Low | Mitochondria-binding dye | 37,38 |
Hoechst 33342 c | Low | DNA-binding dye | 39,40 |
Pyronin Y | Low | RNA-binding dye | 39,40 |
Propidium iodide | Negative to low | Dead-cell exclusion | |
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. a FBM, MPB HSCs express HLA-DR (41,42). b To isolate quiescent HSC. c Substrates for p-glycoprotein, encoded by MDR-1. HSC possess high levels of p-glycoprotein efflux activity. d Recommended for single laser flow cytometry only, lineage marker positive and PI positive cells can be excluded simultaneously. |
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