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.