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.