Antibodies to stem cell marker antigens reduce engraftment of hematopoietic stem cells.
Hematopoietic stem cells (HSCs) have enormous potential for use in transplantation and gene therapy. However, the frequency of repopulating HSCs is often very low; thus, highly effective techniques for cell enrichment and maintenance are required to obtain sufficient cell numbers for therapeutic use and for studies of HSC physiology. Common methods of HSC enrichment use antibodies recognizing HSC surface marker antigens. Because antibodies are known to alter the physiology of other cell types, we investigated the effect of such enrichment strategies on the physiology and lineage commitment of HSCs. We sorted HSCs using a method that does not require antibodies: exclusion of Hoechst 33342 to isolate side population (SP) cells. To elucidate the effect of antibody binding on this HSC population, we compared untreated SP cells with SP cells treated with the Sca-1(+)c-Kit(+)Lin(-) (SKL) antibody cocktail prior to SP sorting. Our findings revealed that HSCs incubated with the antibody cocktail had decreased expression of the stem cell-associated genes c-Kit, Cd34, Tal-1, and Slamf1 relative to untreated SP cells or to cells treated with polyclonal isotype control antibodies. Moreover, SKL antibodies induced cycling in SP cells and diminished their ability to confer long-term hematopoietic engraftment in lethally irradiated mice. Taken together, these data suggest that antibody-based stem cell isolation procedures can have negative effects on HSC physiology.
Gilner
Jennifer B
JB
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Walton
William G
WG
Gush
Kimberly
K
Kirby
Suzanne L
SL
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000494:pharmacology*; D000943:Antigens, Differentiation / Q000235:genetics / Q000276:immunology*; D000067696:Ataxin-1; D000067528:Ataxins; D001562:Benzimidazoles; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D018380:Hematopoietic Stem Cell Transplantation; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000276:immunology; D009687:Nuclear Proteins / Q000276:immunology; D019009:Proto-Oncogene Proteins c-kit / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects* / Q000276:immunology* / Q000528:radiation effects; D014916:Whole-Body Irradiation
D000906:Antibodies; D000943:Antigens, Differentiation; D000067696:Ataxin-1; D000067528:Ataxins; C000593750:Atxn1 protein, mouse; D001562:Benzimidazoles; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D019009:Proto-Oncogene Proteins c-kit; C017807:bisbenzimide ethoxide trihydrochloride