“Functional markers” of hematopoitic stem cells (HSCs), unlike the surface, characterize their metabolic activity.
DNA-binding dye Hoescht 33342 (Ho 33342)
first isolation of HSCs-enriched population in mouse bone marrow (BM):
Baines P, Visser JW. Analysis and separation of murine bone marrow stem cells by H33342 fluorescence-activated cell sorting. Exp Hematol. 1983;11(8):701-8
Characterization of “side population” (SP) HSCs:
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996;183(4):1797-806
mitochondrial-binding dye Rhodamine 123 (Rh123)
Bertoncello I, Hodgson GS, Bradley TR. Multiparameter analysis of transplantable hemopoietic stem cells: I. The separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence. Exp Hematol. 1985;13(10):999-1006
aldehyde dehydrogenase (ALDH) activity
first isolation of mouse HSC based on high expression of ALDH:
Jones RJ, Collector MI, Barber JP, et al. Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity. Blood. 1996;88(2):487-9
first isolation of human HSCs based on high expression of ALDH:
Storms RW, Trujillo AP, Springer JB, Shah L, Colvin OM, Ludeman SM, Smith C. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity.Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9118-23
Study demonstrates that Aldefluor staining on its own is not an effective marker of mouse HSCs:
Levi BP, Yilmaz OH, Duester G, Morrison SJ. Aldehyde dehydrogenase 1a1 is dispensable for stem cell function in the mouse hematopoietic and nervous systems. Blood. 2009;113(8):1670-80
reactive oxygen species (ROS) production
Jang YY, Sharkis SJ. A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche. Blood. 2007;110(8):3056-6
Ho 33342 + Rh123
Wolf NS, Koné A, Priestley GV, Bartelmez SH. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection.
Exp Hematol. 1993;21(5):614-22
Ho 33342 + ALDH for mouse and human HSCs:
Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells.
Pearce DJ, Bonnet D. The combined use of Hoechst efflux ability and aldehyde dehydrogenase activity to identify murine and human hematopoietic stem cells. Exp Hematol. 2007;35(9):1437-46
Purton LE and Scadden DT in review: Limiting factors in murine hematopoietic stem cell assays concluded:
This method (SP and Rh123) should not be assumed to yield stem cells in other tissue types (besides BM) or even in species other than the mouse.
also read: The brief history of phenotype dissection of hematopoietic stem cells