Human and rhesus macaque hematopoietic stem cells cannot be purified based only on SLAM family markers

Abstract

Various combinations of antibodies directed to cell surface markers have been used to isolate human and rhesus macaque hematopoietic stem cells (HSCs). These protocols result in poor enrichment or require multiple complex steps. Recently, a simple phenotype for HSCs based on cell surface markers from the signaling lymphocyte activation molecule (SLAM) family of receptors has been reported in the mouse. We examined the possibility of using the SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We isolated SLAM (CD150(+)CD48(-)) and non-SLAM (not CD150(+)CD48(-)) cells from human umbilical cord blood CD34(+) cells as well as from human and rhesus macaque mobilized peripheral blood CD34(+) cells and compared their ability to form colonies in vitro and reconstitute immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptor(null), NSG) mice. We found that the CD34(+) SLAM population contributed equally or less to colony formation in vitro and to long-term reconstitution in NSG mice compared with the CD34(+) non-SLAM population. Thus, SLAM family markers do not permit the same degree of HSC enrichment in humans and rhesus macaques as in mice.

Figure 1

Cell sorting experiments of SLAM (CD150⁺CD48⁻) and non-SLAM (not CD150⁺CD48⁻) populations. Human G-CSF MPB CD34⁺ cells (A) and CD34⁻ cells (B). Human UCB CD34⁺ cells (C) and CD34⁻ cells (D). Human BM CD34⁺ cells (E) and CD34⁻ cells (F). Rhesus macaque MPB CD34⁺ cells (G) and CD34⁻ cells (H). No significant numbers of SLAM cells are detected in the CD34⁻ population from any hematopoietic source.

Figure 2

Summary of human cell engraftment based on CD45 cell surface expression in the BM of nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptor^null mice transplanted with SLAM (CD150⁺CD48⁻), non-SLAM (not CD150⁺CD48⁻), or unfractionated CD34⁺ cells. Representative flow cytometry analysis after transplantation of 1 × 10⁵ SLAM (A), non-SLAM (B), or unfractionated human CD34⁺ cells (C) derived from G-CSF MPB. (D) Control mice were injected intravenously with an equal volume of saline. (E) Summary of human cell engraftment after transplantation of G-CSF MPB cells from 3 independent donors in NSG mice. Each symbol represents one mouse, and the horizontal lines indicate the mean levels of human cell engraftment (n = 121 mice). Representative flow cytometry experiment after transplantation of 1 × 10⁴ SLAM (F), non-SLAM (G), or unfractionated human CD34⁺ cells (H) derived from 8 pooled cord blood samples. (I) Control animals were transplanted with an equal volume of saline. (J) Summary of human cell engraftment after transplantation of cord blood derived cells in NSG mice (n = 28 mice).