Identification of molecular markers that best facilitate isolation and characterization of stem cell populations has long been a challenge. CD34, a cell surface phosphoprotein, has been generally considered as a hematopoietic stem cell (HSC) marker based on engraftment following bone marrow transplantation.1-3 Use of CD34 as a marker to isolate or define stem cells has since become controversial, however. In 1996, Osawa et al.4 identified CD34- murine HSCs. In 1998, Bhatis et al.5 demonstrated a low level of engraftment and hematopoietic capacity for human CD34- cells. Transplantation studies in fetal sheep also demonstrated repopulating activity in the CD34- population.6 In addition, both murine and human CD34+ cells could be derived from CD34- cells.7,8 Collectively, these reports suggest that HSCs may be CD34+ or CD34- and that selection of cells expressing CD34 might result in exclusion of another population of primitive stem cells.
|Figure 1. The family of ABC transporters is characterized by the presence of an ATP-binding cassette region, which hydrolyzes ATP to support energy-dependent substrate exportation from the intracellular cytoplasm to the extracellular space. Full-length transporters contain two mirror image halves that are separated by a flexible linker region (not shown). Half-transporters, e.g. ABCG2, function as homo- or heterodimers and may be localized to the plasma membrane.|
Discovery of the ability of HSCs to efflux the fluorescent dye Hoechst 33342 has led to its use in highly enriching stem cell fractions.9 Hoechst-negative cells can be isolated by fluorescence-activated cell sorting as a so-called “side-population” (SP). This SP from bone marrow, as well as other tissues, contains stem cells with considerable plasticity.10,11 Isolation of SP cells from human fetal liver, for example, yields a ten-fold enrichment of transplantable stem cells.12
ABCG2 (ATP-binding cassette superfamily G member 2) is a determinant of the SP phenotype and is found in a wide variety of stem cells.13,14 The ABCG2 gene product is a member of the ABC transporter family and was first identified in a breast cancer cell line.15 The family of ABC transporters is classified by expression of an ATP-binding cassette region and by their function to hydrolyze ATP to support energy-dependent substrate exportation from the intracellular cytoplasm to the extracellular space.16 There are two forms of these transporters, the full-length transporters and the half-transporters that function as homo- or heterodimers (Figure 1). ABCG2 is a unique member of the half-transporter group as it is localized to the plasma membrane.17 The expression of ABCG2 appears to be greatest on CD34- cells and is down-regulated with the acquisition of CD34 on the cell surface.13 The down-regulation in ABCG2 expression is also observed in various committed hematopoietic progenitors.18 ABCG2 may therefore serve as a promising marker for primitive HSC isolation and characterization.
The expression pattern of ABCG2 is not limited to HSCs. It represents the Hoechst SP phenotype in cells from diverse sources, including monkey bone marrow, mouse skeletal muscle, and embryonic stem cells.13 Within all cells examined, ABCG2 expression is exclusive to SP cells as compared with non-SP cells. The potential plasticity of SP cells has been demonstrated by studies showing cardiomyocyte and muscle regeneration from transplanted bone marrow-derived SP cells.19,20 Exclusive expression of ABCG2 on SP cells implicates the potential use of ABCG2 as a marker for positive selection of pluripotent stem cells from various adult sources. It is also possible that ABCG2 may play a functional role in developmental stem cell biology.21 The potential functional involvement of ABCG2 in stem cell identity makes ABCG2 even more attractive as a tool in stem cell characterization.