The study of stem cells requires the ability to identify and isolate these cells for experimental research. HSC are generally identified due to their lack of expression of lineage positive cell markers and low staining of side population using DNA and RNA stains which will be discussed in detail in this section.
The discovery of HSC populations expressing particular cell surface markers has allowed for the identification and isolation of these populations (Wognum et al., 2003). There is some overlap between human and mouse HSC populations in terms of identification. For example, in both species HSC are identified through their lack of expression of lineage markers and low staining of DNA and RNA stains. HSC from both mouse and human are identified as existing in a population that are negative for staining of CD markers
commonly expressed on mature lineage cells, including erythroid, granulocyte, B and T cells (defined from onwards as lineage negative). The addition of nucleotide stains, including RNA and DNA stains, has enhanced this population. As an example, Hoechst 33342 was identified as marking HSC populations in 1996 and was subsequently referred to as the side population (SP). Experiments showed that the most primitive HSC effluxed the dye which identified Hoechst 33342 negative cells as stem cells (Goodell et al., 1996, Goodell et al., 1997). ABC/G2 transporters are selectively expressed on stem cell
populations are thought to result in the efflux of Hoechst 33342 solely in stem cell populations (Zhou et al., 2001, Kim et al., 2002).
However, the species will be discussed in more detail separately due to differences in expression of cell surface markers between species.
1.3.3.1 Mouse
Animal studies have provided vast advances in the identification and isolation of HSC and progenitor populations. In 1986, cells which were negative for a cocktail of lineage
markers were identified as a population enriched with mouse cells that had reconstitution potential (Muller-Sieburg et al., 1986). In 1988, cells which were additionally negative for CD90 and positive for Sca-1(ly-6 A/E) were shown to reconstitute haemopoiesis in a proportion of recipients with ablated BM with only the cells positive for Sca-1 having the capacity for in vivo reconstitution (Spangrude et al., 1988). This research was extended to include c-Kit (CD117) as a positive marker for HSC, also known as the cell surface receptor which binds to stem cell factor (SCF) (Ogawa et al., 1991, Ikuta and Weissman, 1992). The combination of lineage negative with Sca-1 and c-Kit positive markers (lineage negative, Sca-1+, c-Kit+; LSK) was identified as the population containing all the HSC activity (Uchida et al., 1994). However, this population is now known to be heterogeneous and contains a mix of stem cell populations with progenitor cells (Bryder et al., 2006).
Subsequently after these initial investigations, research by the Weissman, Jacobsen, Nakauchi and Morrison laboratories collectively identified markers which allowed for the isolation of purer HSC populations. Interestingly, one of these markers, CD34, was found to be a negative marker of LT-HSC in contrast to evidence from the human studies. The addition of LSK with the negative selection of cell surface markers CD34 and Flk-3 was reported to give rise to long term haemopoiesis with the acquisition of these markers selecting for ST-HSC and MPP populations (Osawa et al., 1996, Christensen and Weissman, 2001). More recently and arguably most commonly used in the literature are the cell surface markers CD150 (more commonly referred to as SLAM), CD244 and CD48 (Kiel et al., 2005). CurrentlyLSKCD150+CD48- and LSKCD34-Flk-3- are the most
commonly used sets of markers for LT-HSC identification in the mouse system. The former is arguably the most commonly used method for identification, due to the positive selection of marker CD150. Using this marker showed approximately 50% of the LT-HSC gave rise to BM reconstitution (Kiel et al., 2005). These studies bring us closer to
identifying a HSC population capable of 100% BM reconstitution. This is the purest population currently available for mouse HSC to date. A figure with the most up to date mouse HSC hierarchy is displayed (Figure 1-4). Committed progenitor cell types are well
defined in the mouse system with a combination of cell surface markers including CD127, CD34 and CD16/32 (Doulatov et al., 2012).
Figure 1-4 Mouse haemopoietic hierarchy.
Schematic demonstrates the most recent mouse haemopoietic hierarchy. The boxes denote the cells used to identify human stem/progenitors in this study. The box marked in red identifies the LT-HSC (lineage-c-Kit+Sca-1+CD150+CD48-) used throughout in chapters 4 and 5 and used widely in the literature to separate identify LT-HSC. The ST-HSC and progenitor populations used in this study are described more detail in the materials and methods chapter 2 and were used widely in the literature at the time of doing the
experiments in this thesis. Information in this schematic is based on the literature discussed in section 1.3.3.1.
1.3.3.2 Human
Focusing on human studies, early experiments identified that positive expression of CD34 marked a rare population of BM cells which were enriched for colony formation and capable of in vivo reconstitution of immunocompromised mice (Berenson et al., 1988, Sutherland and Keating, 1992, Civin et al., 1984, Andrews et al., 1989). Collectively, these studies suggested that CD34 marked a population of cells with stem/progenitor activity. In addition, the CD34 protein has also been shown to be expressed on endothelial cells (EC) and embryonic fibroblasts (Krause et al., 1996). To date, human HSC are now commonly identified in the literature as expressing CD34. It is a well known marker of a
heterogeneous stem/progenitor cell population (Stella et al., 1995). Experiments have detected stem cell activity in the CD34- fraction of human cells which indicated that CD34 is not a marker of all stem and progenitor populations (Bhatia et al., 1998, Goodell et al., 1997, Sonoda, 2008). Recently, a study has shown that this population in combination with additional marker CD93 does function as a HSC population and is more primitive than CD34+ cells suggesting CD34- cells are at the pinnacle of the haemopoietic hierarchy (Anjos-Afonso et al., 2013, Danet et al., 2002). However, this research is novel and to date, CD34 is widely used in experimental haematology and clinical haematology in which CD34+ cells are isolated and used for stem cell transplantation (Wognum et al., 2003).
Interestingly, although CD34 is widely used as a human stem/progenitor marker, the function of the CD34 protein is not well understood. It is thought that this is due to a lack of data on functional assays on the protein as discussed in a detailed review (Nielsen and McNagny, 2008). As CD34+ cells are known to represent a heterogeneous population containing stem and progenitor cells, additional surface markers have been sought after in order to further enrich the human stem cell population. Additional marker CD133 has been reported (Yin et al., 1997). However, more recent evidence suggest CD133 does not mark only stem cells but also more mature cell types (Meregalli et al., 2013). CD38 is a cell surface marker known to play roles in immunity, cell adhesion and calcium signalling (Mehta et al., 1996). Experiments have shown that a small proportion of CD34+ cells express the CD38 protein (<10%) therefore representing a rare population of CD34+CD38- cells (Bhatia et al., 1997). The combination of CD34 with CD38 showed that the
CD34+CD38- and CD34+CD38+ fractions differed in terms of cell cycle status and stem cell activity, including reconstitution into immunocompromised mice (Bhatia et al., 1997, Civin et al., 1996). These cell surface combinations are now widely used in studies with the CD34+CD38- fraction representing a more primitive subset of cells. However, further purification of this population can enrich the stem cell population, for example by using the combination of CD34 and CD38 with CD45RA and CD90 (Thy-1). In addition, rhodamine123 has been used as a dye to mark stem cells which are negative for the dye.
Briefly, rhodamine123 is a dye that labels mitochondria with increasing intensity
proportional to cellular activation (Kim and Broxmeyer, 1998). CD45RA is a member of the CD45 family that is highly expressed on naive T lymphocytes; whereas CD90 is commonly used to identify thymocytes, but has also been implicated in a variety of different processes (Streuli et al., 1987, McKenzie et al., 2007, Mayani et al., 1993, Baum et al., 1992). Recently the combination of markers was used to identify a population with stem cell activity (CD34+CD38-CD45RA-CD90+) however the population containing CD90- cells also showed engraftment in serial transplantation assays (Notta et al., 2011,
Majeti et al., 2007). More recently, the addition of CD49f was conclusively shown to be a specific HSC marker and it was shown that the MPP population lost expression (Notta et al., 2011). CD49f is a member of the integrin family which associates with either integrin β1 or β4 to form receptors for laminin and Kalinin. It is expressed on a monocytes, T cells,
platelets, endothelial and epithelial cells, and is involved in adhesion or co-stimulation for T cell activation/proliferation (Hughes, 2001). Although some controversy still exists, collectively CD34+CD38-CD45RA-CD90+CD49f+ cells represent the highest reported purity of human HSC to date and a figure is displayed in Figure 1-5. Humans have well defined cell surface marker expression committed progenitor cell types using CD135, CD10 and CD7 (Doulatov et al., 2012). Although human HSC identification has
progressed, human markers of the stem and progenitor populations in haemopoiesis are not as well identified as in the mouse system.
Figure 1-5 Human haemopoietic hierarchy.
Schematic demonstrates the most recent human haemopoietic hierarchy. The boxes denote the cells used to identify human stem/progenitors in this study. The boxes marked in red identify HSC (CD34+CD38-) with MPP (CD34+CD38+) used throughout in chapter 3 and used widely in the literature to separate HSC with more mature progenitor populations.
The green boxes denote HSC (CD34+CD38-CD90+) with MPP (CD34+CD38-CD90-) and more committed progenitors (CD34+CD38+) used in chapter 5 which is used widely in the literature. Information in this diagram is based on the literature discussed in section 1.3.3.2.
Due to the identification of cell surface markers expressed by human and mouse HSC populations, flow cytometry cell sorting has emerged as the best technique for the isolation of HSC populations. Cell sorting using flow cytometry allows for the isolation of
individual cells which is ideal for stem cell biology in which these cells are so rare. This also allows for the study of the stem cell behaviour at a single cell level. The ability to identify and isolate HSC from their environment allows for their study and this approach has enabled us to understand their behaviour.