The definition of a true stem cell is the ability to elicit three main functions; self renewal, differentiation and the capacity to reconstitute a tissue in vivo (Roobrouck et al., 2008). To understand self renewal and differentiation, cell division must be discussed.
Mitosis is the process of cell division in which two daughter cells are generated. HSC can undergo symmetric and asymmetric cell divisions, which ultimately decides the daughter’s cell fate (Morrison and Kimble, 2006, Domen and Weissman, 1999). Symmetric cell division involves the production of two identical daughter cells after mitosis. These identical cells can either be two HSC or two more differentiated daughter cells (Weissman and Shizuru, 2008). In this way, HSC can be produced which will either maintain the stem cell pool or more differentiated progeny can be produced which will ultimately provide mature cell types to replenish lost blood cells. In addition to symmetric cell division, HSC can also undergo asymmetric cell divisions in which progeny are produced that are not identical. Asymmetric division produces one stem cell and one differentiated cell. The combination of symmetric and asymmetric cell division is ultimately responsible for the maintenance of the haemopoietic system with the production of mature cell types when required while maintaining a functional stem cell pool (Figure 1-1) (Weissman and Shizuru, 2008).
Figure 1-1 Symmetric versus asymmetric cell division.
HSC can undergo symmetric and asymmetric cell division in which identical progeny or two different progeny are generated respectively. Symmetric cell division can produce two identical HSC or more differentiated progenitors. Asymmetric cell division results in the production of one HSC and one progenitor. The combination of symmetric and asymmetric cell divisions ultimately controls haemopoiesis through the maintenance of a stem cell pool and the production of a cascade of more mature progenitors.
The majority of research on asymmetric cell division has been carried out using
developmental model organisms including the Drosophila melanogaster (Morrison and Kimble, 2006, Gomez-Lopez et al., 2013). Studies have shown that both intrinsic and extrinsic mechanisms govern cell division. As an example, cell polarity and the distribution of cell components/proteins occurs prior to cell mitosis and governs subsequent cell fate.
This has been shown in stem cells including in HSC, in which several proteins were found to segregate differentially after cell division (Gonczy, 2008, Beckmann et al., 2007). In addition, external mechanisms also control cell division with evidence that cell location alters the ultimate cell fate (Morrison and Kimble, 2006). The decision of symmetric versus asymmetric division is important not only for cell fate in normal development, but also in disease as previous studies have highlighted that asymmetric divisions are
deregulated in cancer (Morrison and Kimble, 2006, Gomez-Lopez et al., 2013).
Self renewal is a fundamental property of HSC and it is thought that self renewal capacity is reduced as cell division occurs. Therefore more mature cells, including progenitor cells, show a reduced capacity to self renew. Differentiation is described as the production of a more mature, specialised cell down a particular lineage. HSC balance the processes of self renewal and multilineage differentiation to maintain a pluripotent HSC population poised to give rise to appropriate cell types when required including in response to blood loss,
infection or exposure to cytotoxic agents and oxidative stress (Seita and Weissman, 2010, Wilson et al., 2008).
Self renewal and differentiation are key to HSC function and can be measured experimentally. For simplicity, mouse and human experiments will be discussed separately.
1.3.1.1 Mouse
Both in vitro and in vivo assays can be used to experimentally to examine self renewal and differentiation. In vitro colony formation assays (colony formation cell, CFC assay) are widely used. This assay involves the culture of cell populations with the addition of particular cytokines designed to drive proliferation and differentiation. Cells are cultured for a period of time and the resulting colonies formed can be scored based on enumeration and classification of colonies. This gives an indication of proliferation and differentiation capacity. This assay is considered an assay for more mature progenitor cells, however, the colonies derived from a primary plating assay can be replated into a secondary plating assay in which the growth of colonies can act as an indicator for self renewal capacity.
However, a CFC assay is relatively short term and is therefore more indicative of progenitor cell activity. The long-term culture initiating cell (LT-CIC) assay allows the detection and enumeration of the HSC population (Woehrer et al., 2013). In this assay, cells are plated on a layer of stromal cells (designed to mimic in vivo conditions) which support the survival, self renewal and differentiation of HSC. These cultures are
maintained long term to identify true HSC populations in comparison to shorter
experiments which identify progenitor populations only. In addition, limiting dilution of cell populations are used to give an indication of the frequency of LT-CIC per population.
The cobblestone area forming cell (CAFC) assay involves the culture of a test population on a stromal layer and particular areas of HSC growth termed ‘cobblestones’ are scored based on stem/progenitor growth over a period of time (Breems et al., 1994, de Haan and Ploemacher, 2002).
However, in vivo experiments have provided unique insights into stem cell behaviour and are arguably more accurate in terms of quantifying HSC than in vitro assays (Domen and Weissman, 1999, Perry and Li, 2010). The CFU-S assays (as described previously) can provide an in vivo indication of stem/progenitor cell activity. The production of distinct colonies grown on the spleen of irradiated animals refers to the clonogenicity activity of
transplanted cells. Spleens are typically analysed on days 8 and 12 with the latter referring to a more primitive clonogenic cell than the former (Weissman and Shizuru, 2008).
However, this assay is thought to involve more mature progenitor cells as opposed to stem cells due to the short time frame of the assay. The gold standard technique for assaying HSC function is the reconstitution of an ablated/diminished haemopoietic system (Harrison, 1980). A cell population can be transplanted into mouse models in which endogenous haemopoiesis has been ablated using irradiation or chemotherapy drug treatment. A true HSC will be capable of reconstituting the BM and providing progenitor and subsequent mature cells therefore rescuing haemopoiesis. Competitive repopulation involves the transplantation of donor test cells along with support BM cells which ensures host survival and markers can be used to elegantly track donor transplanted populations over time. Examples for host versus donor distinction are sex, expression of reporter genes or arguably the most commonly used, expression of cluster of differentiation (CD) cell surface markers including isoforms of CD45 (van Os et al., 2001, Domen and Weissman, 1999). CD45 was originally known as the leukocyte common antigen as is expressed on all leukocytes (Trowbridge and Thomas, 1994). Two alleles of CD45 are available and mouse strains have been developed with each allele on a C57/BL6 background (van Os et al., 2001). The generation of monoclonal antibodies against these alleles allowed for the distinction between donor versus host in competitive transplantation assays (Weissman and Shizuru, 2008). Limiting dilution experiments using a titration of the number of
transplanted HSC are used and are more reliable in terms of quantifying the number of true stem cells in a population (Perry and Li, 2010). HSC transplantation assays not only give an indication of mutlilineage reconstitution, however self renewal can be experimentally examined through the ability of test populations to rescue haemopoiesis in secondary and tertiary recipient mice and these are the most stringent assays to report stem cell self renewal and therefore activity.
1.3.1.2 Human
Similar to in vitro assays described for mouse studies, the CFC, LT-CIC and CAFC assays can be used to assay human stem/progenitor behaviour (Domen and Weissman, 1999, Liu et al., 2013, Sarma et al., 2010, de Haan and Ploemacher, 2002). More recently, literature is emerging in which 3-dimensional structures are used to model the BM niche (Sharma et al., 2012). As discussed with the mouse in vitro assays, these assays are arguably not measuring true HSC activity.
The ability to transplant HSC into irradiated hosts and compare their differentiation and self renewal capacity has become a standard technique. However, studying human HSC is more complex. The use of immunocompromised mouse models as hosts for human HSC was a groundbreaking discovery in HSC research (Meyerrose et al., 2003). Original research showed that human haemopoietic tissue could engraft in immunocompromised (severe combined immunodeficient, SCID) mice which have a mutation resulting in defects in B and T cell development (Lapidot et al., 1992, Mosier et al., 1988). However, these mice still have natural killer cells which can attack foreign cells and therefore hinder the experiment. In order to study immune responses, there are other models available however further immune compromised mouse models have since been generated which work well as HSC xenograft models. Since this research, a variety of models have been described including non obese diabetic (NOD) SCID animals. These animals have additional defects in natural killer cell, macrophage and complement. Additional strains including, but not limited to, NOD/SCID/β-2-Microglobulin (β2M), NOD/SCID/IL-2R-γ-/- mice or RAG2-/- / IL-2R-γ-/- models are used which show more immunodeficiency than the NOD/SCID animals (Park et al., 2008, van der Loo et al., 1998, Wermann et al., 1996).
Such models are commonly used in the literature and allow for the tracking of human HSC activity without the complications arising from rejection of the foreign transplanted cells by the host immune system (Domen and Weissman, 1999). However, disadvantages of using these mice include a shortened lifespan and their extreme sensistivity due to their compromised immune systems (Meyerrose et al., 2003). A diagram is displayed in Figure 1-2 for examples of the different assays available.
Figure 1-2 Commonly used methods for assaying HSC activity.
Schematic is adapted from published literature and based on the literature described above (Domen and Weissman, 1999). Various techniques have been developed to assay HSC activity. In vitro techniques include the culture of cell populations in particular growth conditions in which resulting colonies can be identified to give an indication of
proliferation and differentiation status (CFC). LT-CIC and CAFC assays monitor cell growth over a longer time period in a co-culture with stromal cells to assess more primitive HSC activity. In vivo techniques involve the transplantation of cells into irradiated
recipients where colonies grown on the spleen (CFU-S) or the capacity for multilineage reconstitution is evaluated. Donor cell activity can be tracked using various methods to distinguish host versus donor cells. Self renewal activity can be examined using the serial transplantation of cells into secondary and tertiary irradiated recipients. Mouse models for the transplantation of human HSC involve various immunocompromised mouse models of which the most common are stated. (Dr Francesca Pellicano and Dr Arunima
Mukhopadhyay should be acknowledged for the CFU-S and LT-CIC images respectively).