Research into Cancer Stem Cells

Research into pubic louse theme Cells pubic louses atomic look 18 composed of a heterogeneous mix of prison prison cubiclephoneular phoneular telephoneular phonephoneular teleph aceular telephoneular teleph unmatch fittings with take leaveing speciality, proliferation and neoplasmigenic properties. In vivo studies know demonstrate that inside a endurecerous neoplastic disease cosmos, solo percentage of carrels atomic number 18 able to start out tumour development 1. It is widely believed that the heterogeneous groups of electric carrels include a sharp universe of crabby person carrells with chaff cadre properties the crabmeat origin jail cubicle (CSC). These prison cells leave the content to self-re in the buff and break unsymmetrical everyy and throw off leaven to majority worlds of nontumourigenic cancer cells. Current cancer treatments whitethorn eradicate the tumour bag but sp ar the populations of shank cells which argon able to restore tumour meander causing recurrence of the cancer. This may explain why initial tumour regression does non necessarily translate to amend persevering survival in many a nonher(prenominal) clinical trials. Identification and characterisation of these stand cells may offer means of targeting cancer at its root.Cancer husk Cell DefinitionThe AACr acidifyshop in 2006 delimitate a cancer arrest cell as A cell indoors a tumour that possesses the expertness to self-re sensitive and to cause the heterogeneous lineages of cancer cells that comprise the tumour. Cancer groundwork cells can thus only be defined by means of an experiment by their expertness to recapitulate the generation of a continuously bestridement tumour.2 Therefore the nucleotide cell definition requires that cell possess 2 fundamental properties. Self transposition, the process whereby at least one young woman cell of a dividing solution cell declares stand cell properties Potency, the ability of cells to differentiate into diverse cells that comprise the tumour. 3. It was agreed that defined CSCs may not necessarily derive from typical t step up al-Qaida cells, indeed one all important(p) and unanswered question is whether tumours derive from organ infrastructure cells that retain self renewal properties or whether tumour waist cells are proliferative progenitors that meet self-renewal qualification 2.Normal Tissue HeterogeneityThe continuous replacement of place, serviceable cells by proliferation of to a greater extent than primitive cells in valet de chambre tissue is a normal homeostatic process. Organs are composed of collections of differentiated cells that perform discrete regions 4. The total cell population is regarded as constituting a cell division hierarchy 5. The beginning cell is central in the renewal hierarchy and has devil functions at bottom this lay. It can act as the initiating cell in a cell division and differentiation process, pro ducing a jumbo family of differentiated desc demolitionants, a process known clonal expansion. Another function is for the cells to undergo division to produce two prow cell daughters indistinguishable to the initial stem cell and to replace the stem cells utilise in clonal expansion. This process is called self-renewal 6 and is shown diagrammatically in Figure 1. As cells make a motion squander the hierarchy they acquire the differentiated features associated with tissue function and the symmetricalness of differentiated cells increases. In this way the stem cell has the ability to fight down organ life 4. This concept predicts the worldly concern of three categories of cell within the population Proliferating, self renewing stem cells Proliferating non-renewing cut throughional cells ( move through amplifying) Non-proliferating, differentiated end cells. undermentioned division the stem cell can give rise to a transit amplifying cell that will undergo further steadfas t proliferation to produce offspring which expand the populations of cells arising from the initial division and progressively move irreversibly to differentiation a tenacious one or several lineages4. An important feature of a stem cell is their ability to undergo asymmetric cell division giving rise to a progenitor cell and to a new stem cell. Somatic SCs reside in confine tissue compartments referred to as the niche. Here the microenvironment suppresses SC proliferation, resulting in a inactive SC population. This population maybe triggered to plow and differentiate in solution to injury (Ghotra, 2009). Seven common land and distinguishing features of stem cells harbor been describe 4Stem cells comprise a sharp subpopulation of a prone tissue.Stem cells are ultra-structurally unspecialized, with a bouffant nuclear-to-cytoplasmic ratio and few organellesStem cells can be pluripotentStem cells are slow up cycle but may be induced to proliferate more(prenominal) rapidly in reply to trustworthy stimuliStem cells have a proliferatve reserve that exceeds an singulars lifetimeAn intermendiate group of transit amplifying cells existsThe microenvironment plays a critical role in the homeostasis of the stem cell and the differentiation of its progeny.The stem cell is capable of division and clonal expansion. As cells differentiate they regress their proliferative potential. The stem cell can self renewal or divide to produce proliferative transitional cells.Tumour HeterogeneityIt has been experienced for many years that tumours exhibit morphologic heterogeneousness but they are alike functionally heterogeneous in legal injury of cell proliferation and tumour forming capacity based on transplantation assays 7. Heterogeneity within tumours is seen within individual tumours in terms of sound structure, cell jump bulls eyes, cell proliferation kinetics and answer to therapy. In vitro and in vivo observations suggest that more or less cancer cells do not proliferate and that liberation of capacity to divide is a feature of the tumour. sole(prenominal) a teensy-weensy dimension of cells have the ability to form tumours in vivo, referred to as tumourgenicity. The cancer stem cell theory posits that neoplasms, like physiological tissue can be hierarchically organised, and that CSCs at the apex of this of this cellular hierarchy and seem to comprise of only a subpopulation of tumour cells are essential for its fount 8, 9. Two casts have been proposed to explain tumours heterogeneousness random and power structure, summarised in Figure 2. Both positions account for the existence of a cell with stem cell properties, but only the hierarchy model predicts the existence of a stem cell at the top of a hierarchy, which the potential to produce all other cell tokens within the tumour.Stochastic stickThe stochastic model predicts that a tumour is biologically homogeneous and the demeanour of the cancer cells is influenc ed by inalienable (eg signalling pathways, levels of transcription factors) or extrinsic factors (eg entertain factors, immune response, and microenvironment). It is suggested that the randomness and unpredictability of these factors result in heterogeneity in many aspects of marker expression and tumours initiation capacity 10. A severalize requirement of the stochastic model is that all cells are equally crank to such influences and that the cells can revert from one state to another. For this model to be functional all tumour cells are not changelessly un inhering and all have equal capacity to be induced to one state or another and the changes upon the cell are not permanent 11. A ontogeny separate of pecking order ModelThe second model is the hierarchy model which predicts that the tumour is a caricature of normal tissue development and a hierarchy where the stem cell is at the go past is maintained (Pierce) 7. The cancer stem cell maintains itself and its clones by se lf-renewal. The cells also advanced to produce differentiated offspring which form the bulk of the tumour and miss stem cell properties. As in normal tissue only a small percentage of the tumour population maintain the capacity for long term proliferation while most cells proceed forward down the differentiation pathway resulting in aberrant terminal differentiation 4. collectable to diversions in characteristics, stem cells can be selected and enriched for. Variations in tumour result rates may be due the effects of normal homeostatic mechanisms that regulate stem cells and transit amplifying cell re toil or alterations of the stem cell niche microenvironment 4. Much of the evidence for this comes from clonogenic and tumourgenic assays, which will be discussed further.Hierarchy model contains cells that are composed of biologically distinct cells including cancer stem cells which are all have different functional properties. The stochastic model predicts that all cells are eq ual the cell heterogeneity is due to intrinsic and extrinsic influences upon the cells which result in heterogeneity of cell function.Experimental demonstrateEarly WorkThe depression evidence for the existence of cancer stem cells came from functional cell proliferation studies in the1940s 1960s. Radiolabelling cells and autoradiography enabled measurements into the proliferation, lifespan and hierarchical relationships in normal and neoplastic tissues 10, 12. From these studies came the proposal that tumours are caricatures of normal development including the existence of stem cells 7. Much early work was on the cancer of the haematopoietic system. In the 1970s Clarkson and other groups carried out pioneering studies that established cancers exhibited functional heterogeneity 10, 13. These include cytokinetic studies carried out in cell lines, murine models of the acute leukaemias and in vivo examination of leukaemia blast proliferation kinetics in valet AML and ALL patients. T he in organisation showed that the majority of leukemic blasts were post mitotic and needed to be continuously replenished from a comparatively small proliferative fraction. Only a small number of leukemic blast cells were cycling in vivo and of these two proliferative fractions were observed a larger, fast cycling subset with a 24 hour cell cycle time and a smaller, slow cycling, with a dormancy of weeks to months. From this data it was suggested that the slow cycling fraction was generating the fast cycling fraction thought to be the leukemic stem cell population because they had akin kinetic properties to those observed for normal haematopoietic stem cells. This was a clear suggestion that tumours exhibit functional heterogeneity in terms of proliferative potential. Following the naming of these slow cycling cells it was predicted the inability to kill the leukaemic stem cells (LSCs) was the cause of relapse and failure of chemotherapeutic therapies. Whilst combining treatment with in vivo cytokinetic studies, investigators observed that LSCs respond to the depletion of the of the leukemic cell mass by go into cycle after chemotherapy. It was suggested the way to eliminate dormant LSCs was to find the windowpane when they are cycling. Identifying and assaying the potential LSCs was a major stumbling block and characterising them was impossible. This was when attention cerebrate on the clonogenic assay was adapted by several groups to assay AML which place phenotype of AML cultures in vitro with differing proliferative potential, providing the further proof for hierarchy in AML 14-16.ClonogenicityDefinition of a cloneA clone is an operationally defined as a group of cells derived from a private ancestor cell. Clonogenicity is the ability of a given cell population, when plated as wiz cells, to produce one or more clones. This can be measured by the clonogenic assay which can limit the proportion of dependance forming cells, as a percentage of plated population, referred to as village forming efficiency (CFE). It has been suggested that colonisation-forming cells possess two fundamental properties of progenitor cells the ability to give rise to differentiated descendents and the capacity for self-perpetuation 17. Therefore the ability to measure the capacity of cells to form clones is a useful marionette in the nurture of the cancer stem cell concept.Quantitative measurement of clonogenicity Development of the clonogenic assay.Puck and Marcus The introductory clonogenic assayIn 1956 Puck and Marcus published a paper describing a cell culture technique for sound judgement of colonization forming ability of maven mammalian cells 18. Plated in culture dishes with a suitable mass long suit humanity cervical carcinoma cells (HeLa) were supplemented with a large number of irradiated feeder cells and the number of colonies form was counted. Their technique was a simple rapid method for growing star mammalian cells into ma croscopic colonies with a dependence forming efficiency of 80 degree Celsius% . The authors essential this assay further to enable quantification of the effects of high qualification re on cell populations in vitro 18-20. They plated HeLa cells and measured their response to x-rays, producing the first in vitro radiation cell survival curve 21. This assay has since been used for a wide variety of studies with many cell types using im heard culture conditions, and for the interrogation of many potential chemotherapeutic agents.Till and McCullochFollowing the work of Puck and Marcus, Till and McCulloch generated the first in vivo survival curves 22, 23. They showed that when crawl bone aggregate cells were injected into receiver mice that had been given total body slam to suppress endogenous haematopoiesis, visible colonies developed in the spleens that derived from cells in the graft. This work demonstrated that the cells injected into the mice were capable of self-renewa l and it was speculated that these cells were stem cells. The evidence for this conclusion was that the curve from the number of marrow cells transplanted proportional to the number of colonies developed within the spleen. In addition, the radiation survival curve of cells that form colonies closely resembled survival curves developed by Puck and Marcus for in vitro cells 21. This, thus far, was only indirect evidence and did not prove that the colonies originated from single cells, so the group carried out further experiments to notice the single cell origin on the colonies within the spleens 24. Heavily irradiated bone marrow was transplanted into heavily irradiated recipient mice. The fancy was that some cells containing genetic abnormalities caused by irradiation in the donor bone marrow cells would retain the ability to proliferate and produce clones containing this abnormality 24. This worked to some extent, with a small number of colonies containing cells which all showed the same chromosome abnormality within that colony. It was hypothesised that if the capacity to form colonies is to be considered as a criterion to rate stem cells, then cells must lose this capacity upon undergoing differentiation. This hypothesis was tested by applying hypoxia as a differentiating stuff to mouse bone marrow, which resulted in a reduction in colony formation in the spleens of hypoxic mice 17. They described how the number of colonies form in the spleens of mice in hypoxic conditions is reduced. This was thought to be due to hypoxia stimulating erythropoiesis which stimulates erythropoietin, indicating that erythropoietin reducing colony forming production in the spleen. This data suggested that an increased demand for differentiated cells reduces the number of stem cells, resulting in the reduction of colony forming ability.Later DevelopmentsSince its development, the in vitro clonogenic assay has scram a valuable tool in the study of cell growth and differentia tion. 25. Several adaptations to the original method have been made including immobilising cells in a top layer of 0.3% agar to avoid formation of tumour cell aggregates by random movement which might be confused with colony growth 26. Agar has also been replaced by some groups with agarose, which is easier to handle (Laboise 1981) or methylcellulose which allows better recovery of the colony for replating. Others have simplified the culture medium and omitted the need for feeder cells. The exact protocol depends largely on cell type, but the basic system remains the same. The development of a protocol for secondary plating efficiency has turn up a useful tool for the measurement of self-renewal and has the advantage of being able to identify cells that are able to undergo a large number of cell divisions 26. This involves selecting specific colonies to determine their proliferative potential over a number of passages.Clonogenicity and Cell Renewal HierarchyClonogenic assays have been used to identify and morphologically characterise the three cell types above. Barrandon and Greens 27 work place the clonal types of keratinocytes and linked this to their capacity for multiplication. They defined colonies as Holoclone, Meroclone or Paraclone. The Holoclone was described as a colony with a larger smooth nearly circular perimeter containing many small cells, which it has been suggested that these cells represent the proliferating self renewing stem cells. Paraclones were described as differentiated end cells which are more elongated and flattened in appearance, however paraclones can divide quite rapidly therefore classification of clonal type cannot be deduced form the study of growth rates completely or morphology alone. Meroclones were described as a combination of holoclones and paraclones. Relating morphology and colony size to clonogenicity can be used to further identify potential stem cells within the clonogenic assay and give more head to the fate o f their descendents. The differences in growth unit size may mull over several properties including different proliferative capacities and clonogenic cell kinetics. However, clonogenicity in vitro alone, does not define a stem cell, and other subpopulations, such as transit amplifying cells may also be able to produce a colony size of 32 or more cells. Although ability of a cell to form a colony implies substantial proliferative capacity, this does not unambiguously identify a stem cell 28.neoplasm Cell Heterogeneity and HierarchyCertain characteristics have emerged from clonogenic studies on cells derived from human tumors. It was noticed that a few cells in to each one tumor were able to give rise to colonies in culture, whilst some colonies contained transit amplyifing cells undergoing a limited number of terminal divisions. Other cells (usually the majority) were non-proliferating stem cells. facial expression at CFE and colony size of human tumors and replating experiments h as demonstrated the heterogeneity of a wide range of tumor types including neoplastic human urothelium 29, melanoma 30, 31 and squamous carcinoma 32. This supports the idea that cells within hard tumors consist of cellular hierarchies, which will be discussed further.The cancer stem cell model accounts for heterogeneity within a primary coil cancer by proposing that each cancer consists of a small population of cancer stem cells and a much larger population of cells which have lost their self-renewal capacity 5. The clonogenic assay has been used explore this cellular heterogeneity present in human tumors, lending support to the stem cell model of tumor growth. Multiple myeloma has served as a valuable model in early clonogenic assay development. This was studied by Hamburger and pink-orange in 1977 33, who created an essentially discriminating system which restrict proliferation to cells capable of anchorage ground independent growth, thought to be a characteristic of stem cells 34. They described an in vitro bioassay for human myeloma colony-forming units in culture which was applied to the study of patients with multiple myeloma and related monoclonal bone marrow derived B cell neoplasm. Bone marrow samples from patients with multiple myeloma and normal volunteers were cultured in the bearing of an agar feeder layer prepared by either human type O+ washed erythrocytes or adherent spleen cells of BALB/c mice. They found a linear relationship amidst colony formation and the number of nucleated bone marrow cells plated. Multiple myeloma patients exhibited much high numbers of colonies formed compared to normal volunteers. It was shown that the number of colonies was proportional to the number of colonies plated, suggesting that colonies were derived from single myeloma stem cells. This was the development of the human tumor stem cells assay.The Human Tumor Stem Cell Assay clonogenicity and cancer stem cellsThe ability to grow human solid tumors in two-lay er soft agar culture was developed for the clinical application of testing in vitro tumor sensitiveness or resistance to chemotherapeutic agents. It is a possible means by which antitumor drugs can be selected for activity against tumor cells from a patient 35 as a way of tailoring chemotherapeutic regimes to individual patients and of testing new cytostatic agents 36.The assay assesses treatment effects of stem cells by a testing their ability to reproduce and form a colonies of cells. Using semi-solid agar with enriched medium supports colony growth from cell suspensions from a variety of human tumors. A semi-solid medium suppresses the growth of most normal cells and there is evidence of the malignant nature of these colonies 33 . An important consideration is the relationship between the response of clonogenic cells to drugs in vitro and the response of the tumor to the same drug in the patient 10. The stem cell model of human cancer suggests that cure or season of remission a fter clinical treatment should correlate only with sidesplitting of stem cells. Assessment of treatment effects on an unselected cell population (eg on the basis or morphological criteria) is therefore possible to be misleading since the effects on a small population of stem cells will be masked by those on the large population of stem cells.Human tumors of a single histological type appear to have a pattern of response in vitro that is similar to their clinical behaviour. Within a histological type, tumours are heterogeneous in response both in vitro and in vivo. Studies directly comparing the response in vitro with the subsequent clinical response have shown important correlations. The proportion of human tumors that grow with a plating efficiency sufficient for assessment of drug activity (30 colonies per 500,000 cells plated is frequently less that 50%. unremarkably only a proportion of these tumors will manifest in vitro sensitivity 37. There have been a wide range of predic tive rate positives reported for the human clonogenic tumour cell assay when applied to a patient population with an expected clinical response rate of 15-49% 38. This determine could be misleading and in practice may only be workable for cytotoxicity testing for only one third of specimens tested. The limitation exits that not all samples will produce clones in vitro so those that do may exhibit a treatment bias 35. Other problems with the use and definition of human tumor clonogenic assays include low plating efficiency and small proportion of tumors available for testing difficulty in preparing single cell suspensions, production of only small quantities of data, and problems defining drug sensitivity and response criteria 35.Factors influencing size of sub-populationsIt has been proposed that as in normal cell populations, human tumor cell populations are also heterogeneous and comprise stem cells, non-stem transitional cells with limited proliferative capacity and end cells 6. MacKillop suggested that four factors may influence the relation back size of these subpopulationsThe probability of self-renewal (Psr) of stem cells (producing two daughter stem cells). The diffusion of cells within the system can be treated mathematically by assuming probability functions.The potential of the transitional cells for further cell division, as defined by clonal expansion number (n=number of generations between the first generation non-stem cells and the end cells.)The relative effect of cell loss on each subpopulation (Stem cells, transit amplifying, end cells) as described by cell loss factors (s, t ec).The number of generations of cell proliferation following initiation of the tumor cell population for individual stem cell.Stem cell division in normal tissue must provide a supply of differentiated functional cells to compensate for physiological losses and at the same time maintain a constant stem cell population. A probability of self-renewal in which two st em cells daughters Psr =0.5, would yield a steady state 28. If no cell loss occurs, it has been modelled that the number of stem cells will increase exponentially with Psr 0.5 6. For the simplest case in which all non-stem cells are end cells (n=0) the proportion of stem cells increases linearly with increasing Psr. and the proportion of stem cells in a tumor decreases as the extent of multiplication of the transitional cell compartment. This results in the stem cell being the less common cell type numerically than transit amplifying and differentiated end cells. These scenarios are affected by cell loss which may occur through necrosis, migration or differentiation, of which only differentiation is selective of cell type. A selective loss through differentiation increases the population of stem cells.The modelling of tumor cell growth has implications for the use of clonogenic assays as predictors of the stem cell fraction on human tumors, especially in regards to cut-off points i n terms of colony size and determining which cells represent the stem cell fraction 6. mingled with studies there are differences between how colonies are scored morphologically and numerically and how long cells are allowed to grow 31 and considering this evidence may be an important issue when comparing data between different studies.Clonogenicity in cell lines and stem cells in cell linesClonogenicity has freshly been used to identify stem cell properties of cells in long term culture cancer cell lines. The colony forming efficiency and secondary plating efficiency of carcinoma derived cell lines including head and make out squamous, breast 39 and prostate 39-42 were investigated and considered to contain potential stem cells. These studies show that cell lines show clear differences between clonal types (holoclone, meroclone, paraclone) and have similar properties in this respect to normal epithelial cells 39. The proportions of clonal types between the carcinoma cell lines v ary greatly. DU145 colonies were evenly spread in number between the clonal types, whereas PC3 cells produced primarily meroclones and LNCaP cells produced mainly paraclones 41, all based on colony morphology.These studies have also looked at the relationship between potential cancer stem cell markers and clonogenicity. CD133 enriched DU145 cells were assayed for clonogenicity, but no difference was found between the positive and disallow cells 41, but when isolated CD44+ integrin 21+ CD133+ sorted cells were compared against CD44+ integrin 21low CD133low a higher CFE was observed in conjunction with a marked difference in morphology to CD44+ integrin 21-/low CD133- in DU145 MACS sorted cells 40. Immunocytochemistry demonstrated that different clonal types showed varying levels of expression of CD44, 21 integrin and -catenin in PC-3 42 and DU145 clones 39. There is further evidence to suggest the presence of cells with stem cell behaviour such as dye-exclusion and higher clonogenic ity, in several human epithelial cell lines 39, 43-45, which further supports the idea that cell lines contain stem cells. The advantage of cancer cell lines that contain cells displaying stem cell characteristics would expedite the study of molecular pathways and the properties that define the cancer stem cells in vitro. new-fashioned DevelopmentsMuch progress has been made in the modelling of the leukemic diseases, where the level of heterogeneity was first and most thoroughly explored. Human cells fulfilling the properties expected of drug rebarbative cancer stem cells were initially isolated from blood cancers 2. Improvements in the genetics of recipient mice have led to the definition SCID-repopulating cell (SRC). Many improvements to the nod/SCID murine model continue to be made by using recipient mice that are engineered to be deficient in natural killer (NK) and macrophage activity part of that innate immune system. It has been demonstrated that a small subpopulation of a cute myeloid leukaemia cells with an immature immunophenotype possess the ability annex immune deficient NOD/SCID mice to give rise to more differentiated leukaemia cells and to recapitulate the heterogeneous phenotype of the bulk tumour 46. The phenotypically more mature cells failed to engraft in mice, suggesting the presence of an identifiable tumour cell hierarchy. These cells are referred to as tumour initiating cells.Cancer Stem Cell IdentificationCSCs have been defined on the basis of their ability to seed tumours in wolf hosts, to self renew and to spawn differentiated progeny (non-CSCs)47. Pioneering work in this area originated from studies on leukaemia stem cells and later included demonstrations of CSCs in solid tumours, particularly breast and caput cancers. However, work in solid tumours has proved challenging. The frequency of CSCs in solid tumours is highly variable 48.Difficulties with tumour CSC denominationEvidence for the existence of cancer stem cells in sol id tumours has been more difficult than in the haematopoietic system to obtain for several reasons 1) The cells within the tumour are less accessible. Tissue has to undergo mechanical or enzymatic digestion to obtain a single cell suspension which can be analysed. 2) There is a lack of functional assays suitable for observe and quantifying normal stem cells from many organs. 3) Only a few cell surface markers have been identified and characterised. Of these there is no one marker which is specific for a stem cell or cancer stem cells and for selection they often have to be used in combination.Cancer Stem Cell MarkersStem cells are most commonly identified by staining for cell surface markers, exclusion of fluorescent dyes or labelling with tritiated thymidine 3 . The technology to develop monoclonal antibodies to specific molecules and flow-cytometery based sorting and analysis has been a big driving force in recent CSC developments. Much work has been done to define cell surface markers. It has been shown that two distinct subpopulations can be separated from a single tumour that differ in their cell surface markers and their ability to seed new tumours in vivo. Most of the currently used markers do not recognise functional stem cell activity. By using combinations of cell surface markers, the homogenous purification of stem cells can be obtained 3. Table 1 below reviews the current suggested markers for some tumour types. The use of animal models has allowed identification and assessment of markers that are expressed by cancer stem cells. The most convincing demonstration of identity CSC selected by biomarkers comes from serial transplantation of cellular populations into animal models. The CSC containing fraction should re-establish the phenotypic characteristics of the original tumour 48. In 1997 thug et al showed that the ability to transfer human leukaemias into NOD/SCID mice was retained by a small proportion of cells with the CD34+, CD38- phenotype 46. The CD44 and CD133 markers have emerged as potential markers of immature epithelial cells for isolating CSCs in several tissue types including brain and prostate. Cells have been isolated from several tumour types and serially transplanted in heterograft models Breast CD44+ CD24-/low established tumours in recipient mice. Brain CD133+ enriched cells. prostate gland Side population CD44+ enriched. In these experiments small numbers of selected cells produced tumours in recipient mice. In this instance CSCs can on