Glucose-responsive insulin-producing cells from stem cells

Recent success with immunosuppression following islet cell transplantation offers hope that a cell transplantation treatment for type 1 (juvenile) diabetes may be possible if sufficient quantities of safe and effective cells can be produced. For the treatment of type 1 diabetes, the two therapeutically essential functions are the ability to monitor blood glucose levels and the production of corresponding and sufficient levels of mature insulin to maintain glycemic control. Stem cells can replicate themselves and produce cells that take on more specialized functions. If a source of stem cells capable of yielding glucose-responsive insulin-producing (GRIP) cells can be identified, then transplantation-based treatment for type 1 diabetes may become widely available. Currently, stem cells from embryonic and adult sources are being investigated for their ability to proliferate and differentiate into cells with GRIP function. Human embryonic pluripotent stem cells, commonly referred to as embryonic stem (ES) cells and embryonic germ (EG) cells, have received significant attention owing to their broad capacity to differentiate and ability to proliferate well in culture. Their application to diabetes research is of particular promise, as it has been demonstrated that mouse ES cells are capable of producing cells able to normalize glucose levels of diabetic mice, and human ES cells can differentiate into cells capable of insulin production. Cells with GRIP function have also been derived from stem cells residing in adult organisms, here referred to as endogenous stem cell sources. Independent of source, stem cells capable of producing cells with GRIP function may provide a widely available cell transplantation treatment for type 1 diabetes.     

Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells

By using two-color phenotypic analysis with fluorescein isothiocyanate- anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM- 102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein- 1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre- B cells in the presence of IL-7, but coculture of plasma cells with KM- 102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF- beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL- 7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.     

Differentiation of mesodermal cells from pluripotent stem cells

The pluripotency of embryonic stem cells has been well demonstrated by a vast variety of studies showing the induction of differentiation into desired cell types that have the potential to be used not only in basic studies but also in medical applications. The induction of mesodermal cells, especially blood cells, from embryonic stem cells is notable from the point of view of transplantation, and the methods for this induction have improved over the last few years, with more defined culture conditions in place. Concurrently, the generation of induced pluripotent stem cells from somatic cells opens the possibility of autologous transplantation. In fact, there are a growing number of reports demonstrating that several mesodermal cells can be differentiated from induced pluripotent stem cells using the same methods used for embryonic stem cells. This review summarizes recent advances in the differentiation of mesodermal cells from embryonic stem cells and induced pluripotent stem cells.     

Insulin expressing cells from differentiated embryonic stem cells are not beta cells

Aim/hypothesis Embryonic stem (ES) cells have been proposed as a potential source of tissue for transplantation for the treatment of Type 1 diabetes. However, studies showing differentiation of beta cells from ES cells are controversial. The aim of this study was to characterise the insulin-expressing cells differentiated in vitro from ES cells and to assess their suitability for the treatment of diabetes.Methods ES cell-derived insulin-expressing cells were characterised by means of immunocytochemistry, RT-PCR and functional analyses. Activation of the Insulin I promoter during ES-cell differentiation was assessed in ES-cell lines transfected with a reporter gene. ES cell-derived cultures were transplanted into STZ-treated SCID-beige mice and blood glucose concentrations of diabetic mice were monitored for 3 weeks.Results Insulin-stained cells differentiated from ES cells were devoid of typical beta-cell granules, rarely showed immunoreactivity for C-peptide and were mostly apoptotic. The main producers of proinsulin/insulin in these cultures were neurons and neuronal precursors and a reporter gene under the control of the insulin I promoter was activated in cells with a neuronal phenotype. Insulin was released into the incubation medium but the secretion was not glucose-dependent. When the cultures were transplanted in diabetic mice they formed teratomas and did not reverse the hyperglycaemic state.Conclusions/Interpretation Our studies show that insulin-positive cells in vitro-differentiated from ES cells are not beta cells and suggest that alternative protocols, based on enrichment of ES cell-derived cultures with cells of the endodermal lineage, should be developed to generate true beta cells for the treatment of diabetes.Abbreviations ES Embryonic stem - LIF leukemia inhibitory factor - ITSF insulin-transferrin-selenite-fibronectin.Bleackley and Korbutt laboratories contributed equally to this paper     

Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells

We have previously reported that stressed apoptotic tumor cells are more immunogenic in vivo than nonstressed ones. Using confocal microscopy we have confirmed our previous observation that heat-stressed apoptotic 12B1-D1 leukemia cells (BCR-ABL(+)) express HSP60 and HSP72 on their surface. To explore how the immune system distinguishes stressed from nonstressed apoptotic tumor cells, we analyzed the responses of dendritic cells to these 2 types of apoptotic cells. We found that nonstressed and heat-stressed apoptotic 12B1-D1 cells were taken up by dendritic cells in a comparable fashion. However, when stressed apoptotic 12B1-D1 cells were coincubated with immature dendritic cells for 24 hours, this resulted in greater up-regulation of costimulatory molecules (CD40, CD80, and CD86) on the surface of dendritic cells. Moreover, stressed apoptotic 12B1-D1 cells were more effective in stimulating dendritic cells to secrete interleukin-12 (IL-12) and in enhancing their immunostimulatory functions in mixed leukocyte reactions. Furthermore, we demonstrated that immunization of mice with stressed apoptotic 12B1-D1 cells induced the secretion of T helper-1 (T(H)1) profile of cytokines by spleen cells. Splenocytes from mice immunized with stressed apoptotic cells, but not nonstressed ones, were capable of lysing 12B1-D1 and the parental 12B1 line, but not a B-cell leukemia line, A20. Our data indicate that stressed apoptotic tumor cells are capable of providing the necessary danger signals, likely through increased surface expression of heat shock proteins (HSPs), resulting in activation/maturation of dendritic cells and, ultimately, the generation of potent antitumor T-cell responses.     

Endothelial cells derived from human embryonic stem cells

Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.     

Multistep differentiation of GH-producing cells from their immature cells

In order to study GH cell differentiation, we used the clonal cell lines called MtT/E and MtT/S cells, which were derived from a rat mammotrophic pituitary tumor. Although MtT/E cells are non-hormone-producing ones, Pit-1 protein is present in their nuclei, which suggests that MtT/E cells are progenitor cells of the Pit-1 cell lineage and have the potential to differentiate into hormone-producing cells. On the other hand, MtT/S cells produce GH; however, the responsiveness to GH-releasing hormone (GHRH) is weak and only a small number of secretory granules are present in their cytoplasm, which suggests that MtT/S cells are premature GH cells. In order to differentiate into GH cells from MtT/E cells as a progenitor cell, we examined several differentiation factors and found that retinoic acid (RA) induced the differentiation of MtT/E cells into GH-producing cells. RA-induced GH cells partially matured with the glucocorticoid treatment; however, the responsiveness to GHRH on GH secretion was incomplete. In order to elucidate the mechanism underlying full differentiation of GH cells, we used MtT/S cells. We treated MtT/S cells with glucocorticoid and found that they differentiated into mature GH cells with many secretory granules in their cytoplasm and they responded well to GHRH. These results suggested that MtT/E and MtT/S cells are progenitor or premature GH cells, and show different responses to differentiation factors. Our data also suggested that GH cells differentiate from their progenitor cells through multistep processes.     

Natural killer cells and natural killer T cells

NK cells are important in protecting against viral infections, and they may regulate the immune response. They are activated by hematopoietic blasts and pose a barrier to bone marrow transplantation. They are also abundant in the pregnant uterine decidua, although their role there is unknown. NK cells are normally inhibited from responding to host cells by inhibitory receptors that recognize self class I MHC antigens. There is evidence that NK cells may be important in the regulation of autoimmunity, but there is even stronger evidence that NKT cells regulate autoimmunity. The mechanisms by which these cells are activated and by which they regulate other cells are now being understood at the molecular level.     

Endothelial cells as cytotoxic effector cells: cytokine-activated rat islet endothelial cells lyse syngeneic islet cells via nitric oxide

Summary In vivo, each beta cell is located in proximity to at least one capillary islet endothelial cell. Rat aorta and islet endothelial cells can be activated in vitro to express inducible nitric oxide synthase by a cytokine mixture of tumour necrosis factor-α, gamma-interferon, and interleukin-1β and to produce high concentrations of nitric oxide. We have performed co-culture experiments with rat islet endothelial cells together with isolated syngeneic islet cells at low target : effector ratios with or without previous cytokine challenge of endothelial cultures. Co-cultures were always free of exogenous cytokines, which were removed prior to addition of islet cells. We found that pre-activated, in contrast to resident islet endothelial cells, at a target : effector ratio as low as 1 : 1 almost completely lysed syngeneic beta and non-beta cells within 24 h of co-culture. Lysis by pre-activated islet endothelial cells was found to be preceded by DNA damage found in 46 % of islet cells after 8 h of co-culture with pre-activated vs 7 % with resting islet endothelial cells. Lysis was blocked to control levels in the presence of the nitric oxide synthase inhibitor N^G-methyl-L-arginine. With the results presented here, we demonstrate for the first time, that activated endothelial lining cells can express effector cell activity and thus can contribute to local tissue destruction, especially in organs that are densely capillarized such as pancreatic islets. [Diabetologia (1997) 40: 150–155] Received: 2 September 1996 and in revised form: 24 October 1996     

Phenotypic characteristics of T cells interacted with synovial cells

We demonstrated the phenotypic characteristics of T cells interacted with synovial fibroblast-like cells. A small percentage of peripheral blood T cells adhered to synovial fibroblast-like cells. When synovial cells were treated with interferon-gamma or interleukin-1 beta, the percentage of T cells that adhered to the treated cells markedly increased in comparison with the value for untreated synovial cells. The kinesis of T cell adherence to treated synovial cells differed from that of HLA-DR antigen expression on synovial cells. T cell adherence was not blocked by mouse monoclonal anti-HLA-DR and anti-HLA-ABC antibodies. The phenotypes of the adherent and nonadherent T cells were investigated with a flow cytometer. The CD29 + subset was more adhesive than the CD45RA + subset to IL-1 beta-stimulated synovial cells. The proportions of high density lymphocyte function associated antigen (LFA)-1 alpha and LFA-1 beta were greater in the adherent than in the nonadherent T cells, and the mean fluorescence intensities of LFA-1 alpha, LFA-1 beta and CD2 molecules on adherent T cells were significantly higher than those on nonadherent T cells. Our results support the concept that an interaction between infiltrating lymphocytes and synovial cells occurs in the synovium, resulting in the initiation and perpetuation of immune responses in synovial tissue in rheumatoid arthritis.     

侧群细胞与肝癌干细胞

肝癌是严重威胁人类生命和健康的一种疾病.其病因和发病机制尚不完全清楚,治疗缺少有效靶点.对肝癌恶性生长、转移及复发机制的研究正在逐渐深入.近年来的研究认为,肿瘤中存在一小群具有自我更新和分化潜能的细胞,即肿瘤干细胞,可能是肿瘤转移和复发的根源.肝癌中应同样存在这样的一群细胞.侧群(side population,SP)细胞是肿瘤细胞中一小部分,具备干细胞的多种特性且易于分离.肝癌组织中SP细胞的鉴定和分离有可能找到肝癌干细胞,有助于肝癌的转移和复发机制的研究,并为肝癌治疗提供有效治疗靶点.     

B-lymphoid cells with attributes of dendritic cells regulate T cells via indoleamine 2,3-dioxygenase

A discrete population of splenocytes with attributes of dendritic cells (DCs) and coexpressing the B-cell marker CD19 is uniquely competent to express the T-cell regulatory enzyme indoleamine 2,3-dioxygenase (IDO) in mice treated with TLR9 ligands (CpGs). Here we show that IDO-competent cells express the B-lineage commitment factor Pax5 and surface immunoglobulins. CD19 ablation abrogated IDO-dependent T-cell suppression by DCs, even though cells with phenotypic attributes matching IDO-competent cells developed normally and expressed IDO in response to interferon γ. Consequently, DCs and regulatory T cells (Tregs) did not acquire T-cell regulatory functions after TLR9 ligation, providing an alternative perspective on the known T-cell regulatory defects of CD19-deficient mice. DCs from B-cell–deficient mice expressed IDO and mediated T-cell suppression after TLR9 ligation, indicating that B-cell attributes were not essential for B-lymphoid IDO-competent cells to regulate T cells. Thus, IDO-competent cells constitute a distinctive B-lymphoid cell type with quintessential T-cell regulatory attributes and phenotypic features of both B cells and DCs.     

Recipient elimination of allogeneic lymphoid cells: donor CD4(+) cells are effective alloantigen-presenting cells

The encounter with allogeneic major histocompatibility complex (MHC) molecules expressed on donor leukocytes during transfusion of blood products has been shown to impact the recipient's immune responses in a number of settings. To better understand the responses induced by the transfer of allogeneic cells, a murine model was used to characterize the recipient responses that control the fate of the allogeneic lymphoid cells. Recipient CD8(+) cells could rapidly eliminate a large number of donor cells within 3 days after injection. When elimination responses were studied in the absence of CD8(+) cells, it was found that alloantibody production was the secondary elimination mechanism. Optimal recipient CD8(+) and B cell responses in this model required help from CD4(+) cells that could be provided by 3 different pathways. Although recipient CD4(+) cells could provide help when activated by direct recognition of allogeneic MHC class II molecules expressed on donor cells or by indirect recognition of processed alloantigen presented on recipient antigen-presenting cells (APCs), the most rapid recipient responses were generated by help provided by donor CD4(+) cells. Purified donor CD4(+) cells were also able to induce these rapid responses, indicating that activated donor CD4(+) cells expressing allogeneic MHC molecules were able to effectively stimulate responses by both recipient CD8(+) and B cells.     

Stem cells in human normal endometrium and endometrial cancer cells: characterization of side population cells

Recently, adult stem cells have been identified in several mature tissues. The human endometrium is responsive to sex steroid hormone. It undergoes extraordinary growth in a cyclic manner and is shed and regenerated throughout a woman's lifetime. It has been proposed that the human endometrium may contain a population of stem cells, which are responsible for its remarkable regenerative ability. It is also suggested that stem-like cells exist in cancer tissues. Stem-like cell subpopulations, referred to as "side population" (SP) cells, have been identified in several tissues and tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Recently, we isolated and characterized the SP cells in normal human endometrium and in an endometrial cancer (EC) cell line. Endometrial SP cells can function as progenitor cells. EC SP cells show the following: (1) reductions in the expression levels of differentiation markers; (2) long-term repopulating properties; (3) self-renewal capacity; (4) enhancement of migration and podia formation; (5) enhancement of tumorigenicity; and (6) bipotent developmental potential (tumor cells and stroma-like cells), suggesting that these SP cells have cancer stem-like cell features. We review the articles that show the presence of stem cells in normal endometrium and EC cells and demonstrate the results of our studies.     

Organ-derived dendritic cells have differential effects on alloreactive T cells

Dendritic cells (DCs) are considered critical for the induction of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In addition to their priming function, dendritic cells have been shown to induce organ-tropism through induction of specific homing molecules on T cells. Using adoptive transfer of CFSE-labeled cells, we first demonstrated that alloreactive T cells differentially up-regulate specific homing molecules in vivo. Host-type dendritic cells from the GVHD target organs liver and spleen or skin- and gut-draining lymph nodes effectively primed naive allogeneic T cells in vitro with the exception of liver-derived dendritic cells, which showed less stimulatory capacity. Gut-derived dendritic cells induced alloreactive donor T cells with a gut-homing phenotype that caused increased GVHD mortality and morbidity compared with T cells stimulated with dendritic cells from spleen, liver, and peripheral lymph nodes in an MHC-mismatched murine BMT model. However, in vivo analysis demonstrated that the in vitro imprinting of homing molecules on alloreactive T cells was only transient. In conclusion, organ-derived dendritic cells can efficiently induce specific homing molecules on alloreactive T cells. A gut-homing phenotype correlates with increased GVHD mortality and morbidity after murine BMT, underlining the importance of the gut in the pathophysiology of GVHD.     

Differentiation of human embryonic stem cells and human induced pluripotent stem cells into steroid-producing cells

Although there have been reports of the differentiation of mesenchymal stem cells and mouse embryonic stem (ES) cells into steroid-producing cells, the differentiation of human ES/induced pluripotent stem (iPS) cells into steroid-producing cells has not been reported. The purpose of our present study was to establish a method for inducing differentiation of human ES/iPS cells into steroid-producing cells. The first approach we tried was embryoid body formation and further culture on adherent plates. The resultant differentiated cells expressed mRNA encoding the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, CYP17A1, and CYP19, and secreted progesterone was detected in the cell medium. However, expression of human chorionic gonadotropin was also detected, suggesting the differentiated cells were trophoblast like. We next tried a multistep approach. As a first step, human ES/iPS cells were induced to differentiate into the mesodermal lineage. After 7 d of differentiation induced by 6-bromoindirubin-3'-oxime (a glycogen synthase kinase-3β inhibitor), the human ES/iPS cells had differentiated into fetal liver kinase-1- and platelet derived growth factor receptor-α-expressing mesodermal lineage cells. As a second step, plasmid DNA encoding steroidogenic factor-1, a master regulator of steroidogenesis, was introduced into these mesodermal cells. The forced expression of steroidogenic factor-1 and subsequent addition of 8-bromoadenosine 3',5'-cyclic monophosphate induced the mesodermal cells to differentiate into the steroidogenic cell lineage, and expression of CYP21A2 and CYP11B1, in addition to steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, CYP11A1, and CYP17A1, was detected. Moreover, secreted cortisol was detected in the medium, but human chorionic gonadotropin was not. These findings indicate that the steroid-producing cells obtained through the described multistep method are not trophoblast like; instead, they exhibit characteristics of adrenal cortical cells.     

Erythroid-like cells from neural stem cells injected into blastocysts

OBJECTIVE: In contrast to embryonic stem (ES) cells, which are able to give rise to all cell types of the body, somatic stem cells have been thought to be more limited in their differentiation potential in that they are committed to generate only cells of their tissue of origin. Unexpectedly, some recent data suggest that somatic stem cells isolated from one tissue can also generate cells of heterologous tissues and organs, implying that somatic stem cells have a greater potential for differentiation. METHODS: To explore further the developmental potential of murine neural stem cells (NSCs) we injected cultured NSCs as neurospheres into preimplantation blastocysts and determined the seeding by donor cells in tissues of developing chimeric fetal and adult animals. RESULTS: We frequently detected progeny of injected NSCs both in embryos and in adult animals. In embryos we observed transient seeding of donor cells to hematopoietic tissues and generation of NSC-derived cells that express globin genes and an erythroid-specific cell-surface marker. In adults progeny of NSCs were mostly detected in neural tissues. The observed low level of chimerism of wild-type NSCs was increased if we injected stem cells expressing a bcl-2 transgene, without changing the seeding pattern. CONCLUSION: These results suggest that cultured NSCs, following their injection into blastocysts, generate at mid-gestation erythroid-like cells but later, in adult chimeric mice, engraftment mainly persisted in neural tissues.     

Stem cells

Stem cells derived from adult and embryonic sources have great therapeutic potential, but much research is still needed before their clinical use becomes commonplace. There is debate about whether adult stem cells can be used instead of those derived from embryos. Rationalisation is needed but can be exercised only once the various cells have been carefully compared and contrasted under appropriate experimental conditions. Some characteristics that might help resolve the issue of cell source can already be applied to the debate. Accessibility is important; some adult cells, such as neural stem cells, are difficult to obtain, at least from living donors. Other factors include the frequency and abundance of adult stem cells and their numbers and potency, which might decline with age or be affected by disease. For embryonic stem cells, ethical concerns have been raised, and the proposed practice of therapeutic cloning tends to be misrepresented in the lay media. For both adult and embryonic stem cells, stability, potential to transmit harmful pathogens or genetic mutations, and risk of forming unwanted tissues or even teratocarcinomas have yet to be fully assessed.     

Cancer stem cells

There is an increasing evidence supporting the cancer stem cell hypothesis. Normal stem cells in the adult organism are responsible for tissue renewal and repair of aged or damaged tissue. A substantial characteristic of stem cells is their ability for self-renewal without loss of proliferation capacity with each cell division. The stem cells are immortal, and rather resistant to action of drugs. They are able to differentiate and form specific types of tissue due to the influence of microenvironmental and some other factors. Stem cells divide asymmetrically producing two daughter cells -- one is a new stem cell and the second is progenitor cell, which has the ability for differentiation and proliferation, but not the capability for self-renewal. Cancer stem cells are in many aspects similar to the stem cells. It has been proven that tumor cells are heterogeneous comprising rare tumor initiating cells and abundant non-tumor initiating cells. Tumor initiating cells -- cancer stem cells have the ability of self-renewal and proliferation, are resistant to drugs, and express typical markers of stem cells. It is not clear whether cancer stem cells originate from normal stem cells in consequence of genetic and epigenetic changes and/or by redifferentiation from somatic tumor cells to the stem-like cells. Probably both mechanisms are involved in the origin of cancer stem cells. Dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. Isolation and identification of cancer stem cells in human tumors and in tumor cell lines has been successful. To date, the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia, in breast cancer, in brain tumors, in lung cancer and gastrointestinal tumors. Cancer stem cell model is also consistent with some clinical observations. Although standard chemotherapy kills most cells in a tumor, cancer stem cells remain viable. Despite the small number of such cells, they might be the cause of tumor recurrence, sometimes many years after the "successful" treatment of primary tumor. Growth of metastases in distinct areas of body and their cellular heterogeneity might be consequence of cancer stem cell differentiation and/or dedifferentiation and asymmetric division of cancer stem cells. Further characterization of cancer stem cells is needed in order to find ways to destroy them, which might contribute significantly to the therapeutic management of malignant tumors.