Molecular phenotype of airway side population cells

Lung epithelial-specific stem cells have been localized to discrete microenvironments throughout the adult conducting airway. Properties of these cells include pollutant resistance, multipotent differentiation, and infrequent proliferation. Goals of the present study were to use Hoechst 33342 efflux, a property of stem cells in other tissues, to purify and further characterize airway stem cells. Hoechst 33342 effluxing lung cells were identified as a verapamil-sensitive side population by flow cytometry. Lung side population cells were further subdivided on the basis of hematopoietic (CD45 positive) or nonhematopoietic (CD45 negative) origin. Nonhematopoietic side population cells were enriched for stem cell antigen-1 reactivity and expressed molecular markers specific to both airway and mesenchymal lineages. Analysis of the molecular phenotype of airway-derived side population cells indicates that they are similar to neuroepithelial body-associated variant Clara cells. Taken together, these data suggest that the nonhematopoietic side population isolated from lung is enriched for previously identified airway stem cells.     

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.     

'Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells

Stem cells in various somatic tissues (bone marrow, skeletal muscle) can be identified by the 'Side Population' marker based on Hoechst 33342 efflux. We show that mouse testicular cells also display a 'Side Population' that express Bcrp1 mRNA, the ABC transporter responsible for Hoechst efflux in hematopoietic cells. Inhibition of Hoechst efflux by specific BCRP1 inhibitor Ko143 show that germinal 'Side Population' phenotype is dependent on BCRP1 activity. Analysis of two well-defined models of altered spermatogenesis (W/Wv mutants and cryptorchid male mice) and RNA expression studies of differentiation markers demonstrate that germinal 'Side Population' contains spermatogonial cells. In addition, alpha 6-integrin and Stra8 germinal stem cell markers, are expressed in the 'Side Population'. In vivo repopulation assay clearly establishes that testis 'Side Population' in adult mice is highly enriched in male germ stem cells.     

Expression of Integrin beta3 is correlated to the properties of quiescent hemopoietic stem cells possessing the side population phenotype

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin beta(3) (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin beta(3) is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage(-) (Lin(-))/integrin beta(3)(high) (beta(3)(high)) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit(+)/Sca-1(+)/Lin(-) SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin beta(3) subunit that is present in SP cells having the properties of HSCs, is associated with integrin alpha(v) (CD51). Therefore, our results demonstrate that high expression of integrin beta(3) is correlated to the properties of quiescent HSCs and suggest that the integrin beta(3) subunit is available as a common surface marker of tissue-specific stem cells.     

Murine fertilized ovum,blastomere and morula cells lacking SP phenotype

In the field of stem cell research, SP (side population) phenotype is used to define the property that cells maintain a high efflux capability for some fluorescent dye, such as Hoechst 33342. Recently, many researches proposed that SP phenotype is a phenotype shared by some stem cells and some progenitor cells, and that SP phenotype is regarded as a candidate purification marker for stem cells. In this research, murine fertilized ova (including conjugate and single nucleus fertilized ova), 2-cell stage and 8-cell stage blastomeres, morulas and blastocysts were isolated and directly stained by Hoechst 33342 dye. The results show that fertilized ovum, blastomere and morula cells do not demonstrate any ability to efflux the dye. However, the inner cell mass (ICM) cells of blastocyst exhibit SP phenotype, which is consistent with the result of embryonic stem cells (ESCs) in vitro. These results indicate that the SP phenotype of ICM-derived ESCs is an intrinsic property and independent of the culture condition in vitro, and that SP phenotype is one of the characteristics of at least some pluripotent stem cells, but is not shared by totipotent stem cells. In addition, the result that the SP phenotype of ICM cells disappeared when the inhibitor verapamil was added into medium implies that the SP phenotype is directly associated with ABCG2. These results suggest that not all the stem cells demonstrate SP phenotype, and that SP phenotype might act as a purification marker for partial stem cells such as some pluripotent embryonic stem cells and multipotent adult stem cells, but not for all stem cells exampled by the totipotent stem cells in the very early stage of mouse embryos.     

Zebrafish kidney marrow contains ABCG2-dependent side population cells exhibiting hematopoietic stem cell properties

Zebrafish (Danio rerio) has emerged as a powerful genetic model for the study of vertebrate hematopoiesis. However, methods for detection and isolation of hematopoietic stem cells (HSCs) have not yet been reported. In mammals, the combination of Hoechst 33342 staining with flow cytometry can be used for separation of a bone marrow side population (SP), which is highly enriched for HSCs. We applied a similar procedure to hematopoietic kidney marrow cells from adult zebrafish, and identified a segregated cohort of SP cells, which demonstrate a set of features typical of stem cells. SP cells show extremely low scatter characteristics, and are small in size with a minimum of cytoplasm. Treatment of zebrafish kidney marrow cells with reserpine or fumitremorgin C, which inhibit the ABCG2 transporter responsible for Hoechst 33342 efflux, caused a clear reduction in the number of SP cells. Consistent with the quiescent state of HSCs, the SP cells are strongly resistant to the myelosuppressive agent 5-fluorouracil. In addition, SP cells specifically demonstrate higher expression of genes known to be markers of HSCs of mammals. Hence, our results show that the SP phenotype is conserved between mammals and teleosts, and the properties of the zebrafish SP cells indicate a significant enrichment for HSCs. These rapid flow cytometric methods for purification of HSCs from zebrafish may greatly facilitate genetic analysis of stem cells using the advantages of this vertebrate model.     

Murine fertilized ovum, blastomere and morula cells lacking SP phenotype

In the field of stem cell research, SP (side population) phenotype is used to define the property that cells maintain a high efflux capability for some fluorescent dye, such as Hoechst 33342. Recently, many researches proposed that SP phenotype is a phenotype shared by some stem cells and some pro- genitor cells, and that SP phenotype is regarded as a candidate purification marker for stem cells. In this research, murine fertilized ova (including conjugate and single nucleus fertilized ova), 2-cell stage and 8-cell stage blastomeres, morulas and blastocysts were isolated and directly stained by Hoechst 33342 dye. The results show that fertilized ovum, blastomere and morula cells do not demonstrate any ability to efflux the dye. However, the inner cell mass (ICM) cells of blastocyst exhibit SP phenotype, which is consistent with the result of embryonic stem cells (ESCs) in vitro. These results indicate that the SP phenotype of ICM-derived ESCs is an intrinsic property and independent of the culture condition in vitro, and that SP phenotype is one of the characteristics of at least some pluripotent stem cells, but is not shared by totipotent stem cells. In addition, the result that the SP phenotype of ICM cells disap- peared when the inhibitor verapamil was added into medium implies that the SP phenotype is directly associated with ABCG2. These results suggest that not all the stem cells demonstrate SP phenotype, and that SP phenotype might act as a purification marker for partial stem cells such as some pluripo- tent embryonic stem cells and multipotent adult stem cells, but not for all stem cells exampled by the totipotent stem cells in the very early stage of mouse embryos.     

Growth and differentiation of progenitor/stem cells derived from the human mammary gland

Estrogen is necessary for the full development of the mammary gland and it is also involved in breast cancer development. We set out to identify and characterise progenitor/stem cells in the human mammary gland and to explore the role of estrogen in their proliferation and differentiation. Three candidate stem cell populations were isolated: double positive (DP) cells co-expressed the luminal and myoepithelial markers, EMA and CALLA, respectively, whereas double negative (DN) cells did not express these cell surface markers; side population (SP) cells were characterised by their differential ability to efflux the dye Hoechst 33342. The ABC transporter, breast cancer resistance protein (BCRP) was more highly expressed in SP cells than in non-SP cells and a specific BCRP inhibitor, Ko143, reduced SP formation, suggesting that BCRP confers the SP phenotype in mammary epithelial cells, as has been demonstrated in other tissues. Interestingly, SP cells were double negative for the EMA and CALLA antigens and therefore represent a separate and distinct population to DP cells. Single cell multiplex RT-PCR indicated that the SP and DN cells do not express detectable levels of ERalpha or ERbeta, suggesting that estrogen is not involved in their proliferation. DP cells expressed ERalpha but at a lower level than differentiated luminal cells. These findings invoke a potential strategy for the breast stem/progenitor cells to ignore the mitogenic effects of estrogen. All three cell populations generated mixed colonies containing both luminal and myoepithelial cells from a single cell and therefore represent candidate multipotent stem cells. However, DN cells predominately generated luminal colonies and exhibited a much higher cloning efficiency than differentiated luminal cells. Further characterisation of these candidate progenitor/stem cells should contribute to a better understanding of normal mammary gland development and breast tumorigenesis.     

Akt signaling regulates side population cell phenotype via Bcrp1 translocation

Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp(-/-) mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1.     

A putative human breast stem cell population is enriched for steroid receptor-positive cells

Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen and progesterone receptor (ERalpha and PR), we examined the biology of these ERalpha/PR-positive cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing the steroid receptors ERalpha and PR. Using DNA radiolabelling in human tissue implanted into athymic nude mice, a population of label-retaining cells were shown to be enriched for the putative stem cell markers p21(CIP1) and Msi-1, the human homolog of Drosophila Musashi. Steroid receptor-positive cells were found to co-express these stem cell markers together with cytokeratin 19, another putative stem cell marker in the breast. Human breast epithelial cells with Hoechst dye-effluxing "side population" (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated "intermediate" cells by lack of expression of myoepithelial and luminal apical membrane markers. These SP cells were 6-fold enriched for ERalpha-positive cells and expressed several fold higher levels of the ERalpha, p21(CIP1) and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered steroid receptor-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells.     

Characterization of Side Populations in HNSCC: Highly Invasive,Chemoresistant and Abnormal Wnt Signaling

Side Population (SP) cells, a subset of Hoechst-low cells, are enriched with stem cells. Originally, SP cells were isolated from bone marrow but recently have been found in various solid tumors and cancer cell lines that are clonogenic in vitro and tumorigenic in vivo. In this study, SP cells from lymph node metastatic head and neck squamous cell carcinoma (HNSCC) cell lines were examined using flow cytometry and Hoechst 3342 efflux assay. We found that highly metastatic HNSCC cell lines M3a2 and M4e contained more SP cells compared to the low metastatic parental HNSCC cell line 686LN. SP cells in HNSCC were highly invasive in vitro and tumorigenic in vivo compared to non-SP cells. Furthermore, SP cells highly expressed ABCG2 and were chemoresistant to Bortezomib and etoposide. Importantly, we found that SP cells in HNSCC had abnormal activation of Wnt/β-catenin signaling as compared to non-SP cells. Together, these findings indicate that SP cells might be a major driving force of head and neck tumor formation and metastasis. The Wnt/β-catenin signaling pathway may be an important target for eliminating cancer stem cells in HNSCC.     

一种新型干细胞--侧群细胞

侧群细胞(side population cell)是利用Hoechst染料和流式细胞术进行造血千／祖细胞分离时发现的一群特殊细胞,广泛分布于多种成体组织、胚胎和某些肿瘤细胞系中;它既具有类似千细胞的自我更新和多向分化潜能,还具有独特的表型标记和生物学特征,代表了一种新的千细胞类型。对侧群细胞的研究,不仅有助于人们增加对千细胞增殖、分化及其发育调控机制的理解,同时还提供了一种从不同组织中分离纯化和利用多能干细胞的新策略,为组织工程和细胞治疗提供新的千细胞材料来源。现就侧群细胞的组织分布、生物学特征、表型标记、信号转导机制及其与肿瘤发生相关性等方面的研究进展进行了综述,并对侧群细胞的进一步研究和应用作了展望。     

Identification of side population cells in mouse primordial germ cells and prenatal testis

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.     

Kinetic analyses as a critical parameter in defining the side population (SP) phenotype

The side population (SP) phenotype has been reported as a method to identify hematopoietic stem cells in the bone marrow based upon differential staining with the fluorescent dye, Hoechst 33342. This technique has drawn great interest in the stem cell community, as it may provide a simple approach to the enrichment of progenitor cells from a variety of normal and malignant tissues. The frequency of these cells and their performance in functional assays has varied considerably within the literature. To investigate mechanisms that may contribute to the SP phenotype, we measured the fluorescence emission of Hoechst-stained bone marrow cells as a function of both time and dye concentration using a custom flow cytometer and data acquisition software. These measurements demonstrate that all nucleated cells within the bone marrow undergo an identical staining pattern at varying rates, even under conditions previously reported to abrogate the SP. Therefore, the SP phenotype is not unique to stem cells, but rather represents a transient feature of marrow cells exposed to Hoechst 33342 for varying amounts of time. We propose that heterogeneity of SP-defined populations may be a consequence of the rate at which differing cell populations accumulate Hoechst 33342. Further, we suggest that dye uptake kinetics will likely be an important factor for optimal use of Hoechst 33342 in isolating stem cells.     

Isolation and characterization of human mammary stem cells

Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.