Determination of somatic and cancer stem cell self-renewing symmetric division rate using sphere assays

Representing a renewable source for cell replacement, neural stem cells have received substantial attention in recent years. The neurosphere assay represents a method to detect the presence of neural stem cells, however owing to a deficiency of specific and definitive markers to identify them, their quantification and the rate they expand is still indefinite. Here we propose a mathematical interpretation of the neurosphere assay allowing actual measurement of neural stem cell symmetric division frequency. The algorithm of the modeling demonstrates a direct correlation between the overall cell fold expansion over time measured in the sphere assay and the rate stem cells expand via symmetric division. The model offers a methodology to evaluate specifically the effect of diseases and treatments on neural stem cell activity and function. Not only providing new insights in the evaluation of the kinetic features of neural stem cells, our modeling further contemplates cancer biology as cancer stem-like cells have been suggested to maintain tumor growth as somatic stem cells maintain tissue homeostasis. Indeed, tumor stem cell's resistance to therapy makes these cells a necessary target for effective treatment. The neurosphere assay mathematical model presented here allows the assessment of the rate malignant stem-like cells expand via symmetric division and the evaluation of the effects of therapeutics on the self-renewal and proliferative activity of this clinically relevant population that drive tumor growth and recurrence.     

A molecular view of stem cell and cancer cell self-renewal

With the recent advances in cell biology and molecular genetics, scientists were able to isolate and culture tissue-specific stem cells from various sources and define their properties. The challenge has now shifted to understanding the genetic programs controlling the stem cell state, i.e. self-renewal and multipotential. Cracking the molecular codes that govern the stem cell state turns out to be a difficult task. This is in part because a single gene may exhibit distinct activities when expressed in different cell types. Comprehending the cell-context dependent readout of any given gene requires an integrated knowledge of the complex cellular machinery, a platform which can be provided by the research on stem cells. This review is an attempt to formulate a model for the self-renewal machinery operating in stem cells and cancer cells. Insight into this issue at the molecular and cellular levels will no doubt facilitate the realization of the stem cell potential in both regenerative medicine and anticancer therapy.     

The cancer stem cell niche(s): The crosstalk between glioma stem cells and their microenvironment

Background

The initiation and progression of various types of tumors, including glioma, are driven by a population of cells with stem cell properties. Glioma stem cells (GSCs) are located in specialized microenvironments (niches) within tumors. These niches represent the hallmarks of malignant gliomas (vascular proliferations, hypoxia/necrosis) and bear analogy to the microenvironments in which physiological stem cells in the brain are found.

Scope of the review

Here we review the progress that has been made towards uncovering the function of the perivascular and the hypoxic niche and the molecular pathways that control the properties of GSCs within them. We propose models of how the different niches and GSC pools in them interact with each other.

Major conclusions

GSCs are not merely passive residents of their niches, but actively contribute to the shaping of the niches through a complex crosstalk with different components of the microenvironment. For example, GSCs play a dominant role in promoting new blood vessel formation through a variety of mechanisms, including the hypoxia dependent stimulation of angiogenesis, recruitment of endothelial progenitor cells and direct transdifferentiation into endothelial cells. Recent work has also revealed that GSCs can recruit and modulate the function of various immune cells to suppress anti-tumor immune responses and to foster tumor-promoting inflammation, which in turn could support the maintenance of GSCs.

General significance

These findings underscore the central role of the GSC microenvironment in driving glioma progression making the GSC niche a prime therapeutic target for the design of therapies aimed at eradicating GSCs.This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Cancer,stem cell misplacement and cancer stem cells

The cell of origin of cancer as well as cancer stem cells is still a mystery. In a recent issue of JCMM, Wang et al. challenged the conventional somatic genetic mutation model of multi‐stage carcinogenesis of breast cancer and proposed that ‘Invasive cancers are not necessary from preformed in situ tumours—an alternative way of carcinogenesis from misplaced stem cells’. If this stem cell misplacement theory could withstand future experimental evaluation, it may provide a paradigm shift in the prevention and management of cancer in the clinic.     

Human umbilical cord Wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Wharton's jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC‐CM) and cell lysate (hWJSC‐CL)] on breast adenocarcinoma (MDA‐MB‐231), ovarian carcinoma (TOV‐112D), and osteosarcoma (MG‐63) cells. The cells were treated with either hWJSC‐CM (50%) or hWJSC‐CL (15 µg/ml) for 48–72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC‐CL and hWJSC‐CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2–6% and 30–60%, while Transwell migration assay showed inhibition by 20–26% and 31–46% for hWJSC‐CM and hWJSC‐CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub‐G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV‐FITC and TUNEL positive cells seen in TOV‐112D and MDA‐MB‐231 suggested that inhibition was via apoptosis while the presence of anti‐BECLIN1 and anti‐LC3B antibodies seen with MG‐63 indicated autophagy. Upregulation of pro‐apoptotic BAX and downregulation of anti‐apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG‐63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts. J. Cell. Biochem. 113: 2027–2039, 2012. © 2012 Wiley Periodicals, Inc.     

A glassy-winged sharpshooter cell line supports replication of Rhopalosiphum padi virus (Dicistroviridae)

Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.     

Understanding cancer stem cell heterogeneity and plasticity

Heterogeneity is an omnipresent feature of mammalian cells in vitro and in vivo. It has been recently realized that even mouse and human embryonic stem cells under the best culture conditions are heterogeneous containing pluripotent as well as partially committed cells. Somatic stem cells in adult organs are also heterogeneous, containing many subpopulations of self-renewing cells with distinct regenerative capacity. The differentiated progeny of adult stem cells also retain significant developmental plasticity that can be induced by a wide variety of experimental approaches. Like normal stem cells, recent data suggest that cancer stem cells (CSCs) similarly display significant phenotypic and functional heterogeneity, and that the CSC progeny can manifest diverse plasticity. Here, I discuss CSC heterogeneity and plasticity in the context of tumor development and progression, and by comparing with normal stem cell development. Appreciation of cancer cell plasticity entails a revision to the earlier concept that only the tumorigenic subset in the tumor needs to be targeted. By understanding the interrelationship between CSCs and their differentiated progeny, we can hope to develop better therapeutic regimens that can prevent the emergence of tumor cell variants that are able to found a new tumor and distant metastases.     

A trans-platinum(II) complex induces apoptosis in cancer stem cells of breast cancer

Recent accumulating evidence has supported the notion that tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (CSCs), are responsible for tumor initiation, maintenance as well as drug resistance. Therefore, targeting the CSCs along with the other cancer cells has been the most important topic during the last decade. In the present study, we evaluated the cytotoxic activity of trans-[PtCl₂(2-hepy)₂] [2-hepy = 2-(2-hydroxyethyl) pyridine] complex and the mechanism of cell death in breast CSCs. Stemness markers, Oct-4 and Sox2, were determined in mammospheres by western blotting. Cytotoxicity was assessed using the ATP viability assay. Cell death was fluorescently visualized and further confirmed by flow cytometry as well as gene expression analysis. The Pt(II) complex significantly reduced the cell viability, prevented mammosphere formation and disrupted mammosphere structures in a dose-dependent manner (0–100 μM). The mode of cell death was apoptosis and it was shown by the presence of caspase 3/7 activity, Annexin V-FITC positivity, decreased mitochondrial membrane potential and increased expressions of pro-apoptotic genes (TNFRSF10A and HRK). Interestingly, necroptosis was also observed by the evidence of increased MLKL expression. In conclusion, the Pt(II) complex seems to be a highly promising anticancer compound due to its promising cytotoxic activity on CSCs. Therefore, it deserves in vivo further studies for the proof-of-concept.     

肿瘤干细胞研究进展

目前,癌症是导致人类死亡的主要因素之一。尽管在癌症治疗方面取得了巨大进展,但是,其较高的复发率还是会导致死亡。连续治疗失败的一个可能原因是,残留的恶性细胞有类似干细胞的分化潜能,这样就能再次形成肿瘤和造成病灶转移。肿瘤干细胞(cancer stem cell,CSC)假说认为,肿瘤组织中存在具有自我跟新能力,无限增殖和肿瘤形成能力的一小部分肿瘤细胞,近年来,随着在血液肿瘤和实体瘤中相继发现CSC存在的相关证据,对CSC的生物学特性的认识不断深入,对肿瘤的复发、病灶转移、耐药性形成也有了新的观点和研究方向,目前的研究主要集中在其分离鉴定阶段,本文就近年来该方面的研究进展作一综述。     

肿瘤干细胞的认知历程与研发热点

肿瘤组织中存在一小群能够自我更新、增殖和分化,对肿瘤的发生、发展、复发、转移起决定作用的细胞,即肿瘤干细胞（cancer stem cells,CSCs）。在传统理论方法已不能攻克癌症的情况下,肿瘤干细胞理论为我们重新认识肿瘤的起源和本质提供了新的方向和视角。从20世纪50年代至今,随着生物技术的发展,肿瘤干细胞理论经历了从设想到验证的漫长历程。但该理论自提出之日起便受到来自各方面不同观点的质疑。当今针对肿瘤干细胞癌症治疗主要集中在靶向问题上。因此,寻找特异的肿瘤干细胞标志物,探索肿瘤干细胞与周围微环境间的复杂关系以及发现调控其功能的关键信号通路成为当前研究的热点。     

Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.     

Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division

Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture.     

Regulating the balance between symmetric and asymmetric stem cell division in the developing brain

Stem cells proliferate through symmetric division or self-renew through asymmetric division whilst generating differentiating cell types. The balance between symmetric and asymmetric division requires tight control to either expand a stem cell pool or to generate cell diversity. In the Drosophila optic lobe, symmetrically dividing neuroepithelial cells transform into asymmetrically dividing neuroblasts. The switch from neuroepithelial cells to neuroblasts is triggered by a proneural wave that sweeps across the neuroepithelium. Here we review recent findings showing that the orchestrated action of the Notch, EGFR, Fat-Hippo, and JAK/STAT signalling pathways controls the progression of the proneural wave and the sequential transition from symmetric to asymmetric division. The neuroepithelial to neuroblast transition in the optic lobe bears many similarities to the switch from neuroepithelial cell to radial glial cell in the developing mammalian cerebral cortex. The Notch signalling pathway has a similar role in the transition from proliferating to differentiating stem cell pools in the developing vertebrate retina and in the neural tube. Therefore, findings in the Drosophila optic lobe provide insights into the transitions between proliferative and differentiative division in the stem cell pools of higher organisms.     

Regulating the balance between symmetric and asymmetric stem cell division in the developing brain

Stem cells proliferate through symmetric division or self-renew through asymmetric division whilst generating differentiating cell types. The balance between symmetric and asymmetric division requires tight control to either expand a stem cell pool or to generate cell diversity. In the Drosophila optic lobe, symmetrically dividing neuroepithelial cells transform into asymmetrically dividing neuroblasts. The switch from neuroepithelial cells to neuroblasts is triggered by a proneural wave that sweeps across the neuroepithelium. Here we review recent findings showing that the orchestrated action of the Notch, EGFR, Fat-Hippo, and JAK/STAT signalling pathways controls the progression of the proneural wave and the sequential transition from symmetric to asymmetric division. The neuroepithelial to neuroblast transition in the optic lobe bears many similarities to the switch from neuroepithelial cell to radial glial cell in the developing mammalian cerebral cortex. The Notch signalling pathway has a similar role in the transition from proliferating to differentiating stem cell pools in the developing vertebrate retina and in the neural tube. Therefore, findings in the Drosophila optic lobe provide insights into the transitions between proliferative and differentiative division in the stem cell pools of higher organisms.     

A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level. Cinzia Cocola, Sveva Sanzone and Simonetta Astigiano have contributed equally to this work.     

Matrix metalloproteinases in stem cell regulation and cancer

Since Gross and Lapiere firstly discovered matrix metalloproteinases (MMPs) as important collagenolytic enzymes during amphibian tadpole morphogenesis in 1962, this intriguing family of extracellular proteinases has been implicated in various processes of developmental biology. However, the pathogenic roles of MMPs in human diseases such as cancer have also garnered widespread attention. The most straightforward explanation for their role in cancer is that MMPs, through extracellular matrix degradation, pave the way for tumor cell invasion and metastasis. While this notion may be true for many circumstances, we now know that, depending on the context, MMPs may employ additional modes of functionality. Here, we will give an update on the function of MMPs in development and cancer, which may directly regulate signaling pathways that control tissue homeostasis and may even work in a non-proteolytic manner. These novel findings about the functionality of MMPs have important implications for MMP inhibitor design and may allow us to revisit MMPs as drug targets in the context of cancer and other diseases.     

Critical appraisal of the side population assay in stem cell and cancer stem cell research

The "Side Population" (SP) discrimination assay is a flow cytometry method used to detect stem cells based on the dye efflux properties of ABC transporters. We discuss the SP assay and its applications in stem cell?biology, with an emphasis on the technical challenges related to sample preparation, data acquisition, analysis, and interpretation. We highlight the value of multicolor phenotyping, the impact of DNA ploidy, and the importance of distinguishing graft versus host cells for an appropriate SP discrimination. To improve the consistency and reliability of data between laboratories, we propose a set of recommendations for SP assay data reporting.