Genesis Of Stem Cells Research Articles

Claudin-6 increases SNAI1, NANOG and SOX2 gene expression in human gastric adenocarcinoma AGS cells.

Gastric cancer is a heterogeneous disease associated to deregulated gastric epithelia tight junction barrier function and di novo expression of claudin-6; these changes are associated with epithelial-mesenchymal transition, enhanced invasiveness, metastatic progression, resistance to chemotherapy, and poor prognosis. Gastric cancer stem cells represent a rare population of cells within the tumor implicated in tumor growth and higher tumorigenic capacity. The possible relation between claudin-6 expression and the expression of some markers associated to epithelial mesenchymal transition and cancer stem cells in gastric cancer cells have never been explored. CD44, CD24, Twist, Villin, DCLK1, claudin-6, NANOG, E-Cadherin, SOX2, and SNAI1 expression was evaluated by immunofluorescence and cytofluorometry in wild type and Claudin-6 transfected AGS cells. Cell migration assays were also performed. Differentially expressed genes and biological processes analysis was performed to determine gene preponderance. The results showed that claudin-6 overexpression enriched the CD44 + /CD24- subpopulation with an overall increase in the expression and the number of CD44 + cells. A significant increase in NANOG, SOX2 and SNAI1 expression and enhanced cell migration was observed in claudin-6 transfected cells. Transcriptome analysis revealed 271 genes involved in enhanced biological processes with only 31 with a significantly p value; thirteen of those genes are closely associated to epithelial mesenchymal transition processes and folding and unfolding processes of proteins in the endoplasmic reticulum. The pro-tumorigenic effect of claudin-6 in gastric cancer could be associated to dedifferentiation of epithelial cells and an increase in di novo cancer stem cell genesis.

Slug is a novel molecular target for head and neck squamous cell carcinoma stem-like cells.

The acquisition of stem-like phenotype is partly attributed to the induction of epithelial-mesenchymal transition (EMT). Thus, the activation of factors involved in EMT can be linked to cancer stem cell genesis. However, the underlying mechanisms in head and neck squamous cell carcinoma (HNSCC) remain largely unknown. Herein, we investigate whether slug, one of the major effectors of EMT, affects the stemness of HNSCC cells. We performed in vitro experiments to determine whether slug gene manipulation can influence the stemness phenotypes, including the capacity for self-renewal, expression of putative stemness markers, chemoresistance, and invasion in HNSCC cells. Further, we identified whether Slug knockout attenuates tumorigenicity of HNSCC cells in vivo. Finally, we examined whether prognosis of HNSCC patients after curative treatment may be affected by the level of slug expression. Overexpression of slug promoted self-renewal of HNSCC cells via activation of sphere formation, the expression of stem cell markers, and induction of chemoresistance to cisplatin. Also, slug overexpression increased the migration and invasion of HNSCC cells in vitro and was mainly observed during the invasion in HNSCC xenograft mouse model. By contrast, slug expression knockdown abrogated their self-renewal capacity, stemness-associated gene expression, and cisplatin chemoresistance. Furthermore, high levels of slug expression correlated with poor prognosis of patients with HNSCC. Inhibition of slug expression may represent a novel therapeutic strategy targeting HNSCC stem-like cells.

Hypothesis: Bacterial induced inflammation disrupts the orderly progression of the stem cell hierarchy and has a role in the pathogenesis of breast cancer

BackgroundThe hierarchical model of stem cell genesis is based on the idea that the number of cell divisions between the zygote and fully differentiated epithelial cells is kept close to the minimum, which is log to the base 2 of the total number of cells produced in a human lifetime. The model assumes the orderly progression of stem cell divisions requires precise control at every stage in development. If the orderly progression is maintained then cancer will be rare. A prediction of the model is that if the orderly progression of the stem cell hierarchy is disturbed by trauma, ulceration or inflammation then cancer will occur. HypothesisBacterial induced inflammation in breast ducts disturbs the stem cell hierarchy and is a cause of breast cancer. EvidenceMammalian milk is not sterile. It contains a range of bacteria, derived endogenously by the entero-mammary circulation. The dominant flora consists of lactose fermenting bacteria. Pregnancy and breast feeding reduce the risk of subsequent breast cancer. The implication is that a lactose fermenting bacterial flora in breast ducts is protective. Malignant and benign breast tissue contains bacteria derived endogenously, but studies so far have not revealed a specific flora associated with malignancy. Periodontitis is associated with oral, oesophageal, colonic, pancreatic, prostatic and breast cancer. The pathogenic bacteria which cause periodontitis spread endogenously to cause inflammation at other epithelial sites. Meta-analysis of epidemiological studies shows that the consumption of yoghurt is associated with a reduction in the risk of breast cancer. ConclusionThe hypothesis, although not proven, is supported by the available evidence. Lactose fermenting bacteria protect but pathogenic bacteria which induce inflammation raise the risk of breast cancer. The consumption of yoghurt also appears to be protective.

GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis.

SummaryHematopoietic stem cells (HSCs) originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5) enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs) are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.

Hematopoietic Signaling Mechanism Revealed from a Stem/Progenitor Cell Cistrome

Thousands of cis-elements in genomes are predicted to have vital functions. Although conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the gold-standard test. A GATA-2-binding, Gata2 intronic cis-element (+9.5) required for hematopoietic stem cell genesis in mice is mutated in a human immunodeficiency syndrome. Because +9.5 is the only cis-element known to mediate stem cell genesis, we devised a strategy to identify functionally comparable enhancers ("+9.5-like") genome-wide. Gene editing revealed +9.5-like activity to mediate GATA-2 occupancy, chromatin opening, and transcriptional activation. A +9.5-like element resided in Samd14, which encodes a protein of unknown function. Samd14 increased hematopoietic progenitor levels/activity and promoted signaling by a pathway vital for hematopoietic stem/progenitor cell regulation (stem cell factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes. Thus, the hematopoietic stem/progenitor cell cistrome revealed a mediator of a signaling pathway that has broad importance for stem/progenitor cell biology.

The hierarchical model of stem cell genesis explains the man mouse paradox, Peto’s paradox, the red cell paradox and Wright’s enigma

The central dogma of carcinogenesis is that deleterious mutations accumulate in regularly cycling stem cells and eventually one of the cells will acquire a specific set of mutations which leads to uncontrolled cell proliferation. Each mutation is rare and the specific set is extremely rare so that even though there are millions of stem cells in a small area of mucosa the specific set of mutations to initiate the process of malignancy will only arise in one stem cell at most; hence neoplasia is clonal. But this model predicts that men, who are 1000 times larger than mice and live 30 times as long, should have a vastly increased risk of cancer compared with mice, but they don’t (man–mouse paradox). The model also predicts that the prevalence of cancer in men should rise as power function of age and mutagen dose, the former is correct but not the latter (Peto’s paradox). Furthermore there are more mitotic divisions in red cell precursors than in all other stem cells combined and yet erythroleukaemia is rare (red cell paradox). The central dogma is also challenged by Wright’s enigma; the observation that some gastro-intestinal neoplasms are polyclonal in origin. The problem with the central dogma is the concept of a regularly cycling stem cell. In fact it is possible to produce all the cells that arise in a human lifetime with fewer than 60 rounds of DNA replication separating the zygote from mature differentiated cells in extreme old age. This hierarchical model of stem cell genesis leads to a very low prevalence of cancer, unless the orderly progression of the hierarchy is disturbed by inflammation, ulceration or trauma. This model explains the paradoxes and Wright’s enigma. It is suggested that the number of cell divisions that separate the zygote from stem cells is a key variable in carcinogenesis.

MR-based hypoxia measures in human glioma

Hypoxia plays a central role in tumor stem cell genesis and is related to a more malignant tumor phenotype, therapy resistance (e.g. in anti-angiogenic therapies) and radio-insensitivity. Reliable hypoxia imaging would provide crucial metabolic information in the diagnostic work-up of brain tumors. In this study, we applied a novel BOLD-based MRI method for the measurement of relative oxygen extraction fraction (rOEF) in glioma patients and investigated potential benefits and drawbacks. Forty-five glioma patients were examined preoperatively in a pilot study on a 3T MR scanner. rOEF was calculated from quantitative transverse relaxation rates (T2, T2*) and cerebral blood volume (CBV) using a quantitative BOLD approach. rOEF maps were assessed visually and by means of a volume of interest (VOI) analysis. In six cases, MRI-targeted biopsy samples were analyzed using HIF-1α-immunohistochemistry. rOEF maps could be obtained with a diagnostic quality. Focal spots with high rOEF values were observed in the majority of high-grade tumors but in none of the low-grade tumors. VOI analysis revealed potentially hypoxic tumor regions with high rOEF in contrast-enhancing tumor regions as well as in the non-enhancing infiltration zone. Systematic bias was found as a result of non-BOLD susceptibility effects (T2*) and contrast agent leakage affecting CBV. Histological samples demonstrated reasonable correspondence between MRI characteristics and HIF-1α-staining. The presented method of rOEF imaging is a promising tool for the metabolic characterization of human glioma. For the interpretation of rOEF maps, confounding factors must be considered, with a special focus on CBV measurements in the presence of contrast agent leakage. Further validation involving a bigger cohort and extended immuno-histochemical correlation is required.

Interferon regulatory factor 7 regulates glioma stem cells via interleukin-6 and Notch signalling

Inflammatory microenvironment signalling plays a crucial role in tumour progression (i.e. cancer cell proliferation, survival, angiogenesis and metastasis) in many types of human malignancies. However, the role of inflammation in brain tumour pathology remains poorly understood. Here, we report that interferon regulatory factor 7 is a crucial regulator of brain tumour progression and heterogeneity. Ectopic expression of interferon regulatory factor 7 in glioma cells promotes tumorigenicity, angiogenesis, microglia recruitment and cancer stemness in vivo and in vitro through induction of interleukin 6, C-X-C motif chemokine 1 and C-C motif chemokine 2. In particular, interferon regulatory factor 7-driven interleukin 6 plays a pivotal role in maintaining glioma stem cell properties via Janus kinase/signal transducer and activator of transcription-mediated activation of Jagged-Notch signalling in glioma cells and glioma stem cells derived from glioma patients. Accordingly, the short hairpin RNA-mediated depletion of interferon regulatory factor 7 in glioma stem cells markedly suppressed interleukin 6-Janus kinase/signal transducer and activator of transcription-mediated Jagged-Notch-signalling pathway, leading to decreases in glioma stem cell marker expression, tumoursphere-forming ability, and tumorigenicity. Furthermore, in a mouse model of wound healing, depletion of interferon regulatory factor 7 suppressed tumour progression and decreased cellular heterogeneity. Finally, interferon regulatory factor 7 was overexpressed in patients with high-grade gliomas, suggesting its potential as an independent prognostic marker for glioma progression. Taken together, our findings indicate that interferon regulatory factor 7-mediated inflammatory signalling acts as a major driver of brain tumour progression and cellular heterogeneity via induction of glioma stem cell genesis and angiogenesis.

The neural stem cell fate determinant TLX promotes tumorigenesis and genesis of cells resembling glioma stem cells.

A growing body of evidence indicates that deregulation of stem cell fate determinants is a hallmark of many types of malignancies. The neural stem cell fate determinant TLX plays a pivotal role in neurogenesis in the adult brain by maintaining neural stem cells. Here, we report a tumorigenic role of TLX in brain tumor initiation and progression. Increased TLX expression was observed in a number of glioma cells and glioma stem cells, and correlated with poor survival of patients with gliomas. Ectopic expression of TLX in the U87MG glioma cell line and Ink4a/Arf-deficient mouse astrocytes (Ink4a/Arf(-/-) astrocytes) induced cell proliferation with a concomitant increase in cyclin D expression, and accelerated foci formation in soft agar and tumor formation in in vivo transplantation assays. Furthermore, overexpression of TLX in Ink4a/Arf(-/-) astrocytes inhibited cell migration and invasion and promoted neurosphere formation and Nestin expression, which are hallmark characteristics of glioma stem cells, under stem cell culture conditions. Our results indicate that TLX is involved in glioma stem cell genesis and represents a potential therapeutic target for this type of malignancy.

Spirulina promotes stem cell genesis and protects against LPS induced declines in neural stem cell proliferation.

Adult stem cells are present in many tissues including, skin, muscle, adipose, bone marrow, and in the brain. Neuroinflammation has been shown to be a potent negative regulator of stem cell and progenitor cell proliferation in the neurogenic regions of the brain. Recently we demonstrated that decreasing a key neuroinflammatory cytokine IL-1β in the hippocampus of aged rats reversed the age-related cognitive decline and increased neurogenesis in the age rats. We also have found that nutraceuticals have the potential to reduce neuroinflammation, and decrease oxidative stress. The objectives of this study were to determine if spirulina could protect the proliferative potential of hippocampal neural progenitor cells from an acute systemic inflammatory insult of lipopolysaccharide (LPS). To this end, young rats were fed for 30 days a control diet or a diet supplemented with 0.1% spirulina. On day 28 the rats were given a single i.p. injection of LPS (1 mg/kg). The following day the rats were injected with BrdU (50 mg/kg b.i.d. i.p.) and were sacrificed 24 hours after the first injection of BrdU. Quantification of the BrdU positive cells in the subgranular zone of the dentate gyrus demonstrated a decrease in proliferation of the stem/progenitor cells in the hippocampus as a result of the LPS insult. Furthermore, the diet supplemented with spirulina was able to negate the LPS induced decrease in stem/progenitor cell proliferation. In a second set of studies we examined the effects of spirulina either alone or in combination with a proprietary formulation (NT-020) of blueberry, green tea, vitamin D3 and carnosine on the function of bone marrow and CD34+ cells in vitro. Spirulina had small effects on its own and more than additive effects in combination with NT-020 to promote mitochondrial respiration and/or proliferation of these cells in culture. When examined on neural stem cells in culture spirulina increased proliferation at baseline and protected against the negative influence of TNFα to reduce neural stem cell proliferation. These results support the hypothesis that a diet enriched with spirulina and other nutraceuticals may help protect the stem/progenitor cells from insults.

A Glutamine-Rich Factor Affects Stem Cell Genesis in Leech

Leech embryogenesis is a model for investigating cellular and molecular processes of development. Due to the unusually large size of embryonic stem cells (teloblasts: 50–300 μm) in the glossiphoniid leech, Theromyzon tessulatum, and the presence of identifiable stem cell precursors (proteloblasts), we previously isolated a group of genes upregulated upon stem cell birth. In the current study, we show that one of these genes, designated Theromyzon proliferation (Tpr), is required for normal stem cell genesis; specifically, transient Tpr knockdown experiments conducted with antisense oligonucleotides and monitored by semiquantitative RT-PCR, caused abnormal proteloblast proliferation leading to embryonic death, but did not overtly affect neuroectodermal or mesodermal stem cell development once these cells were born. Tpr encodes a large glutamine-rich (∼34%) domain that shares compositional similarity with strong transcriptional enhancers many of which have been linked with trinucleotide repeat disorders (e.g., Huntington's).

Changes in gene expression at the precursor --> stem cell transition in leech.

The glossiphoniid leech, Theromyzon trizonare, displays particularly large and accessible embryonic precursor/stem cells during its early embryonic cleavages. We dissected populations of both cell types from staged embryos and examined gene expression profiles by differential display polymerase chain reaction methodology. Among the approximately 10,000 displayed cDNA fragments, 56 (approximately 0.5%) were differentially expressed at the precursor --> stem cell transition; 29 were turned off (degraded, precursor-specific); and 27 were turned on (transcribed, stem cell-specific). Several putative differentially expressed cDNAs from each category were confirmed by Northern blot analysis on staged embryos. DNA sequencing revealed that 19 of the cDNAs were related to a spectrum of genes including the CCR4 antiproliferation gene, Rad family members, and several transcriptional regulators, while the remainder encoded hypothetical (10) or novel (27) sequences. Collectively, these results identify dynamic changes in gene expression during stem cell formation in leech and provide a platform for examining the molecular aspects of stem cell genesis in a simple invertebrate organism.