Identification of CD166 as a Surface Marker for Enriching Prostate Stem/Progenitor and Cancer Initiating Cells

New therapies for late stage and castration resistant prostate cancer (CRPC) depend on defining unique properties and pathways of cell sub-populations capable of sustaining the net growth of the cancer. One of the best enrichment schemes for isolating the putative stem/progenitor cell from the murine prostate gland is Lin(-);Sca1(+);CD49f(hi) (LSC(hi)), which results in a more than 10-fold enrichment for in vitro sphere-forming activity. We have shown previously that the LSC(hi) subpopulation is both necessary and sufficient for cancer initiation in the Pten-null prostate cancer model. To further improve this enrichment scheme, we searched for cell surface molecules upregulated upon castration of murine prostate and identified CD166 as a candidate gene. CD166 encodes a cell surface molecule that can further enrich sphere-forming activity of WT LSC(hi) and Pten null LSC(hi). Importantly, CD166 could enrich sphere-forming ability of benign primary human prostate cells in vitro and induce the formation of tubule-like structures in vivo. CD166 expression is upregulated in human prostate cancers, especially CRPC samples. Although genetic deletion of murine CD166 in the Pten null prostate cancer model does not interfere with sphere formation or block prostate cancer progression and CRPC development, the presence of CD166 on prostate stem/progenitors and castration resistant sub-populations suggest that it is a cell surface molecule with the potential for targeted delivery of human prostate cancer therapeutics.     

Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells,Oligodendrocyte Precursor Cells,and Endothelial Progenitor Cells

Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9) has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs). However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs), and endothelial progenitor cells (EPCs). Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM), and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types.     

Lgr5 Identifies Progenitor Cells Capable of Taste Bud Regeneration after Injury

Taste buds are composed of a variety of taste receptor cell types that develop from tongue epithelium and are regularly replenished under normal homeostatic conditions as well as after injury. The characteristics of cells that give rise to regenerating taste buds are poorly understood. Recent studies have suggested that Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) identifies taste bud stem cells that contribute to homeostatic regeneration in adult circumvallate and foliate taste papillae, which are located in the posterior region of the tongue. Taste papillae in the adult anterior region of the tongue do not express Lgr5. Here, we confirm and extend these studies by demonstrating that Lgr5 cells give rise to both anterior and posterior taste buds during development, and are capable of regenerating posterior taste buds after injury induced by glossopharyngeal nerve transection.     

HCV core通过活化Wnt/β-catenin信号通路诱导肝前体细胞向肝癌干细胞分化

丙型病毒性肝炎（hepatitis C virus,HCV）慢性感染及肝前体细胞向肝癌干细胞分化是原发性肝癌(hepatocellular carcinoma,HCC)的重要致病因素。且Wnt信号参与维持肝癌干细胞特性,但HCV能否通过活化Wnt信号诱导肝前体细胞向肝癌干细胞分化尚不清楚。本室研究发现,在HCV core诱导分化的肝前体细胞中,HCV core抑制成熟肝细胞标志物Alb、CK18的表达,糖原储存能力显著下降（P<0.05）,且上调肝癌干细胞标志物EpCAM、CD133、CD44等表达。另外,HCV core增强β-联蛋白活性及表达水平,促使β-联蛋白向核内聚集,上调其下游靶基因EpCAM、细胞周期蛋白D1、C-myc的表达,沉默β-联蛋白后,Wnt/β-catenin通路其下游靶基因表达明显受到抑制,糖原储存能力部分恢复,荧光共聚焦显示HCV core与β-联蛋白在细胞核内存在共定位。因此,HCV core可能与β-联蛋白相互作用,直接活化Wnt/β-catenin通路,上调其下游靶基因EpCAM等表达,诱导肝前体细胞向肝癌干细胞分化。     

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

Background

Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro.

Methods and Findings

To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene.

Conclusion

VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.     

Emblica officinalis Extract Induces Autophagy and Inhibits Human Ovarian Cancer Cell Proliferation,Angiogenesis, Growth of Mouse Xenograft Tumors

Patients with ovarian cancer (OC) may be treated with surgery, chemotherapyand/or radiation therapy, although none of these strategies are very effective.Several plant-based natural products/dietary supplements, including extractsfrom Emblicaofficinalis (Amla), havedemonstrated potent anti-neoplastic properties. In this study we determined thatAmla extract (AE) has anti-proliferative effects on OC cells under bothin vitro and in vivo conditions. We alsodetermined the anti-proliferative effects one of the components of AE,quercetin, on OC cells under in vitro conditions. AE did notinduce apoptotic cell death, but did significantly increase the expression ofthe autophagic proteins beclin1 and LC3B-II under in vitroconditions. Quercetin also increased the expression of the autophagic proteinsbeclin1 and LC3B-II under in vitro conditions. AE alsosignificantly reduced the expression of several angiogenic genes, includinghypoxia-inducible factor 1α (HIF-1α) in OVCAR3 cells. AE acted synergisticallywith cisplatin to reduce cell proliferation and increase expression of theautophagic proteins beclin1 and LC3B-II under in vitroconditions. AE also had anti-proliferative effects and induced the expression ofthe autophagic proteins beclin1 and LC3B-II in mouse xenograft tumors.Additionally, AE reduced endothelial cell antigen – CD31 positive blood vesselsand HIF-1α expression in mouse xenograft tumors. Together, these studiesindicate that AE inhibits OC cell growth both in vitro andin vivo possibly via inhibition of angiogenesis andactivation of autophagy in OC. Thus AE may prove useful as an alternative oradjunct therapeutic approach in helping to fight OC.     

ARLTS1 and Prostate Cancer Risk - Analysis of Expression and Regulation

Prostate cancer (PCa) is a heterogeneous trait for which several susceptibility loci have been implicated by genome-wide linkage and association studies. The genomic region 13q14 is frequently deleted in tumour tissues of both sporadic and familial PCa patients and is consequently recognised as a possible locus of tumour suppressor gene(s). Deletions of this region have been found in many other cancers. Recently, we showed that homozygous carriers for the T442C variant of the ARLTS1 gene (ADP-ribosylation factor-like tumour suppressor protein 1 or ARL11, located at 13q14) are associated with an increased risk for both unselected and familial PCa. Furthermore, the variant T442C was observed in greater frequency among malignant tissue samples, PCa cell lines and xenografts, supporting its role in PCa tumourigenesis. In this study, 84 PCa cases and 15 controls were analysed for ARLTS1 expression status in blood-derived RNA. A statistically significant (p = 0.0037) decrease of ARLTS1 expression in PCa cases was detected. Regulation of ARLTS1 expression was analysed with eQTL (expression quantitative trait loci) methods. Altogether fourteen significant cis-eQTLs affecting the ARLTS1 expression level were found. In addition, epistatic interactions of ARLTS1 genomic variants with genes involved in immune system processes were predicted with the MDR program. In conclusion, this study further supports the role of ARLTS1 as a tumour suppressor gene and reveals that the expression is regulated through variants localised in regulatory regions.     

免疫微环境对肿瘤干细胞影响研究进展

肿瘤干细胞是肿瘤组织中一小群特殊的未分化的细胞,由于其对化疗药耐受及致瘤潜能,被认为是造成肿瘤发生、复发和转移的根源,所以深入了解肿瘤干细胞特性对提高肿瘤治疗效率有着重要临床意义.肿瘤微环境中的免疫细胞及其分泌的分子与肿瘤干细胞之间存在复杂的相互作用,可以维持肿瘤干细胞的干性及自我更新能力.目前,肿瘤免疫微环境对肿瘤干细胞的影响、肿瘤干细胞对免疫微环境的塑造作用以及靶向肿瘤干细胞或免疫微环境等研究,是肿瘤干细胞研究领域的热点问题.本文就免疫微环境对肿瘤干细胞的影响、靶向肿瘤干细胞及微环境治疗的研究进展进行了综述.     

EPS8 Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10⁻⁷) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10⁻⁵). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of EPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.     

V-Myc Immortalizes Human Neural Stem Cells in the Absence of Pluripotency-Associated Traits

A better understanding of the molecular mechanisms governing stem cell self-renewal will foster the use of different types of stem cells in disease modeling and cell therapy strategies. Immortalization, understood as the capacity for indefinite expansion, is needed for the generation of any cell line. In the case of v-myc immortalized multipotent human Neural Stem Cells (hNSCs), we hypothesized that v-myc immortalization could induce a more de-differentiated state in v-myc hNSC lines. To test this, we investigated the expression of surface, biochemical and genetic markers of stemness and pluripotency in v-myc immortalized and control hNSCs (primary precursors, that is, neurospheres) and compared these two cell types to human Embryonic Stem Cells (hESCs) and fibroblasts. Using a Hierarchical Clustering method and a Principal Component Analysis (PCA), the v-myc hNSCs associated with their counterparts hNSCs (in the absence of v-myc) and displayed a differential expression pattern when compared to hESCs. Moreover, the expression analysis of pluripotency markers suggested no evidence supporting a reprogramming-like process despite the increment in telomerase expression. In conclusion, v-myc expression in hNSC lines ensures self-renewal through the activation of some genes involved in the maintenance of stem cell properties in multipotent cells but does not alter the expression of key pluripotency-associated genes.     

Osterix-Cre Labeled Progenitor Cells Contribute to the Formation and Maintenance of the Bone Marrow Stroma

We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma.     

In Vitro Characterization of Circulating Endothelial Progenitor Cells Isolated from Patients with Acute Coronary Syndrome

Background

The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

Methodology/Principal Findings

By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7–14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

Conclusion/Significance

Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.     

Withania somnifera Root Extract Has Potent Cytotoxic Effect against Human Malignant Melanoma Cells

In Ayurveda, Withania somnifera is commonly known as Ashwagandha, its roots are specifically used in medicinal and clinical applications. It possesses numerous therapeutic actions which include anti-inflammatory, sedative, hypnotic and narcotic. Extracts from this plant have been reported for its anticancer properties. In this study we evaluated for the first time, the cytotoxic effect of Withania root extract on human malignant melanoma A375 cells. The crude extract of Withania was tested for cytotoxicity against A375 cells by MTT assay. Cell morphology of treated A375 cells was visualized through phase contrast as well as fluorescence microscopy. Agarose gel electrophoresis was used to check DNA fragmentation of the crude extract treated cells. Crude extract of Withania root has the potency to reduce viable cell count in dose as well as time dependent manner. Morphological change of the A375 cells was also observed in treated groups in comparison to untreated or vehicle treated control. Apoptotic body and nuclear blebbing were observed in DAPI stained treated cells under fluorescence microscope. A ladder of fragmented DNA was noticed in treated cells. Thus it might be said that the crude water extract of Withania somnifera has potent cytotoxic effect on human malignant melanoma A375 cells.