Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear, however, whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells, among which is E2F2, a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1, in addition to proliferation of hESC, indicated by a higher proportion of cells in G1, fewer cells in G2/M phase, and a reduced capacity to generate hESC colonies in vitro. Nonetheless, E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly, E2F2 knockdown in hESC significantly inhibited tumor growth in vivo, which was considerably smaller than tumors generated from control hESC, although displaying typical teratoma traits, a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness, providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells.
Gliomatosis cerebri is a rare diffuse glioma that is neither mass-forming nor necrotic, and does not disrupt existing structures. Gliomatosis occurring in the cerebellum is known as gliomatosis cerebelli, and only three such cases examined by biopsy have been reported. Here we describe the first autopsy findings of a patient who was diagnosed as having gliomatosis in the cerebellum. Neuropathological examination identified the tumor cells as being positive for glial fibrillary acidic protein, vimentin and nestin, with atypical nuclei that were cashew-nut- or dishcloth-gourd-shaped. These tumor cells were dense in the right cerebellum, but also spread broadly throughout the brain including the left cerebrum and optic nerve. Mitotic figures were frequently seen in the cerebellum, brain stem and cerebrum. Scherers secondary structures were evident not only in the cerebellum but also the cerebrum. No necrosis, microvascular proliferation or destruction of anatomical structures was detected in the whole brain. Differences in the origin of the tumors of the gliomatoses cerbri and cerebelli suggests these tumors are different types of brain tumors. Thus the findings support that the gliomatosis cerebelli is a novel type of brain tumor classification. Furthermore, by the similarities of the histological features among the tumors, it appears appropriate to establish a novel category of "gliomatosis encephali" which includes both gliomatosis cerebri and gliomatosis cerebelli.
Spinocerebellar ataxia type 31 (SCA31) is an autosomal dominant form of pure cerebellar ataxia that is caused by a disease-specific insertion containing penta-nucleotide repeats (TGGAA)n . Neuropathologically, cerebellar Purkinje cells are preferentially affected and reduced in number in SCA31, and they are often surrounded by halo-like amorphous materials. In the present study, we performed neuropathological analyses on two SCA31 brains, and discussed the serial morphological changes of Purkinje cells in SCA31.We found that bent, elongated, often folded nuclei were observed frequently in degenerating Purkinje cells with the halo-like structure. Conversely, Purkinje cells without this structure developed marked atrophy with severely slender and condensed nuclei. On the basis of these pathological findings, we propose two different processes for Purkinje cell degeneration in SCA31, namely, shrinkage of Purkinje cells with or without the halo-like amorphous materials. The former, but not the latter, was considered to be specific to SCA31. Correspondingly, fragmentation of the Golgi apparatus was observed more frequently in Purkinje cells with the halo-like structure than in those without this structure. We consider that the profound nuclear deformity and fragmentation of the Golgi apparatus are closely linked with the formation of the halo-like structure in SCA31.
Despite the fact that most maternal deaths are preventable, maternal mortality remains high in many developing countries. Target A of Millennium Development Goal (MDG) 5 calls for a three-quarters reduction in the maternal mortality ratio (MMR) between 1990 and 2015.
The SUN (Sad1-UNC-84 homology) domain is conserved in a number of nuclear envelope proteins involved in nuclear migration, meiotic telomere tethering, and antiviral responses. The LINC (linker of nucleoskeleton and cytoskeleton) complex, formed by the SUN and the nesprin proteins at the nuclear envelope, serves as a mechanical linkage across the nuclear envelope. Here we report the crystal structure of the SUN2 protein SUN domain, which reveals a homotrimer. The SUN domain is sufficient to mediate binding to the KASH (Klarsicht, ANC-1, and Syne homology) domain of nesprin 2, and the regions involved in the interaction have been identified. Binding of the SUN domain to the KASH domain is abolished by deletion of a region important for trimerization or by point mutations associated with nuclear migration failure. We propose a model of the LINC complex, where the SUN and the KASH domains form a higher ordered oligomeric network in the nuclear envelope. These findings provide the structural basis for understanding the function and the regulation of the LINC complex.
Transcriptome regionalization is an essential polarity determinant among metazoans, directing embryonic axis formation during normal development. Although conservation of this principle in mammals is assumed, recent evidence is conflicting and it is not known whether transcriptome asymmetries exist within unfertilized mammalian eggs or between the respective cleavage products of early embryonic divisions. We here address this by comparing transcriptome profiles of paired single cells and sub-cellular structures obtained microsurgically from mouse oocytes and totipotent embryos. Paired microsurgical spindle and remnant samples from unfertilized metaphase II oocytes possessed distinguishable profiles. Fertilization produces a totipotent 1-cell embryo (zygote) and associated spindle-enriched second polar body whose paired profiles also differed, reflecting spindle transcript enrichment. However, there was no programmed transcriptome asymmetry between sister cells within 2- or 3-cell embryos. Accordingly, there is transcriptome asymmetry within mouse oocytes, but not between the sister blastomeres of early embryos. This work places constraints on pre-patterning in mammals and provides documentation correlating potency changes and transcriptome partitioning at the single-cell level.
Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P ? 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients.
PEGylation of recombinant adenovirus (Ad) vectors is a promising approach for not only evasion from neutralizing anti-Ad antibodies and uptake by phagocytic cells, but also prolongation of the blood retention time of Ad vectors after systemic administration. However, the conventional PEGylation leads to significant reduction in the transduction activity of Ad vectors, probably because PEG is nonspecifically conjugated to the Ad capsid protein and inhibits the binding of Ad vectors to the primary receptor, coxsackievirus-adenovirus receptor (CAR). In order to PEGylate an Ad vector without significant reduction in the transduction activity, the biotin-binding peptide (BAP) was inserted into the hypervariable region (HVR) 5 of the hexon, which is not involved in the binding to CAR, and PEG was then specifically conjugated to the hexon HVR5 via avidin-biotin interaction. In vitro transduction experiments demonstrated that the hexon-specific PEGylation did not cause an apparent reduction in the transduction efficiency of the Ad vector, although the insertion of the BAP into the HVR5 itself reduced the transduction efficiency by 50-fold, compared with the conventional Ad vector, in the absence of anti-Ad serum. In the presence of anti-Ad serum, the transduction with the Ad vector with the BAP in the hexon HVR5 was significantly blocked; however, anti-Ad serum only slightly inhibited the transduction with the hexon-specifically PEGylated Ad vector (Ad-BAP/Bio/Avi/Bio-PEG-L2). Intravenous administration of Ad-BAP/Bio/Avi/Bio-PEG-L2 resulted in prolonged blood retention, significant reduction in the transduction in the liver, and accumulation in the tumor; however, unexpectedly, the transduction efficiency of Ad-BAP/Bio/Avi/Bio-PEG-L2 in the tumor was almost at the background level.
The oocytes of vertebrates are typically arrested at metaphase II (mII) by the cytostatic factor Emi2 until fertilization. Regulatory mechanisms in Xenopus Emi2 (xEmi2) are understood in detail but contrastingly little is known about the corresponding mechanisms in mammals. Here, we analyze Emi2 and its regulatory neighbours at the molecular level in intact mouse oocytes. Emi2, but not xEmi2, exhibited nuclear targeting. Unlike xEmi2, separable N- and C-terminal domains of mouse Emi2 modulated metaphase establishment and maintenance, respectively, through indirect and direct mechanisms. The C-terminal activity was mapped to the potential phosphorylation target Tx(5)SxS, a destruction box (D-box), a lattice of Zn(2+)-coordinating residues and an RL domain. The minimal region of Emi2 required for its cytostatic activity was mapped to a region containing these motifs, from residue 491 to the C terminus. The cytostatic factor Mos-MAPK promoted Emi2-dependent metaphase establishment, but Mos autonomously disappeared from meiotically competent mII oocytes. The N-terminal Plx1-interacting phosphodegron of xEmi2 was apparently shifted to within a minimal fragment (residues 51-300) of mouse Emi2 that also contained a calmodulin kinase II (CaMKII) phosphorylation motif and which was efficiently degraded during mII exit. Two equimolar CaMKII gamma isoform variants were present in mII oocytes, neither of which phosphorylated Emi2 in vitro, consistent with the involvement of additional factors. No evidence was found that calcineurin is required for mouse mII exit. These data support a model in which mammalian meiotic establishment, maintenance and exit converge upon a modular Emi2 hub via evolutionarily conserved and divergent mechanisms.
In vertebrates, a rise in intracellular free Ca(2+) (Ca(2+)(i)) levels during fertilization initiates second metaphase (mII) exit and the developmental programme. The Ca(2+) rise has long been considered to be crucial for development, but verifying this contribution would benefit from defining its role during fertilization. Here, we delineate the role of Ca(2+) release during mII exit in wild-type mouse eggs and show that it is dispensable for full-term development. Exit from mII can be induced by Zn(2+)-specific sequestration without Ca(2+) release, eliciting Cyclin B degradation in a manner dependent upon the proteasome pathway and intact microtubules, but not accompanied by degradation of the meiotic regulator Emi2. Parthenogenotes generated by Zn(2+) sequestration developed in vitro with normal expression of Ca(2+)-sensitive genes. Meiotic exit induced by either Ca(2+) oscillations or a single Ca(2+) rise in oocytes containing a signaling-deficient sperm resulted in comparable developmental rates. In the absence of Ca(2+) release, full-term development occurred approximately 50% less efficiently, but at readily detectable rates, with the birth of 27 offspring. These results show in intact mouse oocytes that Zn(2+) is essential for mII arrest and suggest that triggering meiotic exit is the sole indispensable developmental role of Ca(2+) signaling in mammalian fertilization.
Medulloblastoma is a highly malignant primary tumor of the central nervous system. It represents the most frequent type of solid tumor and the leading cause of death related to cancer in early childhood. Current treatment includes surgery, chemotherapy and radiotherapy which may lead to severe cognitive impairment and secondary brain tumors. New perspectives for therapeutic development have emerged with the identification of stem-like cells displaying high tumorigenic potential and increased radio- and chemo-resistance in gliomas. Under the cancer stem cell hypothesis, transformation of neural stem cells and/or granular neuron progenitors of the cerebellum are though to be involved in medulloblastoma development. Dissecting the genetic and molecular alterations associated with this process should significantly impact both basic and applied cancer research. Based on cumulative evidences in the fields of genetics and molecular biology of medulloblastomas, we discuss the possible involvement of developmental signaling pathways as critical biochemical switches determining normal neurogenesis or tumorigenesis. From the clinical viewpoint, modulation of signaling pathways such as TGF?, regulating neural stem cell proliferation and tumor development, might be attempted as an alternative strategy for future drug development aiming at more efficient therapies and improved clinical outcome of patients with pediatric brain cancers.
A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.
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