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In JoVE (1)
Other Publications (11)
Articles by Angel Ayuso-Sacido in JoVE
JoVE General
Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device
1Biomedical Engineering Department, Cornell University, 2Neurosurgical Laboratory for Translational Stem Cell Research, Weill Cornell Brain Tumor Center, Weill Cornell Medical College of Cornell University, 3Cell Morphology Department, Instituto de Investigacion Principe Felipe, 4Department of Chemical and Biomolecular Engineering, Cornell University
We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.
Other articles by Angel Ayuso-Sacido on PubMed
Actinomycetes Isolated from Lichens: Evaluation of Their Diversity and Detection of Biosynthetic Gene Sequences
Actinomycetes, one of the major communities of the microbial population present in soil, can also be found inhabiting a wide diversity of ecological sources. We have explored the use of lichens as an alternative source for the isolation of novel actinomycetes. Lichens are symbiotic mixtures of fungi, green algae and/or cyanobacteria and whereas these symbiotic components have been extensively described, the microbial community inhabiting this niche has not been well characterized. We studied the diversity of the actinomycete population isolated from lichens collected in tropical areas from the Hawaii and Reunion islands and in cold areas from Alaska. The diversity of the microbial population was evaluated using fatty acid analysis and molecular fingerprinting. A PCR approach to screen the isolates for genes associated with secondary metabolite production was applied to evaluate the biosynthetic potential of these strains; profiles obtained for each isolate were compared to the antimicrobial activity exhibited by these isolates in laboratory conditions. Our results demonstrate that lichens represent an extremely rich reservoir for the isolation of a wide diversity of actinomycetes many of them representing still today a rich untapped source of secondary metabolites.
Acceleration of Mesoderm Development and Expansion of Hematopoietic Progenitors in Differentiating ES Cells by the Mouse Mix-like Homeodomain Transcription Factor
The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mix-like (mMix) in gastrulation, its function in mesoderm development remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem cells in which mMix was genetically inactivated. Here we show that conditional induction of mMix in embryonic stem cell-derived embryoid bodies results in the early activation of mesodermal markers prior to expression of Brachyury/T and acceleration of the mesodermal developmental program. Strikingly, increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. Differentiation to primitive (embryonic) and definitive (adult type) blood cells proceeds normally and without an apparent bias in the representation of different hematopoietic cell fates. Therefore, the mouse Mix gene functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages.
The Duality of Epidermal Growth Factor Receptor (EGFR) Signaling and Neural Stem Cell Phenotype: Cell Enhancer or Cell Transformer?
Recruitment of neural stem cells (NSCs) represents an elegant strategy for replacing adult central nervous system (CNS) cells lost to injury or disease. However, except in the rostral migratory stream to the olfactory bulb, the adult CNS harbors a relatively non permissive environment for motility of neural stem cells. This opens the possibility of therapeutic enhancement of NSC motility towards sites of CNS injury or disease. The Epidermal Growth Factor Receptor (EGFR) is involved in the activation of a number of downstream pathways that regulate the phenotype of progenitor cells. Activated EGFR tyrosine kinase activity enhances NSC migration, proliferation, and survival. However, EGFR signaling is also known to play a role in the most malignant and highly invasive of human tumors, glioblastoma multiforme (GBM). Recent evidence supports the theory that GBM derives from a 'cancer stem cell' and that EGFR signals are commonly altered in these precursor cells. This article will review the role of EGFR signaling as it relates to neural stem cell motility and invasion. The duality of altered EGFR signaling in neural progenitor cells is discussed and opportunities for enhancing the recruitment of adult progenitors, and consequences of altering EGFR signaling in progenitor cells will be highlighted.
Use of Human Neural Tissue for the Generation of Progenitors
Accumulating evidence suggests that a better understanding of normal human brain stem cells and tumor stem cells (TSCs) will have profound implications for treating central nervous system disease during the next decade. Neurosurgeons routinely resect excess surgical tissue containing either normal brain stem cells or TSCs. These cells are immediately available for expansion and use in basic biological assays, animal implantation, and comparative analysis studies. Although normal stem cells have much slower kinetics of expansion than TSCs, they are easily expandable and can be frozen for future use in stem cell banks. This nearly limitless resource holds promise for understanding the basic biology of normal brain stem cells and TSCs, which will likely direct the next major shift in therapeutics for brain tumors, brain and spinal cord injury, and neurodegenerative disease. This report reviews the progress that has been made in harvesting and expanding both normal and tumor-derived stem cells and emphasizes the integral role neurosurgeons will play in moving the neural stem cell field forward.
Long-term Expansion of Adult Human Brain Subventricular Zone Precursors
Many common neurosurgical procedures, including anterior temporal lobectomy and endoscopic ventricular puncture, allow neurosurgeons to retrieve portions of the germinal subventricular zone (SVZ). Isolation and maintenance of precursor cells from this zone can be used for hypothesis-driven experiments with a goal of improving our understanding of the basic mechanisms of central nervous system injury or disease and the potential of cell-based therapies to treat them. This article details our ability to reliably harvest, isolate, characterize, and maintain normal adult human brain SVZ precursor cells.
Activated EGFR Signaling Increases Proliferation, Survival, and Migration and Blocks Neuronal Differentiation in Post-natal Neural Stem Cells
Recent evidence supports the notion that transformation of undifferentiated neural stem cell (NSC) precursors may contribute to the development of glioblastoma multiforme (GBM). The over-expression and mutation of the epidermal growth factor receptor (EGFR), along with other cellular pathway mutations, plays a significant role in GBM maintenance progression. Though EGFR signaling is important in determining neural cell fate and conferring astrocyte differentiation, there is a limited understanding of its role in NSC and tumor stem cell (TSC) biology. We hypothesized that EGFR expression and mutation in post-natal NSCs may contribute to cellular aggressiveness including enhanced cellular proliferation, survival and migration. Stable subclones of C17.2 murine NSCs were transfected to over-express either the wild-type EGFR (wtEGFR) or its most common mutated variant EGFRvIII. Activated EGFR signaling in these cells induced behaviors characteristic of GBM TSCs, including enhanced proliferation, survival and migration, even in the absence of EGF ligand. wtEGFR activation was also found to block neuronal differentiation and was associated with a dramatic increase in chemotaxis in the presence of EGF. EGFRvIII expression lead to an increase in NSC proliferation and survival, while it simultaneously blocked neuronal differentiation and promoted glial fate. Our findings suggest that activated EGFR signaling enhances the aggressiveness of NSCs. Understanding the regulatory mechanisms of NSCs may lend insight into deregulated mechanisms of GBM TSC invasion, proliferation, survival and resistance to current treatment modalities.
Effects of MRI Contrast Agents on the Stem Cell Phenotype
The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist, Endorem, Sinerem) was compared. Labeling efficiency with Resovist and Endorem differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.
Reversible Neural Stem Cell Niche Dysfunction in a Model of Multiple Sclerosis
The subventricular zone (SVZ) of the brain constitutes a niche for neural stem and progenitor cells that can initiate repair after central nervous system (CNS) injury. In a relapsing-remitting model of experimental autoimmune encephalomyelitis (EAE), the neural stem cells (NSCs) become activated and initiate regeneration during acute disease, but lose this ability during the chronic phases of disease. We hypothesized that chronic microglia activation contributes to the failure of the NSC repair potential in the SVZ.
Development of a Human Extracellular Matrix for Applications Related with Stem Cells and Tissue Engineering
Cancer-Initiating Enriched Cell Lines from Human Glioblastoma: Preparing for Drug Discovery Assays
Glioblastoma multiforme (GBM) is the most lethal type of brain tumour in the adult humans. The cancer-Initiatingcell (CIC) hypothesis supports the notion that failures in current approaches to GBM treatment might be attributed to the survival of the CIC subpopulation. Recent evidence shows the idea that using CIC-enriched cell lines derived from human GBM as new targets for drug discovery programs, may improve the chance of successfully translating the basic research findingsinto clinical trials. Although this approach appears promising, many important biological and technical issues (characterization of functional CIC markers, inter- and intra-tumoral CIC heterogeneity, and isolation and maintenance inconsistency) need to be resolved.
Genomic Instability of Surgical Sample and Cancer-initiating Cell Lines from Human Glioblastoma
Glioblastoma multiforme (GBM) is the most aggressive brain tumor in the adult human, with an average survival of 16 months. A small population of cells within the GBM termed cancer-initiating cells is responsible for the initiation and maintenance of the tumor mass. The traditional glioblastoma cancer cells, grown with serum containing media, display increased rate of genomic instability events, which in turn renders the cell cultures with little resembling to the original tumor, making doubtful their use as preclinical models for screening therapeutic agents. On the contrary, the cancer-initiating cells grown in serum-free media seems to show lower rate of genomic instability processes. However, considering the diversity of genetic and/or epigenetic background, we will need to evaluate the possibility of using different culture conditions to allow for the isolation and culture of such cancer-initiating cells diversity, keeping, at the same time, the genomic instability rate as the original tumor. We summarized the main genetic and epigenetic mechanisms that are driving genomic instability in cancer-initiating cells from human glioblastoma.
