Navegando por Autor "Ortega, Felipe"

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Continuous live imaging of adult neural stem cell division and lineage progression in vitro
(2011) Costa, Marcos Romualdo; Ortega, Felipe; Brill, Monika S.; Beckervordersandforth, Ruth; Petrone, Ciro; Schroeder, Timm; Götz, Magdalena; Berninger, Benedikt
Little is known about the intrinsic specification of adult neural stem cells (NSCs) and to what extent they depend on their local niche. To observe adult NSC division and lineage progression independent of their niche, we isolated cells from the adult mouse subependymal zone (SEZ) and cultured them at low density without growth factors. We demonstrate here that SEZ cells in this culture system are primarily neurogenic and that adult NSCs progress through stereotypic lineage trees consisting of asymmetric stem cell divisions, symmetric transit-amplifying divisions and final symmetric neurogenic divisions. Stem cells, identified by their astro/radial glial identity and their slow-dividing nature, were observed to generate asymmetrically and fast-dividing cells that maintained an astro/radial glia identity. These, in turn, gave rise to symmetrically and fast-dividing cells that lost glial hallmarks, but had not yet acquired neuronal features. The number of amplifying divisions was limited to a maximum of five in this system. Moreover, we found that cell growth correlated with the number of subsequent divisions of SEZ cells, with slow-dividing astro/radial glia exhibiting the most substantial growth prior to division. The fact that in the absence both of exogenously supplied growth factors and of signals provided by the local niche neurogenic lineage progression takes place in such stereotypic fashion, suggests that lineage progression is, to a significant degree, cell intrinsic or pre-programmed at the beginning of the lineage.
Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations
(2017-12-16) Gómez-Villafuertes, Rosa; Paniagua-Herranz, Lucía; Gascon, Sergio; Agustín-Durán, David de; Ferreras, María de la O; Gil-Redondo, Juan Carlos; Queipo, María José; Menendez-Mendez, Aida; Pérez-Sen, Ráquel; G. Delicado, Esmerilda; Gualix, Javier; Costa, Marcos Romualdo; Schroeder, Timm; Miras-Portugal, María Teresa; Ortega, Felipe
Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.
Live Imaging of Adult Neural Stem Cells in Rodents
(Universidade Federal do Rio Grande do Norte, 2016) Ortega, Felipe; Costa, Marcos Romualdo
The generation of cells of the neural lineage within the brain is not restricted to early development. New neurons, oligodendrocytes, and astrocytes are produced in the adult brain throughout the entire murine life. However, despite the extensive research performed in the field of adult neurogenesis during the past years, fundamental questions regarding the cell biology of adult neural stem cells (aNSCs) remain to be uncovered. For instance, it is crucial to elucidate whether a single aNSC is capable of differentiating into all three different macroglial cell types in vivo or these distinct progenies constitute entirely separate lineages. Similarly, the cell cycle length, the time and mode of division (symmetric vs. asymmetric) that these cells undergo within their lineage progression are interesting questions under current investigation. In this sense, live imaging constitutes a valuable ally in the search of reliable answers to the previous questions. In spite of the current limitations of technology new approaches are being developed and outstanding amount of knowledge is being piled up providing interesting insights in the behavior of aNSCs. Here, we will review the state of the art of live imaging as well as the alternative models that currently offer new answers to critical questions.
Using an adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single-cell level
(2011-11-03) Ortega, Felipe; Costa, Marcos Romualdo; Simon-Ebert, Tatiana; Schroeder, Timm; Götz, Magdalena
A comprehensive understanding of the cell biology of adult neural stem cells (aNSCNSCNSCs) requires direct observation of aNSCNSCNSC division and lineage progression in the absence of niche-dependent signals. Here we describe a culture preparation of the adult mouse subependymal zone (SESEZ), which allows for continuous single-cell tracking of aNSCNSCNSC behavior. The protocol involves the isolation (~3 h) and culture of cells from the adult SESEZ at low density in the absence of mitogenic growth factors in chemically defined medium and subsequent live imaging using time-lapse video microscopy (5–7 d); these steps are followed by postimaging immunocytochemistry to identify progeny (~7 h). This protocol enables the observation of the progression from slow-dividing aNSCNSCNSCs of radial/astroglial identity up to the neuroblast stage, involving asymmetric and symmetric cell divisions of distinct fast-dividing precursors. This culture provides an experimental system for studying instructive or permissive effects of signal molecules on aNSCNSCNSC modes of cell division and lineage progression.

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Navegando por Autor "Ortega, Felipe"