lineage-reprogramming-pericyte-derived-cells-adult-human-brain-into

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. Here we studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, we show that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, we uncovered the origin of each of the peripheral glial cells and linked them to identified central and peripheral neural stem cells.

Identity, origin, and migration of peripheral glial cells in the Drosophila embryo.

Gliazellen sind entscheidend f&uuml;r die richtige Entwicklung und Funktion des Nervensystems. In Drosophila Embryos sind die Gliazellen des peripheren Nervensystems sowohl durch zentrale Neuroblasten und Sinnesorgan Vorstufen generiert. Die meisten periphere Gliazellen m&uuml;ssen entlang axonale Projektionen der motorische und sensorische Neuronen erreichen ihre endg&uuml;ltigen Positionen in der Peripherie zu migrieren. Hier studierte wir das r&auml;umliche und zeitliche Muster, die Identit&auml;t, die Migration und die Herkunft der alle peripheren Gliazellen in den Segmenten vermehrtes Wildtyp-Embryonen. Die Einrichtung von individuellen Identit&auml;ten unter diese Zellen spiegelt sich durch den Ausdruck eines kombinatorischen Kodex f&uuml;r Molekulare Marker. Dies erm&ouml;glicht die Identifizierung von einzelnen Zellen in verschiedenen genetischen Hintergr&uuml;nden. Dar&uuml;ber hinaus mutant Analyse von zwei dieser Markergene, gro&szlig;en Spalt und Castor, erkennen ihre Implikation in peripheren Gliazellen Entwicklung. Anhand confocal Mikroskopie in 4D zu &uuml;berwachen und folgen der peripheren Glia Migration in lebende Embryonen zeigen wir, dass die Positionierung der meisten dieser Zellen ist mit geringf&uuml;gigen Abweichungen vorgegebenen und die Reihenfolge, in der Zellen in der Peripherie zu migrieren, fast behoben ist. Durch das Studium ihrer Linien, wir deckten die Herkunft der einzelnen peripheren Gliazellen und sie identifizierten zentrale und periphere neuronale Stammzellen verbunden.

Identit&auml;t, Herkunft und Migration von peripheren Gliazellen in Drosophila Embryos.

In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines we have clarified the lineages of the lateral glia in the embryonic ventral nerve cord and linked each glial cell to a specific stem cell. For the lineage of the longitudinal glioblast we show that it consists of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm target genes) reveals that individual glial cells express specific combinations of markers. However, cluster analysis uncovers similar combinatorial codes for cells within, and significant differences between the categories of surface-associated, cortex-associated, and longitudinal glia. Glial cells derived from the same stem cell may be homogeneous (though not identical; stem cells NB1-1, NB5-6, NB6-4, LGB) or heterogeneous (NB7-4, NB1-3) with regard to gene expression. In addition to providing a powerful tool to analyse the fate of individual glial cells in different genetic backgrounds, each of these marker genes represents a candidate factor involved in glial specification or differentiation. We demonstrate this by the analysis of a castor loss of function mutation, which affects the number and migration of specific glial cells.

Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression.

Im Taufliege embryonalen ZNS entwickeln mehrere Untertypen von Gliazellen, die ordnen sich an charakteristischen stellen und vermutlich bestimmte Funktionen zu erf&uuml;llen. Die Mechanismen, die zur Spezifikation und Differenzierung von Gliazellen Subtypen sind weitgehend unbekannt. DiI in Glia-spezifische Gal4 Linien gekennzeichnet haben wir die Linien der die seitlichen Glia in der embryonalen ventrale Nerv Schnur gekl&auml;rt und jeder Gliazelle eine bestimmte Stammzellen verbunden. F&uuml;r die Abstammung von den l&auml;ngs-Glioblast zeigen wir, dass es besteht aus 9 Zellen, die mindestens vier verschiedene Identit&auml;ten zu erwerben. Eine gro&szlig;e Sammlung von molekularer Marker (viele von Ihnen repr&auml;sentieren Transkriptionsfaktoren und potenzielle Gcm-Zielgene) zeigt, dass einzelne Gliazellen bestimmte Kombinationen von Markern schnelles. Cluster-Analyse deckt jedoch &auml;hnliche kombinatorische Codes f&uuml;r Zellen und signifikante Unterschiede zwischen den Gruppen von Oberfl&auml;che-assoziierte Kortex-assoziierte und l&auml;ngs Glia. Gliazellen, abgeleitet von der gleichen Stammzelle k&ouml;nnen homogen sein (obwohl nicht identisch; Stammzellen NB1-1, NB5-6, NB6-4, LGB) oder heterogene (NB7-4, NB1-3) in Bezug auf die Genexpression. Neben der Bereitstellung eines m&auml;chtigen Werkzeug um das Schicksal der einzelnen Gliazellen in verschiedenen genetischen Hintergr&uuml;nden zu analysieren, stellt diese Marker-Gene einen Kandidat Faktor Gliazellen Spezifikation oder Differenzierung beteiligt. Wir demonstrieren dies durch die Analyse der Lenkrolle Verlust der Funktion Mutation, die die Anzahl und die Migration von bestimmten Gliazellen betrifft.

Untertypen von Gliazellen in der Taufliege embryonalen ventrale Nerv Schnur in Bezug auf die Abstammung und Gen-Ausdruck.

Until now, limitations in the ability to enrich adult NSCs (aNSCs) have hampered meaningful analysis of these cells at the transcriptome level. Here we show via a split-Cre technology that coincident activity of the hGFAP and prominin1 promoters is a hallmark of aNSCs in vivo. Sorting of cells from the adult mouse subependymal zone (SEZ) based on their expression of GFAP and prominin1 isolates all self-renewing, multipotent stem cells at high purity. Comparison of the transcriptome of these purified aNSCs to parenchymal nonneurogenic astrocytes and other SEZ cells reveals aNSC hallmarks, including neuronal lineage priming and the importance of cilia- and Ca-dependent signaling pathways. Inducible deletion of the ciliary protein IFT88 in aNSCs validates the role of ciliary function in aNSCs. Our work reveals candidate molecular regulators for unique features of aNSCs and facilitates future selective analysis of aNSCs in other functional contexts, such as aging and injury.

In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells.

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.

Continuous live imaging of adult neural stem cell division and lineage progression in vitro.

&Uuml;ber die systeminterne Spezifikation der adulten neuralen Stammzellen (NSCs) ist wenig bekannt, und in welchem Umfang sie ihre lokale Nische h&auml;ngen. Erwachsene NSC-Division und Fortschreiten der Abstammung unabh&auml;ngig von ihrer Nische beobachten, wir Zellen aus der adulten Maus Subependymal Zone (SEZ) isoliert und kultiviert sie bei niedriger Dichte ohne Wachstumsfaktoren. Wir zeigen hier, dass SEZ Zellen in diesem Kultursystem in erster Linie neurogene und Erwachsenen NSCs Fortschritt durch stereotype Linie B&auml;ume bestehend aus asymmetrischen Zellteilungen, symmetrischen Transit-Verst&auml;rkung Divisionen und final symmetrischen neurogene Divisionen Einhalt zu Gebieten. Stammzellen, die durch ihre Astro/radiale Gliazellen Identit&auml;t und ihrer langsam teilenden Natur identifiziert wurden beobachtet, um asymmetrisch zu generieren und schnell teilenden Zellen verwaltet eine Astro/Radial Glia-Identit&auml;t. Diese wiederum f&uuml;hrte zu symmetrisch und schnell teilenden Zellen, die Gliazellen Kennzeichen verloren, aber hatte nicht noch erworbenen neuronale Funktionen. Die Anzahl der Unterteilungen Verstaerkung beschr&auml;nkte sich auf ein Maximum von f&uuml;nf in diesem System. Dar&uuml;ber hinaus fanden wir, dass Zellwachstum korreliert mit der Anzahl der nachfolgenden Abteilungen des SEZ Zellen, mit langsam teilenden Astro/Radial Glia pr&auml;sentiert das wesentliche Wachstum vor der Teilung. Die Tatsache, dass in der Abwesenheit von exogen mitgelieferten Wachstumsfaktoren sowie der Signale durch die lokale Nische neurogene sofern Linie Fortschreiten so Stereotype Weise stattfindet das Fortschreiten der Linie schl&auml;gt ist in erheblichem Ma&szlig;e, Zelle systeminterne oder am Anfang der Linie vorprogrammiert.

Kontinuierliche Leben Bildgebung von adulten neuralen Stammzellen Division und Linie Fortschreiten in-vitro.

Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.

Prospective isolation of adult neural stem cells from the mouse subependymal zone.

We describe the labeling of adult neural stem cells (aNSCs) in the mouse and human dentate gyrus (DG) by the combinatorial expression of glial fibrillary acidic protein (GFAP) and Prominin1, as revealed by immunohistochemistry. Split-Cre-based genetic fate mapping of these double-positive cells in the adult murine DG reveals their NSC identity, as they are self-renewing and contribute to neurogenesis over several months. Their progeny reacts to stimuli such as voluntary exercise with increased neurogenesis. Prominin1+/GFAP+ cells also exist in the adult human DG, the only region in the human brain for which adult neurogenesis has been consistently reported. Our data, together with previous evidence of such double-positive NSCs in the developing murine brain and in neurogenic regions of vertebrates with widespread neurogenesis, suggest that Prominin1- and GFAP-expressing cells are NSCs in a wide range of species in development and adulthood.

Article	Year
Identity, origin, and migration of peripheral glial cells in the Drosophila embryo. Mechanisms of development\| PubMed ID: 18077143	2008
Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression. Mechanisms of development\| PubMed ID: 18296030	2008
In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. Cell stem cell\| PubMed ID: 21112568	2010
Continuous live imaging of adult neural stem cell division and lineage progression in vitro. Development (Cambridge, England)\| PubMed ID: 21343361	2011
Prospective isolation of adult neural stem cells from the mouse subependymal zone. Nature protocols\| PubMed ID: 22094733	2011
In Vivo Targeting of Adult Neural Stem Cells in the Dentate Gyrus by a Split-Cre Approach. Stem cell reports\| PubMed ID: 24527389	2014

Ruth Beckervordersandforth

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