Tracking differentiating neural progenitors in pluripotent cultures using microRNA-regulated lentiviral vectors

Sachdeva, Rohit; Jönsson, Marie E.; Nelander, Jenny; Kirkeby, Agnete; Guibentif, Carolina; Gentner, Bernhard; Naldini, Luigi; Björklund, Anders; Parmar, Malin; Jakobsson, Johan

doi:10.1073/pnas.1006568107

Cited by 40 publications

(35 citation statements)

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“…The procedure for immunohistochemistry has been published in detail elsewhere 12,33 . Primary antibodies were diluted as follows: chicken anti-GFP 1:1,000 (Abcam), rabbit anti-GFAP 1:1,000 (DAKO), mouse anti-NeuN 1:1,000 (Millipore), rabbit anti-Iba1 1:1,000 (WAKO), mouse anti-S100b 1:500 (Sigma), NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2801 ARTICLE mouse anti-O4 1:100 (Millipore), rabbit anti-Ng2 1:200 (Millipore), rabbit-anti-DARPP-32 1:1,000 (Chemicon).…”

Section: Methodsmentioning

confidence: 99%

“…Several recent reports demonstrate the utility of using microRNA (miRNA)-regulated vectors in order to achieve cellspecific transgene expression [10][11][12] . This system is based on the incorporation of complementary miRNA target sites into the transgene cassette, which leads to degradation of the transgene messenger RNA specifically in cells expressing the miRNA resulting in detargeting of transgene expression.…”

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confidence: 99%

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Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

et al. 2013

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Functional studies of resident microglia require molecular tools for their genetic manipulation. Here we show that microRNA-9-regulated lentiviral vectors can be used for the targeted genetic modification of resident microglia in the rodent brain. Using transgenic reporter mice, we demonstrate that murine microglia lack microRNA-9 activity, whereas most other cells in the brain express microRNA-9. Injection of microRNA-9-regulated vectors into the adult rat brain induces transgene expression specifically in cells with morphological features typical of ramified microglia. The majority of transgene-expressing cells colabels with the microglia marker Iba1. We use this approach to visualize and isolate activated resident microglia without affecting circulating and infiltrating monocytes or macrophages in an excitotoxic lesion model in rat striatum. The microRNA-9-regulated vectors described here are a straightforward and powerful tool that facilitates functional studies of resident microglia.

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Section: Methodsmentioning

confidence: 99%

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Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

et al. 2013

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“…Standard immunohistochemistry was used, as published in detail elsewhere (Sachdeva et al, 2010;Thompson et al, 2005). Primary antibodies were diluted as follows: chicken anti-GFP (1:1000; Abcam, GFP # Ab13970), mouse anti-NeuN (1:1000; Millipore, NeuN # MAb377), rabbit anti-GAD67 (1:5000; Chemicon, GAD67 # AB1511), rabbit anti-TH (1:1000, Pelfreeze, TH # P40101-0), goat anti-calretinin (1:2500, Millipore, Calretinin # MAb305), mouse anti-calbindin (1:1000; Sigma, Calbindin # C9848), mouse anti-parvalbumin (1:1000; Sigma, Parvalbumin # P3088) and rabbit anti-Fos (1:1000; Oncogene, c-Fos # PC05).…”

Section: Immunofluorescencementioning

confidence: 99%

microRNA-125 distinguishes developmentally generated and adult-born olfactory bulb interneurons

et al. 2014

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New neurons, originating from the subventricular zone, are continuously integrating into neuronal circuitry in the olfactory bulb (OB). Using a transgenic sensor mouse, we found that adult-born OB interneurons express microRNA-125 (miR-125), whereas the preexisting developmentally generated OB interneurons represent a unique population of cells in the adult brain, without miR-125 activity. Stable inhibition of miR-125 in newborn OB neurons resulted in enhanced dendritic morphogenesis, as well as in increased synaptic activation in response to odour sensory stimuli. These data demonstrate that miR-125 controls functional synaptic integration of adult-born OB interneurons. Our results also suggest that absence of an otherwise broadly expressed miRNA is a novel mechanism with which to achieve neuronal subtype specification. KEY WORDS: microRNA, Neurogenesis, Olfactory bulb, Mouse INTRODUCTIONThe mammalian olfactory bulb (OB) contains two interneuron subpopulations of different temporal and spatial origin. The first population is generated only during embryogenesis and the early postnatal period from local OB progenitor cells (Lemasson et al., 2005;Vergaño-Vera et al., 2006). Generation of the second population starts in the early postnatal period and continues during adulthood. This population emerges from neural stem/progenitor cells (NSPC) located in the subventricular zone (SVZ). NSPC produce neuroblasts that migrate to the OB, where they differentiate to inhibitory interneurons (Doetsch et al., 1999). In the present study, these two populations of OB interneurons are termed, for simplistic reasons, developmentally generated OB (DG-OB) interneurons and adult-born OB (AB-OB) interneurons. These two populations are thought to regulate distinct functions in odour discrimination and can be distinguished by several physiological and morphological characteristics (Alonso et al., 2012;David et al., 2013;Imayoshi et al., 2008;Lemasson et al., 2005;Magavi et al., 2005). Still, the molecular factors that discriminate these two neuronal populations remain largely unknown. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. RESEARCH ARTICLE Received 23 July 2013; Accepted 10 February 2014MicroRNAs (miRNAs) are small non-coding RNAs that mediate post-transcriptional gene regulation. microRNA-125 (miR-125) is the mammalian homolog of lin-4, the first discovered miRNA in Caenorhabditis elegans (Lagos-Quintana et al., 2002;Lee et al., 1993;Wightman et al., 1993). miR-125 is highly expressed in the mammalian brain and cell culture experiments have linked miR-125 to regulation of neuronal differentiation and regulation of synaptic strength and function (Boissart et al., 2012;Edbauer et al., 2010;Le et al., 2009). However, the in vivo role of miR-125 in the brain remains unexplored. In this study, ...

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“…Although possible, these strategies are often complicated to transfer to human cells due to technical issues (9), and only a few successful cases have been described (10,11). To circumvent these difficulties, we have employed an alternative strategy that exploits the cells' endogenous microRNA (miRNA) machinery (12). Our approach is simple to use and offers a reporter system that can be as reliable as BAC transgenesis or knock-in technology.…”

Section: Introductionmentioning

confidence: 99%

“…Based on this strategy, we used miR-292. It is specifically expressed in pluripotent cells, therefore only allowing GFP expression in differentiated cells (12). However, the versatility of the system allows the use of any microRNA of choice, including neuron-specific microRNAs (14).…”

Section: Introductionmentioning

confidence: 99%

Using Endogenous MicroRNA Expression Patterns to Visualize Neural Differentiation of Human Pluripotent Stem Cells

Kirkeby

Parmar

Jakobsson

2011

Springer Protocols Handbooks

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Many existing protocols for neuronal differentiation of human pluripotent cells result in heterogeneous cell populations and unsynchronized differentiation, necessitating the development of methods for labeling specific cell populations. Here we describe how microRNA-regulated lentiviral vectors can be used to visualize specific cell populations by exploiting endogenous microRNA expression patterns. This strategy provides a useful tool for visualization and identification of neural progeny derived from human pluripotent stem cells. We provide detailed protocols for lentiviral transduction, neural differentiation, and subsequent analysis of human embryonic stem cells.

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Tracking differentiating neural progenitors in pluripotent cultures using microRNA-regulated lentiviral vectors

Cited by 40 publications

References 24 publications

Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

microRNA-125 distinguishes developmentally generated and adult-born olfactory bulb interneurons

Using Endogenous MicroRNA Expression Patterns to Visualize Neural Differentiation of Human Pluripotent Stem Cells

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