Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols¹. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.     

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO₂. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.     

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.     

Rapid Internalization and Intracellular Metabolic Processing of Exogenous Ganglioside by Cerebellar Granule Cells Differentiated in Culture

Ganglioside GM1, tritiated at the level of the long chain base (sphingosine) [( Sph-3H]GM1), sialic acid (N-acetylneuraminic acid) [( NeuAc-3H]GM1), or terminal galactose [( Gal-3H]GM1) was supplied to cerebellar granule cells differentiated in vitro, and its metabolic processing was followed with pulse time. Using [Sph-3H]GM1 and [NeuAc-3H]GM1 the formation of radioactive compounds of catabolic origin (GM2, GM3, lactosylceramide, glucosylceramide, and ceramide) started being detectable at 10-15 min of pulse, whereas compounds of biosynthetic origin (GD1a, GD1b, GT1b, O-acetylated GT1b, spingomyelin, and sialoglycoprotein) appeared after 15-30 min of pulse. Using [Gal-3H]GM1 two radioactive substances were formed, GD1a and GT1b, with the former (produced by direct sialosylation of GM1) appearing after 30 min of pulse and the latter (formed by biosynthetic recycling of released galactose) appearing after 2 h. The radioactivity linked to all metabolites increased with increasing pulse time until 4 h. The percentage of GM1 taken up and subjected to metabolic processing was found to increase from 1.8% after 10 min of pulse to 12.5% after 4 h. Cerebellar granule cells were able to release enzymes of lysosomal origin, beta-D-N-acetylhexosaminidase and beta-D-galactosidase, into the culture medium, with the release being markedly decreased by the absence in the medium of fetal calf serum, a condition that was used for studying exogenous GM1 uptake and metabolization. However, these enzymes exerted no activity at the pH of the culture medium, and no radioactive gangliosides, besides GM1, were detected in the culture medium during pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the Ganglioside Long-Chain Base Composition of Rat Cerebellar Granule Cells During Differentiation and Aging in Culture

Abstract: Changes in the ganglioside long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed-phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising ∼8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the "b series" (GD1b, GT1b, and GQ1b) than in the "a series" (GM1 and GD1a). During aging in culture, for 8–22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a.     

Development of Benzodiazepine and Picrotoxin (t-Butylbicyclophosphorothionate) Binding Sites in Rat Cerebellar Granule Cells in Culture

The specific bindings of [3H]flunitrazepam [( 3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, although their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p less than 0.001). Taken together, our data suggest the existence of several gamma-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-mimetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol (THIP) in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.     

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system ^1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex ^6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method ⁹. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice ^10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA ¹². These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.     

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood ¹. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine ^1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation ^3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes ^5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells ⁹. Both approaches, whose basic principles were already described by other video protocols ^10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively ^9,15.These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.     

Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells,an Extrathymic αβT-cell Progenitor

The thymus, the primary organ for the generation of αβ T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of αβT cells. Yet, thymic involution begins early in life leading to a drastically reduced output of naïve αβT cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic αβT cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. γδ T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a γδ T cell subset by showing that the scarce entity of CD4⁺ Vδ1⁺γδ T cells can transdifferentiate into αβT cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. Vδ1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4⁺ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4⁺ Vδ1⁺γδ T cells. This knowledge is prerequisite for studies that relate to this αβT cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.     

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection,Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin—the histone-to-protamine switch.Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.     

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures,Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability ({"type":"clinical-trial","attrs":{"text":"NCT01151124","term_id":"NCT01151124"}}NCT01151124 and {"type":"clinical-trial","attrs":{"text":"NCT02117635","term_id":"NCT02117635"}}NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycER^TAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3’UTRs by dual reporter plasmid transfection and dual luciferase assay.     

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells^1-3.Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines⁴. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors^5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies⁷. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types⁸. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC^2,3.The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)³. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.