Name: three-dimensional organotypic cultures of vestibular and auditory sensory organs
Uploaded: 2018-06-01T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

We thank Dr. A. Jacobo, Dr. J. Salvi, and A. Petelski for their contributions to the original research on which this protocol is based. We also thank J. Llamas and W. Makmura for technical assistance and animal husbandry. We acknowledge NIDCD Training grant T32 DC009975, NIDCD grant R01DC015530, Robertson Therapeutic Development Fund, and the Caruso Family Foundation for funding. Finally, we acknowledge support from Howard Hughes Medical Institute, of which Dr. Hudspeth is an Investigator.

All methods described here have been approved by the Animal Care and Use Committees of Rockefeller University and of the University of Southern California.
1. (Optional) Preparation of Collagen I Solution from Mouse-tail Tendons
Note: Collagen I solutions are available commercially. Follow the manufacturer&#39;s instructions for gel preparation.
<ol>
	<li>Euthanize 5 - 10 young adult (3 - 5 weeks old) mice of any wild type strain with carbon dioxi...

<ol>
	<li>Oesterle, E. C., Tsue, T. T., Reh, T. A., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair-cell+regeneration+in+organ+cultures+of+the+postnatal+chicken+inner+ear.">Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.</a> Hear Res. 70 (1), 85-108 (1993).</li><li>Meyers, J. R., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+change+controls+supporting+cell+proliferation+in+lesioned+mammalian+balance+epithelium.">Shape change controls supporting cell proliferation in lesioned mammalian balance epithelium.</a> J Neurosci Off J Soc Neurosci. 27 (16), 4313-4325 (2007).</li><li>Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+mouse+utricle+as+an+in+vitro+preparation+for+studies+of+ototoxic-drug-induced+sensory+hair+cell+death.">The adult mouse utricle as an in vitro preparation for studies of ototoxic-drug-induced sensory hair cell death.</a> Brain Res. 1091 (1), 277-281 (2006).</li><li>Warchol, M. E., Lambert, P. R., Goldstein, B. J., Forge, A., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+proliferation+in+inner+ear+sensory+epithelia+from+adult+guinea+pigs+and+humans.">Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans.</a> Science. 259 (5101), 1619-1622 (1993).</li><li>Lin, V., Golub, J. S., Nguyen, T. B., Hume, C. R., Oesterle, E. C., Stone, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Notch+activity+promotes+nonmitotic+regeneration+of+hair+cells+in+the+adult+mouse+utricles.">Inhibition of Notch activity promotes nonmitotic regeneration of hair cells in the adult mouse utricles.</a> J Neurosci Off J Soc Neurosci. 31 (43), 15329-15339 (2011).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-regulation+of+the+Notch+and+Wnt+signaling+pathways+promotes+supporting+cell+proliferation+and+hair+cell+regeneration+in+mouse+utricles.">Co-regulation of the Notch and Wnt signaling pathways promotes supporting cell proliferation and hair cell regeneration in mouse utricles.</a> Sci Rep. 6, 29418 (2016).</li><li>Chai, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+induces+proliferation+of+sensory+precursors+in+the+postnatal+mouse+cochlea.">Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea.</a> Proc Natl Acad Sci U S A. 109 (21), 8167-8172 (2012).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5++cells+regenerate+hair+cells+via+proliferation+and+direct+transdifferentiation+in+damaged+neonatal+mouse+utricle.">Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle.</a> Nat Commun. 6, 6613 (2015).</li><li>Doetzlhofer, A., White, P. M., Johnson, J. E., Segil, N., Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+growth+and+differentiation+of+mammalian+sensory+hair+cell+progenitors:+a+requirement+for+EGF+and+periotic+mesenchyme.">In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme.</a> Dev Biol. 272 (2), 432-447 (2004).</li><li>White, P. M., Stone, J. S., Groves, A. K., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+signaling+is+required+for+regenerative+proliferation+in+the+cochlea:+conservation+in+birds+and+mammals.">EGFR signaling is required for regenerative proliferation in the cochlea: conservation in birds and mammals.</a> Dev Biol. 363 (1), 191-200 (2012).</li><li>Gnedeva, K., Jacobo, A., Salvi, J. D., Petelski, A. A., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+force+restricts+growth+of+the+murine+utricle.">Elastic force restricts growth of the murine utricle.</a> eLife. 6, (2017).</li><li>Aragona, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanical+checkpoint+controls+multicellular+growth+through+YAP/TAZ+regulation+by+actin-processing+factors.">A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.</a> Cell. 154 (5), 1047-1059 (2013).</li><li>Dong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+a+universal+size-control+mechanism+in+Drosophila+and+mammals.">Elucidation of a universal size-control mechanism in Drosophila and mammals.</a> Cell. 130 (6), 1120-1133 (2007).</li><li>Low, B. C., Pan, C. Q., Shivashankar, G. V., Bershadsky, A., Sudol, M., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP/TAZ+as+mechanosensors+and+mechanotransducers+in+regulating+organ+size+and+tumor+growth.">YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth.</a> FEBS Lett. 588 (16), 2663-2670 (2014).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+YAP+oncoprotein+by+the+Hippo+pathway+is+involved+in+cell+contact+inhibition+and+tissue+growth+control.">Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.</a> Genes Dev. 21 (21), 2747-2761 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. , (2013).</li><li>Semerci, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lunatic+fringe-mediated+Notch+signaling+regulates+adult+hippocampal+neural+stem+cell+maintenance.">Lunatic fringe-mediated Notch signaling regulates adult hippocampal neural stem cell maintenance.</a> eLife. 6, (2017).</li><li>Tuan, R. S., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology+protocols.">Developmental biology protocols.</a> Methods in molecular biology. , 137 (2000).</li><li>Brandon, C. S., Voelkel-Johnson, C., May, L. A., Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+adult+mouse+utricle+and+adenovirus-mediated+supporting-cell+infection.">Dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection.</a> J Vis Exp JoVE. (61), (2012).</li><li>Gosset, M., Berenbaum, F., Thirion, S., Jacques, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+phenotyping+of+murine+chondrocytes.">Primary culture and phenotyping of murine chondrocytes.</a> Nat Protoc. 3 (8), 1253-1260 (2008).</li><li>Landegger, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+AAV+vector+enables+safe+and+efficient+gene+transfer+to+the+mammalian+inner+ear.">A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear.</a> Nat Biotechnol. 35 (3), 280-284 (2017).</li><li>Burns, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinforcement+of+cell+junctions+correlates+with+the+absence+of+hair+cell+regeneration+in+mammals+and+its+occurrence+in+birds.">Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds.</a> J Comp Neurol. 511 (3), 396-414 (2008).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+polarized+extension+and+planar+cell+polarity+in+the+cochlea+by+the+vertebrate+PCP+pathway.">Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway.</a> Nat Genet. 37 (9), 980-985 (2005).</li><li>Chacon-Heszele, M. F., Ren, D., Reynolds, A. B., Chi, F., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cochlear+convergent+extension+by+the+vertebrate+planar+cell+polarity+pathway+is+dependent+on+p120-catenin.">Regulation of cochlear convergent extension by the vertebrate planar cell polarity pathway is dependent on p120-catenin.</a> Dev Camb Engl. 139 (5), 968-978 (2012).</li><li>Yamamoto, N., Okano, T., Ma, X., Adelstein, R. S., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+II+regulates+extension,+growth+and+patterning+in+the+mammalian+cochlear+duct.">Myosin II regulates extension, growth and patterning in the mammalian cochlear duct.</a> Dev Camb Engl. 136 (12), 1977-1986 (2009).</li><li>Tada, M., Heisenberg, C. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension:+using+collective+cell+migration+and+cell+intercalation+to+shape+embryos.">Convergent extension: using collective cell migration and cell intercalation to shape embryos.</a> Dev Camb Engl. 139 (21), 3897-3904 (2012).</li></ol>

The molecular signals that mediate growth and differentiation in the inner ear during development have been studied extensively5,6,7,8,9,10. However, evidence obtained from the utricular model system suggests that mechanical cues, sensed through cell junctions and the activation of Hippo signaling, also play an important role...

The study of most aspects of mammalian organ development has been facilitated by in vitro systems. Two principal methods are now used for the culture of vestibular sensory organs: free-floating1 and adherent2 preparations. Both methods permit the investigation of hair cell vulnerabilities3 and regeneration1,4&#160;in vitro. In addition, the developmental roles of the Notch5,6, Wnt7,8, and epidermal growth factor receptor (EGFR)9,10 signaling cascades in the inner ear have been established, in part, through the use of in vitro cultures of sensory epithelia. However, cell growth and differentiation are controlled, not only through signaling by morphogens, but also through physical and mechanical cues such as intercellular contacts, the stiffness of extracellular matrix, and mechanical stretching or constriction. The role of such mechanical stimuli is challenging to investigate in the developing inner ear in vivo. Moreover, existing free-floating and adherent culture methods are not suitable for such studies in vitro. Here we describe a method for three-dimensional organotypic culture in collagen I gels of varying stiffness. This method largely preserves the in vivo architecture of the vestibular and cochlear sensory organs and allows investigation of the effects of mechanical force on growth and differentiation11.
Because mechanical stimuli are known to activate downstream molecular events, such as the Hippo signaling pathway12,13,14,15, it is important to be able to combine mechanical stimulation with biochemical and genetic manipulations. The culture method described here permits virus-mediated gene delivery and can therefore be used to study both mechanical and molecular signaling during inner ear development11.

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			<td height="21" style="height:21px;width:252px;">Transferrin</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">T8158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">T6146</td>
			<td></td>
		</tr>
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three-dimensional organotypic cultures of vestibular and auditory sensory organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Vestibular and auditory sensory organs from embryonic ears, cultured in 40-Pa collagen I gels mimicking low stiffness embryonic conditions11, retain relatively normal three-dimensional structures (Figure 1) and maintain hair cells and supporting cells (Figure 2 and Figure 3). Although supporting cell density decreases by over 30% (Student&#39;s t-test: n = 4, p</e...

Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

The overall goal of this procedure is to establish three-dimensional organotypic cultures of the murine vestibular and auditory sensory organs. This method can help answer the key questions in the inner ear research field, such as, what are the roles of mechanical force and tissue stiffness during development of the inner-ear sensory organs. The main advantage of this technique is that it largely preserves innate architecture of vestibular cochlea and allows us to study both molecular and mechanical signaling during inner ear development.Dr.Hudspeth and I first had the idea of this method when we realized that three-dimensional cultures can provide morphysiologic conditions to study vestibular organ growth in-vitro. First, use 70%ethanol to clean and disinfect the working area and all the necessary dissection instruments, including number five forceps and a hair knife. Then, use a sterile scalpel blade to make a longitudinal cut, split the head of the mouse into two halves, and extract the temporal bones.Transfer in 15 milliliters of HBSS buffer in a 60 millimeter Petri dish on ice. Next, use two pairs of fine number five forceps to isolate the inner ears from the temporal bones. Then, transfer three to four ears at a time in a 60 millimeter Petri dish containing ice-cold HBSS buffer.Next, orient the ears with the medial side up to locate the utricle. Use the two pairs of number five forceps to remove the cartilage surrounding the vestibular organs. Next, cut the vestibular nerve, the connection between the utricle and saccule, and the semi-circular canals to free the utricle and the attached ampullae from the ears.Trim excess semi-circular canals if needed. Alternatively, use the two pairs of number five forceps to remove the cartilaginous tissue surrounding the hearing organ. Then, isolate the cochlea.Next, use a 200-microliter pipette fitted with a wide, non-stick tip to transfer the cochlea and the utricles to a 30-millimeter Petri dish filled with the DMEM/F12-based growth medium. Then, transfer the cochlear and the utricle preparations to a tissue culture incubator, gassed with 5%carbon dioxide at 37 degrees Celsius. This is to achieve organ cultures with enclosed lumens.Prepare 100 microliters of the collagen one polymerization solution in a sterile 1.5-milliliter tube. Then, leave the tube on ice. Next, remove the cochlear and utricle preparations from the tissue culture incubator.Prepare the neutralized collagen one solution. To do so, add 400 microliters of collagen one to a chilled 1.5-milliliter tube with 100 microliters of polymerization solution by pipetting several times on ice. Transfer 500 microliters of the neutralized collagen one solution to a chilled 30-millimeter Petri dish with a 10-millimeter glass bottom insert.Immediately transfer the cochlea or the utricles to the neutralized collagen one solution using a 200-microliter pipette fitted with a wide, non-stick tip. Use a sterile hair knife, or a pair of fine forceps to adjust the organs to their desired positions under a binocular dissecting microscope. In order to ensure complete polymerization, incubate the Petri dish at 37 degrees Celsius for 20 minutes.After 20 minutes, add three milliliters of growth medium, supplemented with 0.5%fetal bovine serum in each Petri dish. After adding the growth medium, incubate the culture at 37 degrees Celsius. First, defrost the virus on ice.Then, mix the viral suspension with Trypan blue solution in a 0.5-milliliter conical tube to adjust the final dye concentration of 0.05%After mixing the Trypan blue solution, leave the viral particles on ice. Then, use a binocular dissecting microscope to observe the breaking of the tip of the glass needle, prepared on a micro-pipette puller with clean, fine forceps. First, remove the sensory organ culture from the incubator.Attach the needle to the micro-injector. Fill the needle with two to three microliters of dye and viral mixture. Look through the binocular dissecting microscope while setting the needle into the sensory organ.Keep forwarding the needle tip through the mesenchymal and epithelial layers of the roof of a three-dimensional utricular culture. Keep injecting the viral mixture until the cavities of the utricle and ampullae fill with the blue dye. Then, incubate the organ culture in a 37 degree Celsius tissue culture incubator gassed with 5%carbon dioxide.These are the light microscopic images of the utricle and cochlear culture embedded in the three-dimensional collagen one gel. The utricle and the cochlea are obtained from 17.5 and 14.5 days old embryos, respectively, and cultured for 48 hours. These confocal microscopic images show 18.5 days old embryonic utricle just before and after explantation in the 40PA collagen one gel.After three days in the culture, almost 80%of the Myo7A positive hair cells remain intact. Supporting cell density decreases by over 30%and hair cell density decreases by 60%after three days in utricular cultures. These confocal microscopic images show an embryonic 14.5 days old cochlea just before and after explantation in the 40PA collagen one gel.After three days in culture, Myo7A and Sox2 positive inner and outer hair cells are morphologically distinguishable and are organized in about four to five rows. Here, the images show successful viral infection of the supporting cells at the base of the cochlear and throughout the utricular three-dimensional cultures obtained from 15.5 and 17.5 days of embryos, respectively. After watching this video, you should have a good understanding of how to dissect intact inner-ear sensory organs establish three-dimensional cultures of utricle and cochlea and collagen type one gel, and perform viral transfections in those three-dimensional cultures.Remember that working with viral particles, depending on gene cargo, can be hazardous. Precautions, such as wearing personal protective equipment and proper viral disposal, should always be practiced.

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Research

JoVE Journal

Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

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 (NCT01151124 and 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-mycERTAMconstruct. 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&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...