Name: induction of leptomeningeal cells modification via intracisternal injection
Uploaded: 2020-05-07T14:30:00
Description: Watch this Scientific Journal Video about Induction of Leptomeningeal Cells Modification Via Intracisternal Injection at JoVE.com

This study was supported by grants from the Swedish Research Council, the Swedish Cancer Society, the Swedish Foundation for Strategic Research, Knut och Alice Wallenbergs Stiftelse and the Strategic Research Programme in Stem Cells and Regenerative Medicine at Karolinska Institutet (StratRegen).

The surgical procedures hereby presented were approved by Stockholms Norra Djurf&#246;rs&#246;ksetiska N&#228;mnd and carried out in agreement with specifications provided by the research institute (Karolinska Institute, Sweden).
NOTE: Intracisternal injection can be flexibly adapted for multiple research purposes. We present below a procedure developed to efficiently label leptomeningeal cells for fate mapping based on injection of endoxifen in a transgenic mouse line carryin...

<ol>
	<li>Vanlandewijck, 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+molecular+atlas+of+cell+types+and+zonation+in+the+brain+vasculature.">A molecular atlas of cell types and zonation in the brain vasculature.</a> Nature. 554 (7693), 475-480 (2018).</li><li>Whish, S., 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=The+inner+CSF-brain+barrier:+developmentally+controlled+access+to+the+brain+via+intercellular+junctions.">The inner CSF-brain barrier: developmentally controlled access to the brain via intercellular junctions.</a> Frontiers in Neuroscience. 9, 16 (2015).</li><li>Weller, R. O., Sharp, M. M., Christodoulides, M., Carare, R. O., Mollgard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+meninges+as+barriers+and+facilitators+for+the+movement+of+fluid,+cells+and+pathogens+related+to+the+rodent+and+human+CNS.">The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS.</a> Acta Neuropathologica. 135 (3), 363-385 (2018).</li><li>Choe, Y., Siegenthaler, J. A., Pleasure, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cascade+of+morphogenic+signaling+initiated+by+the+meninges+controls+corpus+callosum+formation.">A cascade of morphogenic signaling initiated by the meninges controls corpus callosum formation.</a> Neuron. 73 (4), 698-712 (2012).</li><li>Siegenthaler, J. A., 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=Retinoic+acid+from+the+meninges+regulates+cortical+neuron+generation.">Retinoic acid from the meninges regulates cortical neuron generation.</a> Cell. 139 (3), 597-609 (2009).</li><li>Bifari, 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=Neurogenic+Radial+Glia-like+Cells+in+Meninges+Migrate+and+Differentiate+into+Functionally+Integrated+Neurons+in+the+Neonatal+Cortex.">Neurogenic Radial Glia-like Cells in Meninges Migrate and Differentiate into Functionally Integrated Neurons in the Neonatal Cortex.</a> Cell Stem Cell. 20 (3), 360-373 (2017).</li><li>De Bock, 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+new+angle+on+blood-CNS+interfaces:+a+role+for+connexins?.">A new angle on blood-CNS interfaces: a role for connexins?.</a> FEBS Letters. 588 (8), 1259-1270 (2014).</li><li>Ramos, 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=Cisterna+Magna+Injection+in+Rats+to+Study+Glymphatic+Function.">Cisterna Magna Injection in Rats to Study Glymphatic Function.</a> Methods in Molecular Biology. 1938, 97-104 (2019).</li><li>Xavier, A. L. 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=Cannula+Implantation+into+the+Cisterna+Magna+of+Rodents.">Cannula Implantation into the Cisterna Magna of Rodents.</a> Journal of Visualized Experiments. (135), (2018).</li><li>Iliff, J. 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=A+paravascular+pathway+facilitates+CSF+flow+through+the+brain+parenchyma+and+the+clearance+of+interstitial+solutes,+including+amyloid+beta.">A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta.</a> Science Translational Medicine. 4 (147), (2012).</li><li>Coureuil, M., Lecuyer, H., Bourdoulous, S., Nassif, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+journey+into+the+brain:+insight+into+how+bacterial+pathogens+cross+blood-brain+barriers.">A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers.</a> Nature Reviews Microbiology. 15 (3), 149-159 (2017).</li><li>Madisen, L., 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=Transgenic+mice+for+intersectional+targeting+of+neural+sensors+and+effectors+with+high+specificity+and+performance.">Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.</a> Neuron. 85 (5), 942-958 (2015).</li><li>Slezak, 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=Transgenic+mice+for+conditional+gene+manipulation+in+astroglial+cells.">Transgenic mice for conditional gene manipulation in astroglial cells.</a> Glia. 55 (15), 1565-1576 (2007).</li><li>Hardy, S. J., Christodoulides, M., Weller, R. O., Heckels, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Neisseria+meningitidis+with+cells+of+the+human+meninges.">Interactions of Neisseria meningitidis with cells of the human meninges.</a> Molecular Microbiology. 36 (4), 817-829 (2000).</li><li>Colicchio, 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=The+meningococcal+ABC-Type+L-glutamate+transporter+GltT+is+necessary+for+the+development+of+experimental+meningitis+in+mice.">The meningococcal ABC-Type L-glutamate transporter GltT is necessary for the development of experimental meningitis in mice.</a> Infection and Immunity. 77 (9), 3578-3587 (2009).</li><li>Ricci, S., 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=Inhibition+of+matrix+metalloproteinases+attenuates+brain+damage+in+experimental+meningococcal+meningitis.">Inhibition of matrix metalloproteinases attenuates brain damage in experimental meningococcal meningitis.</a> BMC Infectious Diseases. 14, 726 (2014).</li></ol>

The protocol outlined here presents a straightforward and reproducible procedure to label leptomeningeal cells for fate mapping. We use intracisternal injection of endoxifen, an active metabolite of Tamoxifen, to induce expression of tdTomato fluorescent reporter in Cx30-CreER; R26R-tdTomato mice12,13.
Compared to other protocols used for gaining access to the cerebrospinal fluid through the cisterna magna9, our...

Leptomeningeal cells are a fibroblast-like population of cells organized in a thin layer overlaying the brain and expressing genes implicated in collagen crosslinking (e.g., Dcn and Lum), and in the establishment of a brain-meningeal barrier (e.g., Cldn11)1,2. Leptomeningeal cells are implicated in a wide range of physiological functions, from strict control over the cerebrospinal fluid drainage3 to guidance of neural progenitors in the developing brain4,5. A recent study has also proposed that leptomeninges in the newborn may harbor radial glia-like cells that migrate into the brain parenchyma and develop into functional cortical neurons6.
Leptomeningeal cells are located in close proximity to surface astrocytes and share with them, as well as other parenchymal astroglia, expression of connexin-30 (Cx30)7. The surgical procedure outlined below allows widespread and specific labelling of these meningeal cells via a one-time delivery of endoxifen into the cisterna magna of transgenic mice conditionally expressing tdTomato in Cx30+ cells (i.e., using a CreER-loxP system for fate mapping). Endoxifen is an active metabolite of Tamoxifen and induces recombination of CreER-expressing cells in the same way as Tamoxifen does. It is, however, the recommended solution for topical application because it dissolves in 5-10% DMSO, instead of high concentrations of ethanol. Additionally, endoxifen does not cross the brain-meningeal barrier, thereby enabling specific recombination of leptomeningeal cells, without labelling of the underlying Cx30+ astroglial population (see Representative Results).
The technique presented here aims at manually and safely injecting the compound in the cerebrospinal fluid, via direct access to the cisterna magna. Unlike other, more invasive procedures requiring craniotomy, this approach allows to infuse compounds without causing damage to the skull or the brain parenchyma. Thus, it is not associated with the induction of inflammatory reactions triggered by activation of parenchymal glia cells. Similar to other injection strategies described before8,9,10, the present approach relies on the surgical exposure of the atlanto-occipital dural membrane covering the cisterna magna, after blunt dissection of the overlaying neck muscles. However, unlike for other procedures, we recommend the use of a needle bent at the tip, which can be stabilized against the occipital bone during administration. This will prevent the risk of the needle penetrating too deep and damaging the underlying cerebellum and medulla.
This surgical procedure is compatible with lineage tracing investigations that aim at mapping changes in cell identities and migration routes through parenchymal layers. It can also be adapted to genetic ablation studies that intend to probe the role of leptomeningeal cells in health and disease, such as their contribution to cortical development5 or the spreading of bacterial meningitis3,11. Finally, it can be utilized to track cerebrospinal fluid movement when combined with delivery of fluorescent tracers in wildtype animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia unit</td>
			<td>Univentor 410</td>
			<td>8323102</td>
			<td>Complete of vaporizer, chamber, and tubing that connects to chamber and mouse head holder</td>
		</tr>
		<tr>
			<td>Anesthesia (Isoflurane)</td>
			<td>Baxter Medical AB</td>
			<td>000890</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Sigma-Aldrich</td>
			<td>PVP1</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Orion Pharma AB</td>
			<td>014920</td>
			<td>Commercial name Rymadil</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Carl Roth</td>
			<td>0258.1</td>
			<td>Use silk 5-0 sutures, in alternative</td>
		</tr>
		<tr>
			<td>Medbond Tissue Glue</td>
			<td>Stoelting</td>
			<td>50479</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>E8284</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Histolab</td>
			<td>01370</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (30G beveled needle)</td>
			<td>Hamilton</td>
			<td>80300</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Aspen Nordic</td>
			<td>520455</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse head holder</td>
			<td>Narishige International</td>
			<td>SGM-4</td>
			<td>With mouth piece for inhalational anhestetics. Alternatively, use a stereotactic frame</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Aesculap</td>
			<td>GT420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile absorption spears</td>
			<td>Fine Science Tools</td>
			<td>18105-01</td>
			<td>Sterile cotton swabs are also a good option</td>
		</tr>
		<tr>
			<td>Surgical separator</td>
			<td>World Precision Instrument</td>
			<td>501897</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>Bausch&#38;Lomb Nordic AB</td>
			<td>541760</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of leptomeningeal cells modification via intracisternal injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

Intracisternal injection of endoxifen in transgenic mice expressing CreER under the Cx30 promoter13 and an inducible fluorescent reporter allows for specific recombination of leptomeningeal cells without labelling the neighboring Cx30-expressing surface and parenchymal astrocytes in the cortex (Figure 1). In order to gain access to the cisterna magna, the anesthetized animal is positioned with its body and its head at an angle of appro...

Watch this Scientific Journal Video about Induction of Leptomeningeal Cells Modification Via Intracisternal Injection at JoVE.com

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

Our surgical procedure enables specific gene editing of leptomeningeal cells to study their function in many physiological and pathological conditions such as neurodevelopment and the spreading of bacterial meningitis. To minimize damage during the intracisternal delivery of endoxifen, we utilize a bent needle that can be secured against the caudal edge of the skull to prevent it from penetrating deeper in the tissue. The procedure can be used to probe the role of genes expressing leptomeningeal cells through loss and gain of function experiments.It can also be adopted to track the cerebrospinal fluid flow. Visual demonstration of this method will ensure successful identification of the injection site for intracisternal injection and understanding on how to secure the needle against the occipital bone. Begin by preparing a Hamilton syringe with a 30 gauge beveled needle for injection.Use forceps to bend the needle to 30 degrees three millimeters from the tip. Next, dilute endoxifen in 10%DMSO to a concentration of one milligram per milliliter and backfill the syringe. Anesthetize the animal in the isoflurane chamber.Then adjust the mouse head holder so that the mouthpiece is approximately at a 30 degree angle from the surface of the surgical table and fix the animal's head onto the holder. To improve accessibility to the cisterna magna, position the animal's body at approximately 30 degrees from the surface of the table with the head tilted downward which will establish an angle of 120 degrees with the rest of the body and extend the back of the neck to facilitate access to the cisterna magna. Once the animal is properly positioned, apply ophthalmic ointment, shave the back of its neck, and sanitize the area with alcohol wipes and Betadine.Use surgical scissors to make a midline incision starting at the level of the occipital bone and extending posteriorly. Gently separate the superficial connective tissue and neck muscles by pulling sideways from the midline with fine tip tweezers which will expose the dural membrane overlying the cisterna magna. Position a small surgical separator to enable visualization of the cisterna magna throughout the procedure.Identify the caudal end of the occipital bone and insert the previously bent needle immediately underneath. Once the dura has been perforated, allow the bent tip of the needle to penetrate underneath the surface by gently pulling the syringe upward and parallel to the animal's body which will ensure better stability. Inject the compound slowly to avoid interference with cerebrospinal fluid's natural flow.After the injection, let the needle rest inside for one minute, then carefully remove it with the help of fine tip forceps. Close the skin with a few drops of cyanoacrylate adhesive and apply local anesthetic at the injection site. Remove the animal from the holder and place it in a clean cage on a heating pad.Then make sure to monitor the animal until it regains consciousness. Intracisternal injection of endoxifen in transgenic mice expressing CreER under the Cx30 promoter, an inducible fluorescent reporter allows for specific recombination of leptomeningeal cells without labeling the neighboring Cx30 expressing surface astrocytes and parenchymal astrocytes in the cortex. This injection protocol takes advantage of the physiological movement of the cerebrospinal fluid so the endoxifen solution is distributed throughout the subarachnoid space to efficiently recombine leptomeningeal cells overlaying the olfactory bulbs, cortex, and cerebellum.The solution does not cross the brain meningeal barrier or come in contact with astroglial cells of the parenchyma as opposed to systemic administration through oral gavage. Recombination of leptomeningeal cells after intracisternal injection was identified through Pdgfr-alpha reactivity while surface and parenchymal astrocytes expressing Gfap remained unlabeled. When attempting this experiment, it is important to locate the injection site for intracisternal delivery.This visual demonstration provides the technical know-how to carry out the procedure while minimizing the risk of damaging the neural tissue under it. Gene ablation studies can be performed with this technique to investigate the role of leptomeningeal cells in corticogenesis and regulation of cerebrospinal fluid formation and to identify additional sites for spreading of bacteria in the subarachnoid space.

induction-leptomeningeal-cells-modification-via-intracisternal

Research

JoVE Journal

Neuroscience

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy

While both living donation and donation after circulatory death provide alternative opportunities for organ transplantation, donation after donor brain death (BD) still represents the major source for solid transplants. Unfortunately, the irreversible loss of brain function is known to induce multiple pathophysiological changes, including hemodynamic as well as hormonal modifications, finally leading to a systemic inflammatory response. Models that allow a systematic investigation of these effects in vivo are scarce. We present a murine model of BD induction, which could aid investigations into the devastating effects of BD on allograft quality. After implementing intra-arterial blood pressure measurement via the common carotid artery and reliable ventilation via a tracheostomy, BD is induced by steadily increasing intracranial pressure using a balloon catheter. Four hours after BD induction, organs may be harvested for analysis or for further transplantation procedures. Our strategy enables the comprehensive analysis of donor BD in a murine model, therefore allowing an in-depth understanding of BD-related effects in solid organ transplantation and potentially paving the way to optimized organ preconditioning.

We present a murine model of brain death induction in order to evaluate the influence of its pathophysiological effects on organs as well as on consecutive grafts in the context of solid organ transplantation.

While both living donation and donation after circulatory death provide alternative opportunities for organ transplantation, donation after donor brain ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Injection of Glioblastoma (GBM) Cells into E5 Optic Tectum In Ovo

To begin, incubate the fertilized chicken eggs in a humidified egg incubator with the pointed end facing down at 37.5 degrees Celsius. On the sixth day of incubation, or E5, sterilize the eggshell by spraying it with 70%ethanol. Using an egg candler, trace along the perimeter of the airspace above the embryo with a pencil and cover the outlined area with transparent tape.Gently cut around the traced area with curved scissors without cutting into the embryonic membranes or blood vessels. Discard the top of the eggshell. Add a few drops of saline or cell culture media onto the airspace membrane to wet it so that it will detach easily.Using fine forceps, carefully pierce the airspace membrane over the top of the embryo. Remove it. Grab the transparent amnion membrane that immediately surrounds the embryo to position the head and inject around 50, 000 cells in five microliters of suitable cell culture media into the optic tectum with a glass micropipette and a Pneumatic PicoPump.Add a few drops of 50 milligrams per milliliter of ampicillin on top of the embryo and cover the hole in the top of the eggs with clear tape. Place them in a humidified egg incubator until E15 for dissection. The tumors that formed in the E5 optic tectum in vivo after the injection of glioblastoma, or GBM stem cells, or GSCs expressing green fluorescent protein at E15 are shown in this figure.GSCs attach to the ventricular surface and form invasive tumors in the brain wall. Four colors were used to identify five features in this experiment, green GSCs, white nuclei, white blood vessels, blue integrin alpha-6, and either red SOX2 or red Nestin. These figures show that the GSCs reside in your blood vessels and appear to be migrating along them.Movies of rotating 3D volume renders to fixed and immunostained slices of in vivo GSC tumors are shown here.

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...