Name: stable and efficient genetic modification of cells in the adult mouse v-svz for the analysis of neural stem cell autonomous and non-autonomous effects
Uploaded: 2016-02-17T14:00:00
Description: Watch this Scientific Journal Video about Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects at JoVE

We acknowledge the help of M.J. Palop and the technical support of the SCSIE of the Universidad de Valencia. We also thank Antonia Follenzi for helpful comments and discussion of the manuscript. I.F is supported by Fundaci&#242;n Bot&#236;n, by Banco Santander through its Santander Universities Global Division, and by grants from Generalitat Valenciana (Programa Prometeo, ACOMP, and ISIC) and Ministerio de Econom&#236;a y Competitividad (MINECO: SAF2011-23331, CIBERNED and RETIC TerCel). This work was also supported by BFU2010-21823 and RETIC TerCel grants from MINECO and the European Research Council (ERC) 2012-StG (311736- PD-HUMMODEL) to A.C. B.M-P. is the recipient of a Spanish FPI fellowship of the MINECO.

ETHICS STATEMENT: This protocol follows the animal care guidelines of the University of Valencia in compliance with European directive 2010/63/EU.
1. Generation of LV for In Vivo Marking Studies (see Figure 1a)
CAUTION: The procedure described herein is biosafety level 2, therefore perform all the following procedures in a biohazard hood. Ensure that research personnel are appropriately qualified and trained in all procedures. Wear personal protective equipment, including gown, d...

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LVs offer important advantages over other viral systems for the genetic modification of adult NSCs16,18. Stereotaxic delivery of lentiviruses to the V-SVZ niche represents an efficient method to label and trace infrequently dividing B1-NSCs overcoming the limitations of other commonly used methods such as BrdU, which is diluted after multiple cell divisions, or retrovirus, which only target cells that are proliferating at the moment of application. LVs, together with adenoviruses, can infect cells independentl...

The murine ventricular-subventricular zone (V-SVZ), in the walls of the lateral ventricle facing the striatum, is a very active germinal region in which a continual process of progenitor cell replication and differentiation results in the persistent production of olfactory bulb (OB) interneurons and corpus callosum oligodendrocytes1. The lifelong generation of these cells appears to be supported by the presence in this region of neural stem cells (NSCs; also called B1 cells), which express the astrocytic antigen glial fibrillary acidic protein (GFAP) and stem cell markers such as nestin, Id1 and Sox22. GFAP-expressing B1 cells generate transit amplifying progenitor (TAP) cells (C cells), which express transcription factors Dlx2 (distal-less homeobox 2) and Ascl1 (mammalian achaete-schute homolog 1) and divide rapidly a few times before they give rise to migrating neuroblasts (A cells) or oligodendroblasts3. Newly-generated proliferative neuroblasts migrate anteriorly, forming the rostral migratory stream (RMS) to the OB, where they integrate into the granular and glomerular layers as differentiated inhibitory interneurons. Migrating young oligodendroblasts move to the CC, where they become immature NG2-positive cells that continue to divide locally or differentiate into mature myelinating oligodendrocytes1,4.
B1 cells, which derive from fetal radial glial cells, retain the elongated and polarized morphology of their predecessors and exhibit a highly specialized relationship with their niche. They span between the ependyma which lines up the ventricle and the network of blood vessels that irrigate the V-SVZ niche. The small apical process of B1 cells intercalates among multiciliated ependymocytes and ends in a single non-motile primary cilium, whereas their basal process extends long distances to approach the planar vascular plexus that irrigates this niche ending in the basal lamina of the plexus capillaries2,5-8 .
The most reliable way to distinguish B1-NSCs from non-neurogenic astrocytes, which are also GFAP+, in the intact V-SVZ niche is based on whole-mount preparations of the ventricle lateral wall and their analysis by 3-D confocal microscopy after immunostaining for GFAP to label the thin B1-NSC apical process, &#946;-catenin to delineate cell membranes, and either &#947;-tubulin as a marker of cilial basal bodies or acetylated &#945;-tubulin to label the extent of each cilium5,8. Observations of these whole-mounts from the ventricular surface have indicated that B1 and ependymal cells are arranged in &#34;pinwheels&#34;5, in which the uniciliated apical processes of one or several GFAP+ B1 cells are encircled by a rosette of multiciliated ependymal cells.
The characteristic morphology of B1 cells correlates with experimental evidence indicating that blood vessels/endothelial cells and ventricular cerebrospinal fluid (CSF) constitute regulated sources of soluble signals acting on NSCs2,6,9-11. At the ventricular surface, homotypic and heterotypic apico-lateral interactions involving ependymal and B1 cells include tight junctions and adherens junctions5,12. Moreover, adhesion molecules implicated in the junctional complexes between B1 and ependymal cells, such as N-cadherin and V-CAM, have been shown to regulate not only the highly organized positioning of B1 in the V-SVZ niche, but also their quiescence12,13. The ependymal-B1 cell monolayer appears to act as a diffusion barrier allowing the regulated flux of water and small molecules from the CSF, but restricting the intercellular passage of large proteins10,11. Experimental evidence indicates that the uniquely positioned B1 cell apical cilium could play a role as a sensor of signaling polypeptides present in the CSF2,5-7. Ependymal cells are, per se, also a source of soluble and membrane-bound signals with a role in the regulation of NSC behavior14,15.
Traceable nucleosides, such as bromo-deoxyuridine (BrdU), or retroviruses have been widely used to label progenitor cells, including NSCs, in vivo. However, these methods are not optimal for long-term fate tracing because BrdU signals dilute through repeated cell divisions and retroviruses appear to preferentially target transiently amplifying cells due to their requirement of cell proliferation for transduction16,17. To examine NSC physiology in vivo, including interactions with niche components, it is crucial to establish a method to label and trace rarely dividing cells, as B1-NSCs are largely quiescent and their neighboring ependymal cells never divide under physiological conditions3. Here, we show that lentiviral vectors (LVs) allow for high-efficiency gene marking and long-term modification of adult NSCs and non-dividing ependymal cells, due most reasonably to their ability to transduce and to integrate into the genome of target cells in a cell cycle-independent way. Moreover, we show how the route of delivery and viral titer help to specifically transduce ependymal cells, but not B1 cells thereby allowing the analysis of niche-dependent, ependymal effects on NSCs.

<table>
	<tbody>
		<tr>
			<td colspan="4">Part 1: Generation of LV for in vivo delivery.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment:</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima XL-100K</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>SW-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>SW-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman Coulter</td>
			<td>358126</td>
			<td>25X89 mm</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman Coulter</td>
			<td>326819</td>
			<td>13X51 mm</td>
		</tr>
		<tr>
			<td>Ultracentrifuge adapters</td>
			<td>Beckman Coulter</td>
			<td>358156</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>SPL</td>
			<td>PLC-30006</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>SPL</td>
			<td>PLC-30024</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>SPL</td>
			<td>PLC-20101</td>
			<td>100x20 style</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Afora</td>
			<td>DE400800</td>
			<td>12x75 mm, 5 ml</td>
		</tr>
		<tr>
			<td>Cup sterile FACS filter</td>
			<td>BD</td>
			<td>340626</td>
			<td>30 &#181;m</td>
		</tr>
		<tr>
			<td>Nitrocellulose filter</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>0.22 &#956;m&#160;</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>LSR Fortessa</td>
			<td>Blue laser 488 nm</td>
		</tr>
		<tr>
			<td>Steritop filter</td>
			<td>Biofil</td>
			<td>FPE-204-500&#160;</td>
			<td>0.22 &#181;m</td>
		</tr>
		<tr>
			<td colspan="4">Reagents:</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12251</td>
			<td>Core packaging plasmid</td>
		</tr>
		<tr>
			<td>pRSV.REV plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12253</td>
			<td>Core packaging plasmid</td>
		</tr>
		<tr>
			<td>pMD2G plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12259</td>
			<td>Envelope plasmid</td>
		</tr>
		<tr>
			<td>pRRL-SIN-PPT.PGK.EGFP.Wpre plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12252</td>
			<td>Transfer vector plasmid</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td>Biowest</td>
			<td>L0101-500</td>
			<td>For HeLa cell culture</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium&#160;</td>
			<td>Life technologies</td>
			<td>12440-053</td>
			<td>For 293T cell culture</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE)</td>
			<td></td>
			<td></td>
			<td>Tris-HCl (sigma, T5941), 0.1 mM EDTA (sigma, E5134), pH 7.6,&#160; DNAse/RNAse-free, 0.2 &#181;m sterile-filtered</td>
		</tr>
		<tr>
			<td>2X HBS</td>
			<td></td>
			<td></td>
			<td>0.28 M NaCl (Sigma, S7653), 0.05 M HEPES (Sigma, H7523), 1.5 mM anhydrous Na2HPO4 (Sigma, S7907) in dH2O (preferably not MilliQ). Adjust pH to 7.0 with NaOH solution (Calbiochem, 567530).</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biowest</td>
			<td>S181B-500</td>
			<td>Stock solution at 100X, used to prepare HeLa and 293T culture medium at a final concentration of 10X.</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513-100</td>
			<td>Stock solution at 200 mM, used to prepare HeLa and 293T culture medium at a final concentration of 6 mM.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life technologies</td>
			<td>11360-039</td>
			<td>Stock solution at 100 mM, used to prepare HeLa and 293T culture medium at a final concentration of 1 mM.</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Life technologies</td>
			<td>35050-061</td>
			<td>Used to prepare 293T culture medium at a final concentration of 1%.</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458</td>
			<td>Stock solution contains 5,000 units/ml penicillin and 5 mg/ml streptomycin. Used to prepare HeLa and 293T culture medium at a final concentration of 1%.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td>With phenol red, contains 2.5 g porcine trypsin and 0.2 g EDTA 4Na/L HBSS.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160; (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td>Without calcium chloride and magnesium chloride, 10X, liquid, sterile-filtered, suitable for cell culture. Stock solution used to prepare 1X PBS in cell culture grade water.</td>
		</tr>
		<tr>
			<td>Polybrene (hexadimethrine bromide)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>Powder. Prepare a 1000X stock solution at 8 mg/ml in dH2O</td>
		</tr>
		<tr>
			<td>Paraformaldehyde EM grade 16%</td>
			<td>EM Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 2: Sterotaxic injection of LV into the SEZ proper or the lateral ventricle.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment:</td>
		</tr>
		<tr>
			<td><a href="http://www.leicabiosystems.com/">Vernier stereotaxic instrument</a></td>
			<td>NeuroLab, Leica</td>
			<td>39463001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cunningham mouse and neonatal rat adaptor</td>
			<td>NeuroLab, Leica</td>
			<td>39462950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe holder</td>
			<td>KD Scientific</td>
			<td>KDS-311-CE</td>
			<td></td>
		</tr>
		<tr>
			<td>33-gauge syringe</td>
			<td>Hamilton</td>
			<td>P/N&#160;&#160;&#160; 84851/00</td>
			<td>#85RN</td>
		</tr>
		<tr>
			<td>Electric drill</td>
			<td>Fine Science Tool</td>
			<td>98096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal blanket</td>
			<td>Ufesa</td>
			<td>AL5512/01</td>
			<td>230-240 V, 100-110 W, type C_AL01</td>
		</tr>
		<tr>
			<td>Shaver&#160;</td>
			<td>Jata</td>
			<td>MP373N</td>
			<td>Model: beauty, 3 V, 300 mA, type HT-03.</td>
		</tr>
		<tr>
			<td colspan="4">Reagents:</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Esteve</td>
			<td>DOMTOR&#160;</td>
			<td>Comercial solution at 1 mg/ml.&#160;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Merial</td>
			<td>Imalgene 500&#160;</td>
			<td>Comercial solution at 50 mg/ml</td>
		</tr>
		<tr>
			<td>Medetomidina/ketamine mixture</td>
			<td></td>
			<td></td>
			<td>Prepare a working mixture of medetomidine at a final concentration of 0.2 mg/ml dilution and ketamine at a final concentration of 15 mg/ml in saline solution. Use as anesthesia injecting a volume to get a final concentration of 0.5-1 mg medetomidina per kg body weight and 50-75 mg ketamine per kg body weight</td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td>Pfizer</td>
			<td>Torbugesic</td>
			<td>Stock solution at 10 mg/ml. Used as analgesia at 1 mg/ml in saline solution.</td>
		</tr>
		<tr>
			<td>Atipamezole</td>
			<td>Esteve</td>
			<td>Antisedan</td>
			<td>Stock solution at 5 mg/ml, used in a final concentration of 0.5 mg/ml in saline solution to exit from anesthesia.</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Braun</td>
			<td>13465412</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl</td>
			<td>Braun</td>
			<td>1050052</td>
			<td>Topical skin adhesive</td>
		</tr>
		<tr>
			<td>HydroGel</td>
			<td>Clear H2O</td>
			<td>70-01-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td>11x21 cm</td>
		</tr>
		<tr>
			<td>Bleach/Virkon</td>
			<td>Dupont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical marker pen</td>
			<td>Staedler</td>
			<td>313-9</td>
			<td>Permanent lumocolor</td>
		</tr>
		<tr>
			<td>Ophthalmic lubricant &#160;</td>
			<td></td>
			<td>SICCAFLUID</td>
			<td>0.5 g/dosis, carbomer 974P</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Betadine</td>
			<td>694109.6</td>
			<td>10% povidone-iodine</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 3: Histological analysis.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment:</td>
		</tr>
		<tr>
			<td>Automatic peristaltic pump</td>
			<td>Cole-Parmer Inst. Co.</td>
			<td>HV-07524-55</td>
			<td>Masterflex L/S variable-speed economy drive, 1.6-100 rpm, 230 V</td>
		</tr>
		<tr>
			<td>Pump head</td>
			<td>Cole-Parmer Inst. Co.</td>
			<td>HV-07518-00</td>
			<td>Masterflex L/S Easy-Load pump head for precision tubing; PSF housing, CRS rotor</td>
		</tr>
		<tr>
			<td>Silicone tube</td>
			<td>Cole-Parmer Inst. Co.</td>
			<td>HV-96410-16</td>
			<td>Platinum L/S 16</td>
		</tr>
		<tr>
			<td>Scalp vein set</td>
			<td>Vygon V-green</td>
			<td>70246.05T</td>
			<td>25G, 30 cm tube length</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FluoView FV10i</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Tehtnica</td>
			<td>SHP-10</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Reagents:</td>
		</tr>
		<tr>
			<td>Phosphate buffer (PB)</td>
			<td></td>
			<td></td>
			<td>0.2 M PB: 0.2 M Na2HPO4 (Sigma, S7907) and 0.2 M NaH2PO4 (Panreac, 141965.1211) in dH2O, adjust pH to 7.2-7.4</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Panreac</td>
			<td>141451.1211</td>
			<td>Prepare fresh every time. Heat dH2O up to 55&#8211;60 &#176;C using a hot plate placed in a fume hood and pour PFA powder while stirring to obtain an 8% solution. The solution is cloudy white as PFA does not dissolve easily. Add 1N NaOH drop by drop just until the solution clears. Cool down, filter through Whatman paper and add an equivalent volume of 0.2 M PB.</td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td></td>
			<td></td>
			<td>0.9% NaCl in dH2O</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>LOCTITE</td>
			<td>767547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Panreac</td>
			<td>122712.1608</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Millipore</td>
			<td>S30-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>Anti-GFP rabbit antibody</td>
			<td>ROCKLAND</td>
			<td>600-401-215</td>
			<td>Use at a 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Molecular probes</td>
			<td>A-21206</td>
			<td>Use at a 1:750 dilution</td>
		</tr>
		<tr>
			<td>6-Diamindino-2-phenylindole dihydrochloride hydrate (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Fluorescent nuclear staining. Use at 2 mg/ml in ddH2O. Keep in the dark at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>EM Sciences</td>
			<td>17984-25</td>
			<td>Mounting medium for fluorescent preparations</td>
		</tr>
	</tbody>
</table>

stable and efficient genetic modification of cells in the adult mouse v-svz for the analysis of neural stem cell autonomous and non-autonomous effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

LV-mediated gene delivery system can be used for the long-term in vivo transduction of cells in the adult mouse V-SVZ, allowing their tracking and genetic modification during proliferation, migration and differentiation. The infection and the expression are highly effective and yield numerous cells that can be easily distinguished among other non-infected cells by the expression of the reporter included. We have thus far visualized transduced cells with GFP fluorescent reporters, driven by the ubiquitously expre...

Watch this Scientific Journal Video about Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects at JoVE

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

The overall goal of this procedure is to deliver low volumes of high titer lentiviral particles carrying specific DNA constructs to the adult mouse ventricular-subventricular zone to infect all of its cell types or to the lateral ventricle to affect ependymal cells only.This method can help answer key questions about interactions that neural stem cells of the adult ventricular-subventricular niche have with their neighboring ependymal cells.The main advantage of this technique is that lentiviral vectors integrate into the genome of target cells in a cell cycle independent way, allowing for long-term diffication of rarely dividing cells.Helping me to demonstrate the procedure will be grad student Beatriz Mart

stable-efficient-genetic-modification-cells-adult-mouse-v-svz-for

Research

JoVE Journal

Developmental Biology

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...