We are grateful to the facilities of the CRTD and Dresden Concept partners for the outstanding support provided, notably K. Neumann and her team at the Stem Cell Engineering Facility, H. Hartmann and her team at the Light Microscopy Facility, A. Gompf and her team at the Flow Cytometry Facility and Hartmut Wolf of the MPI-CBG workshop for the construction of the electroporation chambers. We thank Joshua Schmidt for his feedback on the manuscript. MA acknowledges funding from the Center for Regenerative Therapies TU Dresden, the DFG (Emmy Noether, AL 2231/1-1), and the Schram Foundation.

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

The authors declare that they have no conflicts of interest.

Human cerebral and cortical organoids have become key models to investigate human neocortex development7,16,17. For modeling of human disorders, isogenic hiPSC lines and organoids have become the gold standard68. However, since the generation of novel iPSC lines is time-consuming and costly, the acute manipulation of cortical organoid models for studies of gene function has been widely applied.
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The neocortex refers to the outer covering of the cerebral hemispheres and is a structure that is unique to mammals. The neocortex represents the seat of higher cognitive functions1,2,3,4,5. During development, neural stem and progenitor cells give rise to neurons in a process termed neurogenesis. Functional studies investigating human neocortex development provide the basis for elucidating the mechanisms underlying human neural stem cell regulation, neural pathologies, and human brain evolution2,6,7.
Historically, studies of human brain development relied on descriptive histological approaches using post-mortem tissue, with more recent free-floating tissue culture systems enabling functional investigations with human fetal tissue8,9. Additionally, human fetal brain tissue was shown to have the capacity to self-organize into long-term expanding organoids10. Fetal tissue research has provided important insights into human development11,12, yet restricted tissue availability and ethical considerations limit its widespread application for mechanistic studies of human brain development. In the past decade, protocols have been developed that allow the generation of three-dimensional neural organoids from human pluripotent stem cells (hPSC), including human induced PSC (hiPSC)13,14.
Cerebral organoids display important features of the developing brain, such as the formation of ventricle-like structures, apicobasal polarity, cortical cytoarchitecture, interkinetic nuclear migration during radial glia division, and neuronal migration. Importantly, these new human organoid models recapitulate characteristics of the human developing brain that are not modeled well in the mouse, including evolutionarily relevant key neural progenitor types, in particular basal radial glia (or outer radial glia)7,14,15. Several limitations of early cerebral organoid protocols - such as issues with organoid heterogeneity, limited nutrient supply to the inner core, and varied regional identity-have been addressed in recent protocol advancements and further improvements can be expected in the coming years15,16,17,18,19. Human cerebral and cortical organoids have quickly become key models to study human cortical development20, neurological disorders21,22,23,24 and brain evolution25,26,27,28,29, and to perform large-scale screening approaches30,31,32.
For acute genetic manipulation, two methods have primarily been used in animal models of neocortex development: viral delivery by infection of target cells33 and in utero or in vitro electroporation34,35. Injection of DNA - and, more recently, CRISPR/Cas9 ribonucleoprotein (RNP) complexes36- into the lateral ventricles, followed by electroporation, provides the advantage that specific regions of the brain can be targeted based on the orientation of the electroporation electrodes. Electroporation involves brief electric pulses that temporarily increase cell membrane permeability, allowing the introduction of DNA and other charged molecules into cells. In utero electroporation was first performed in the mouse37, where it rapidly became a widely applied methodology for developmental neurobiology. The method was subsequently also applied to other species, such as the rat38,39 and the ferret40,41,42, a gyrencephalic species used to study neocortex expansion and cortical folding3,43,44,45.
Electroporation has also become an important method in human brain organoid research46. In cerebral organoids, electroporation has been applied to visualize cell morphology and neuronal axons14,47, to deliver gene knockdown reagents22,48,49, and for investigation of gene function by overexpression50. The method is not restricted to human models but has also been applied for genetic modification of primate cerebral organoids50,51. Moreover, the electroporation of cortical organoids generated in a Spin&#160;&#937; spinning bioreactor has been described52.
In this protocol, we outline the electroporation of sliced human cortical organoids15 for studies of gene function in cortical development. Brain region-specific organoids increase reproducibility and consistency, which are critical for the success of quantitative analysis, for example, in disease modeling. In the sliced human cortical organoid protocol15, potent patterning cues are applied during iPSC differentiation to obtain a homogeneous population of dorsal forebrain progenitors, which is followed by the application of culture media that promote tissue growth with fewer instructive signals53,54. Slicing of cortical organoids has been shown to reduce cell death resulting from reduced availability of nutrients and oxygen in the organoid core15. Moreover, slicing supports the development of ventricle-like structures containing abundant basal radial glia, with sustained neurogenesis leading to the formation of an expanded cortical plate-like region15. The repeated slicing in this protocol also makes these cortical organoids particularly suitable to electroporation, as the ventricle lumens can be easily identified and targeted by injection. Electroporation of sliced cortical organoids has been applied to study gene regulatory regions and cortical evolution26,55.
During the electroporation procedure, the injection mix is delivered to the lumen of ventricle-like structures. Upon application of a pulsed electric field, the mix is taken up by apical radial glia that line the ventricle. As apical radial glia divide and give rise to more committed cell types4, the electroporated agents are passed on to basal progenitor cells and neurons. Electroporated progeny are distributed in the ventricular zone (VZ) and subventricular zone (SVZ) after 3 days and span most of the cortical wall, including the cortical plate (CP)-like region, at 7 days post-electroporation during mid-neurogenic stages26,55.
Here, we describe CRISPR/Cas9-mediated gene disruption56 by electroporation in sliced human cortical organoids26. In addition, the electroporation method can also be applied for gene overexpression, visualization of cell morphology and cellular processes by expression of fluorescent proteins, delivery of plasmid libraries for Massive Parallel Reporter Assays (MPRA), delivery of epigenome editing tools and labeling of cells for live imaging.

<table><tbody><tr><td>15 mL PP Centrifuge Tubes, conical</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>4D-Nucleofector Core Unit</td><td>Lonza</td><td></td><td></td></tr><tr><td>4D-Nucleofector X Unit</td><td>Lonza</td><td></td><td></td></tr><tr><td>5 mL polystyrene round-bottom tube with cell-strainer cap</td><td>Corning</td><td>Falcon 352235</td><td></td></tr><tr><td>6-well plate, nunclon treated</td><td>Thermo Fisher</td><td>140675</td><td>For hiPSC culture</td></tr><tr><td>6-well plate, ultra low attachment</td><td>Corning</td><td>3471</td><td>For organoid culture</td></tr><tr><td>A83-01</td><td>STEMCELL Technologies</td><td>72022</td><td>Forebrain medium 1 (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Air compressor</td><td>Aerotec</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Chicken IgY (IgG) (H+L)</td><td>Jackson Immuno Research</td><td>703-545-155</td><td>Secondary antibody, RRID: AB_2340375; dilution 1:1000</td></tr><tr><td>Alexa Fluor 555 Donkey Anti-Rabbit IgG (H+L)</td><td>Invitrogen</td><td>A-31572</td><td>Secondary antibody, RRID: AB_162543; dilution 1:1000</td></tr><tr><td>Alt-R CRISPR-Cas9 crRNA, 2 nmol</td><td>Integrated DNA technologies</td><td></td><td>Custom design (order as 2 nmol)</td></tr><tr><td>Alt-R CRISPR-Cas9 tracrRNA</td><td>Integrated DNA technologies</td><td>1072532</td><td>5 nmol</td></tr><tr><td>Alt-R S.p. HiFi Cas9 Nuclease V3</td><td>Integrated DNA technologies</td><td>1081060</td><td>100 &amp;micro;g, from Streptococcus pyogenes</td></tr><tr><td>Amphotericin B (Fungizone)</td><td>Gibco</td><td>15290018</td><td>Forebrain medium 2, 3, and 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Anti-GFP primary antibody (chicken, polyclonal)</td><td>Abcam</td><td>ab13970</td><td>RRID: AB_300798; dilution 1:2000</td></tr><tr><td>Anti-PCGF4 primary antibody (mouse, monoclonal)</td><td>Millipore</td><td>05-637&amp;nbsp;</td><td>RRID: AB_309865; dilution 1:300</td></tr><tr><td>ApoTome fluorescent microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma Aldrich</td><td>1043003</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>B27-supplement (+ vitamin A)</td><td>Gibco</td><td>17504044</td><td>Forebrain medium 3, 4&amp;nbsp;&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Banana to Micrograbber Cable Kit</td><td>Harvard Apparatus BTX</td><td>45-0216</td><td></td></tr><tr><td>Biometra TRIO-Thermocycler (PCR machine)</td><td>Analytik Jena</td><td>846-2-070-723</td><td></td></tr><tr><td>Capillaries, borosilicate glass with filament, OD 1.2 mm; ID 0.69 mm; 10 cm length</td><td>Science Products</td><td>BF120-69-10</td><td>Need to be pulled to specific thickness</td></tr><tr><td>CHIR-99021</td><td>STEMCELL Technologies</td><td>72052</td><td>Forebrain medium 2&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Collagenase Type IV</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>CRTDi004-A</td><td>Stem Cell Engineering Facility at CMCB DD</td><td>https://hpscreg.eu/cell-line/CRTDi004-A</td><td>Single-cell adapted hiPSC line from healthy donor</td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td>1 mg/mL stock, use 1:1000 diluted for IF</td></tr><tr><td>Dibutyryl-cAMP</td><td>STEMCELL Technologies</td><td>73884</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>DMEM/F-12, HEPES</td><td>Fisher Scientific</td><td>31330095</td><td>Forebrain medium 1, 2, and 3&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Dorsomorphin</td><td>STEMCELL Technologies</td><td>72102</td><td>Forebrain medium 1&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Dumont #55 Forceps, straigth, 11 cm</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>ECM 830 Square Wave Electroporation System</td><td>Harvard Apparatus BTX</td><td>45-2052</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>32205-1L-M</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>EDS-500G</td><td></td></tr><tr><td>Fast Green FCF</td><td>Sigma Aldrich</td><td>F7252</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GE Healthcare&amp;nbsp;</td><td>SH30070.03&amp;nbsp;</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td>For pulling of microcapillaries</td></tr><tr><td>Geneious Prime</td><td>GraphPad Software LLC d.b.a Geneious</td><td>Software version 2024.0.5</td><td></td></tr><tr><td>&lt;em&gt;gLACZ&lt;/em&gt;</td><td>Kalebic et al., 2016; Platt et al., 2014</td><td></td><td>5&#039;-TGCGAATACGCCCACGCGATCGG; underlined nucleotides = PAM</td></tr><tr><td>GlutaMAX</td><td>Gibco&amp;nbsp;</td><td>35050038</td><td>Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15</td></tr><tr><td>Glycine</td><td>Sigma Aldrich</td><td>G8898</td><td></td></tr><tr><td>&lt;em&gt;gPCGF4 &lt;/em&gt;KO1</td><td>this paper</td><td></td><td>5&#039;-TGAACTTGGACATCACAAATAGG (corresponds to the KO images in Figure 3I+J)</td></tr><tr><td>&lt;em&gt;gPCGF4 &lt;/em&gt;KO2</td><td>this paper</td><td></td><td>5&#039;-ACAAATAGGACAATACTTGCTGG (corresponds to the KO images in Figure 3I+J)</td></tr><tr><td>HEPES</td><td>made in house</td><td></td><td>1 M stock</td></tr><tr><td>Horse Serum, heat-inactivated</td><td>Gibco</td><td>26050088</td><td></td></tr><tr><td>Human GDNF Recombinant Protein</td><td>Thermo Fisher</td><td>450-10</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Human/Mouse/Rat BDNF Recombinant Protein</td><td>Thermo Fisher</td><td>450-02</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Sigma Aldrich</td><td>258148</td><td>To make TrisHCl</td></tr><tr><td>ImmEdge (wax) Pen</td><td>Vector Laboraories</td><td>H-4000</td><td></td></tr><tr><td>Insulin solution human</td><td>Sigma Aldrich</td><td>&amp;nbsp;I9278</td><td>Forebrain medium 3&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>BP2618-1</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Gibco&amp;nbsp;</td><td>10828-010</td><td>Forebrain medium 1&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Laser Scanning Confocal 980 Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Low Melting Point Agarose, ultra pure</td><td>Thermo Fisher</td><td>16520100</td><td>for embedding of hCOs during vibratome sectioning</td></tr><tr><td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free</td><td>Corning</td><td>354230</td><td>For organoid culture</td></tr><tr><td>Matrigel hESC-Qualified Matrix, LDEV-free</td><td>Corning</td><td>354277</td><td>For hiPSC culture</td></tr><tr><td>MgCl2, 1 M</td><td>Thermo Fisher</td><td>AM9530G</td><td></td></tr><tr><td>Microinjector PicoPump + Foot Switch</td><td>World Precision Instruments</td><td>SYS-PV820</td><td></td></tr><tr><td>Microloader Pipette Tips 0.5 to 20 &amp;micro;L</td><td>Eppendorf</td><td>5242956003</td><td>For loading of glass capillaries</td></tr><tr><td>Mowiol 4-88</td><td>Sigma Aldrich</td><td>81381</td><td></td></tr><tr><td>mTeSR 1</td><td>STEMCELL Technologies</td><td>85850</td><td>Stem cell medium</td></tr><tr><td>N-2 supplement</td><td>Gibco</td><td>17502048</td><td>Forebrain medium 2, and 3&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S5886</td><td></td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>NEB Next High Fidelity 2x PCR Mastermix&amp;nbsp;</td><td>New England Biolabs</td><td>M0541S</td><td></td></tr><tr><td>Neubauer counting chamber</td><td>Brand</td><td>718605</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco&amp;nbsp;</td><td>11140050</td><td>Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Nuclease-Free Duplex Buffer</td><td>Integrated DNA technologies</td><td>1072570</td><td></td></tr><tr><td>O. C. T. Compound</td><td>Tissue-Tek</td><td>4583</td><td>Embedding medium for frozen tissue specimen</td></tr><tr><td>P3 Primary Cell 4D-Nucleofector X Kit S</td><td>Lonza</td><td>V4XP-3032</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Chemical</td><td>P/0840/53</td><td></td></tr><tr><td>pCAG-GFP</td><td>Addgene</td><td>11150</td><td></td></tr><tr><td>Peel-A-Way Embedding Mold TruncatedT12</td><td>Polysciences Inc.</td><td>18986-1</td><td>For embedding of hCOs for vibratome and cryo-sectioning</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)&amp;nbsp;</td><td>Gibco</td><td>15140122</td><td>Forebrain medium 1, 2, 3, and 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt; and for stem cell medium after nucleofection</td></tr><tr><td>Petri Dish 60 mm x 15 mm, No Vent, Sterile</td><td>Corning</td><td>BP50-02</td><td></td></tr><tr><td>Petri Dish Platinum Electrode Chamber, 5 mm gap</td><td>Harvard Apparatus BTX</td><td>45-0504</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>made in house</td><td></td><td></td></tr><tr><td>Proteinase K</td><td>Sigma Aldrich</td><td>P2308</td><td>From Titrachium album; 10 mg/mL stock</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28706</td><td></td></tr><tr><td>RHO/ROCK pathway inhibitor (Y-27632)</td><td>STEMCELL Technologies</td><td>72308</td><td></td></tr><tr><td>SB-431542</td><td>STEMCELL Technologies</td><td>72232</td><td>Forebrain medium 2&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Sodium Citrate Dihydrate</td><td>Sigma Aldrich</td><td>W302600-1KG-K</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma Aldrich</td><td>S7903</td><td></td></tr><tr><td>SuperFrost Plus adhesion slides</td><td>Fisher Scientific</td><td>10149870</td><td></td></tr><tr><td>SZX10 with a KL 300 LED</td><td>Olympus</td><td>SZX10</td><td>Or alternative stereoscope</td></tr><tr><td>Thermo Shaker</td><td>Grant bio</td><td>PSC24N</td><td></td></tr><tr><td>Triton-X</td><td>Sigma Aldrich</td><td>T9284</td><td>a.k.a. Octoxinol 9</td></tr><tr><td>Trizma base</td><td>Sigma Aldrich</td><td>T1503</td><td>To make TrisHCl</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x)</td><td>Thermo Fisher</td><td>12604021</td><td>Dissociation enzmye to make single-cell suspensions</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td>a.k.a. Polysorbate 20</td></tr><tr><td>Tyrode&#039;s salt</td><td>Sigma Aldrich</td><td>T2145-10x1l</td><td></td></tr><tr><td>Vibrating Microtome (vibratome)</td><td>Leica</td><td>VT1200 S</td><td></td></tr><tr><td>Wide-Bore 1000 &amp;micro;L Universal Fit Filter Tips</td><td>Corning</td><td>TF-1005-WB-R-S</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Gibco&amp;nbsp;</td><td>21985-023</td><td>Forebrain medium 1, 3, and 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr></tbody></table>

electroporation of sliced human cortical organoids for studies of gene function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

CRISPR/Cas9-mediated gene ablation requires the design of gRNAs. Generally, targeting one of the first exons of a gene of interest with a pair of gRNAs spaced 7-11 bp apart (avoiding multiples of 3 bp) works well (Figure 1A). As a first test, the efficiency of template recognition by gRNAs may be interrogated in vitro using a PCR template26,40,62. Efficient targeting and Cas9-mediated cutti...

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

electroporation-sliced-human-cortical-organoids-for-studies-gene

Research

JoVE Journal

Developmental Biology

1.0K Views.  TUD Dresden University of Technology. Mijn groep is geïnteresseerd in de mechanismen die genexpressie reguleren in neurale voorlopercellen van de zich ontwikkelende neocortex. En in het bijzonder richten we ons op epigenetische mechanismen. Deze zijn belangrijk voor het potentieel van neurale voorlopercellen om zich te vermenigvuldigen en te differentiëren, en dus voor een goede ontwikkeling en functie van de hersenen.Corticale organoïden zijn spannende nieuwe modellen waarmee we de ont...

Video: Elektroporatie van gesneden menselijke corticale organoïden voor onderzoek naar de genfunctie

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Neuroscience

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biology

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices

Source: Keener, D. G. et al. <a href="https://www.jove.com/video/61662" target="_blank">Single-Cell Electroporation Across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons.</a> J. Vis. Exp. (2020)
This video demonstrates the protocol for single-cell electroporation of genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice cultures. The technique is useful to examine specific molecular and physiological functions of neurons, including cell autonomous mechanisms and transsynaptic protein interactions.

Source: Keener, D. G. et al. Single-Cell Electroporation Across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific ...

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

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