Name: Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification
Uploaded: 2023-03-24T12:10:00
Description: Watch this Scientific Journal Video about Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification at JoVE.com

Vi undskylder over for alle de forskere, hvis arbejde ikke kunne citeres på grund af pladsbegrænsninger. Vi takker Ulrich Bleyer fra DPZ' tekniske tjenester og Hartmut Wolfs fra værkstedet hos MPI-CBG for konstruktionen af petriskålelektrodekamrene; Stoyan Petkov og Rüdiger Behr for at levere humane (iLonza2.2), rhesusmakak (iRh33.1) og silkeaber (cj_160419_5) iPSC'er; Sabrina Heide til kryosektionering og immunofluorescensfarvning; og Neringa Liutikaite og César Mateo Bastidas Betancourt for kritisk at have læst manuskriptet. Arbejdet i W.B.H.'s laboratorium blev støttet af et ERA-NET NEURON (MicroKin) tilskud. Arbejdet i M.H.'s laboratorium blev støttet af et ERC-starttilskud (101039421).

1. Kultur af primater iPSC'er
BEMÆRK: På grund af sin robusthed kan den metode, der præsenteres her, anvendes på enhver primat iPSC-linje. I denne artikel beskriver vi cerebral organoidproduktion fra humane (iLonza2.2)29, chimpanser (Sandra A)30, rhesusmakak (iRh33.1)29 og almindelige silkeaber (cj_160419_5)31 iPSC-linjer. Kulturbetingelserne er opsummeret i tabel 1</stron...

Genetic Modification of Primate Cerebral Organoids

<ol>
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De procedurer, der er beskrevet her, repræsenterer en samlet protokol til generering af cerebrale organoider fra forskellige primatarter med en målrettet elektroporationsmetode. Dette muliggør ektopisk ekspression af en GOI i et modelsystem, der efterligner primater (herunder menneskelig) (pato)fysiologisk neocortexudvikling. Denne samlede protokol til generering af primater cerebrale organoider bruger de samme materialer (f.eks. Medier) og protokoltrin for alle fire præsenterede primatarter. Udviklingsforskelle mell...

Undersøgelse af den (pato)fysiologiske udvikling og udvikling af hjernebarken er en formidabel opgave, der hæmmes af manglen på egnede modelsystemer. Tidligere var sådanne undersøgelser begrænset til todimensionelle cellekulturmodeller (såsom primære neurale stamfader eller neuronale cellekulturer) og evolutionært fjerne dyremodeller (såsom gnavere)1,2. Mens disse modeller er nyttige til at løse visse spørgsmål, er de begrænsede til modellering af kompleksiteten, celletypesammensætningen, cellulær arkitektur og genekspressionsmønstre for den udviklende humane neocortex i sunde og syge tilstande. Disse begrænsninger fører f.eks. til dårlig oversættelighed af musemodeller af menneskelige sygdomme til den menneskelige situation, som beskrevet for visse tilfælde af mikrocefali (f.eks. Zhang et al.3). For nylig er transgene ikke-menneskelige primater, som er en evolutionært, funktionelt og morfologisk tættere model for menneskelig neocortexudvikling, kommet i fokus 4,5,6,7,8, da de overvinder mange begrænsninger af cellekultur- og gnaverbaserede modeller. Anvendelsen af ikke-menneskelige primater i forskning er imidlertid ikke kun meget dyr og tidskrævende, men giver også anledning til etiske betænkeligheder. For nylig har udviklingen af hjerneorganoidteknologi 9,10 vist sig som et lovende alternativ, der løser mange af begrænsningerne i tidligere modeller 11,12,13,14,15,16.
Hjerneorganoider er tredimensionelle (3D), multicellulære strukturer, der efterligner hovedtræk ved cytoarkitekturen og celletypesammensætningen af en eller flere hjerneområder i et defineret udviklingstidsvindue 11,12,13,14,17. Disse 3D-strukturer genereres enten fra inducerede pluripotente stamceller (iPSC'er) eller, hvis de er tilgængelige for den pågældende art, fra embryonale stamceller (ESC'er). Generelt kan der skelnes mellem to typer hjerneorganoider baseret på den anvendte metode: ustyrede og regionaliserede (guidede) hjerneorganoider18. Ved generering af sidstnævnte type organoider tilvejebringes små molekyler eller faktorer, der styrer differentieringen af de pluripotente stamceller til organoider i et bestemt hjerneområde (f.eks. Forhjerneorganoider)18. I modsætning hertil styres differentieringen i ikke-styrede organoider ikke af tilsætning af små molekyler, men er udelukkende afhængig af den spontane differentiering af iPSC'erne/ESC'erne. De resulterende hjerneorganoider består af celletyper, der repræsenterer forskellige hjerneområder (f.eks. cerebrale organoider)18. Hjerneorganoider kombinerer mange nøglefunktioner i hjernens udvikling med relativt omkostnings- og tidseffektiv generering fra enhver art af interesse, for hvilke iPSC'er eller ESC'er er tilgængelige 11,12,13,14. Dette gør hjerneorganoider til en fremragende model for mange slags neurobiologiske undersøgelser, lige fra evolutionære og udviklingsmæssige spørgsmål til sygdomsmodellering og lægemiddeltest15,16. Imidlertid afhænger adressering af sådanne spørgsmål ved hjælp af hjerneorganoider stærkt af tilgængeligheden af forskellige metoder til genetisk modifikation.
Et centralt aspekt ved at studere neocortex (pato)fysiologisk udvikling og dens udvikling er den funktionelle analyse af gener og genvarianter. Dette opnås normalt ved (ektopisk) ekspression og / eller ved knock-down (KD) eller knock-out (KO) af disse gener. Sådanne genetiske modifikationer kan klassificeres i stabil og forbigående genetisk modifikation samt i, at modifikationerne er tidsmæssigt og rumligt begrænsede eller ikke begrænsede. Stabil genetisk modifikation defineres ved indførelse af en genetisk ændring i værtsgenomet, der overføres til alle efterfølgende cellegenerationer. Afhængigt af tidspunktet for genetisk modifikation kan det påvirke alle celler i en organoid eller kan begrænses til bestemte cellepopulationer. Oftest opnås stabil genetisk modifikation i hjerneorganoider på iPSC/ESC-niveau ved anvendelse af lentivirus, transposonlignende systemer og CRISPR/Cas9-teknologien (gennemgået af f.eks. Fischer et al.17, Kyrousi et al.19 og Teriyapirom et al.20). Dette har den fordel, at alle celler i hjerneorganoid bærer den genetiske modifikation, og at den ikke er tidsmæssigt eller rumligt begrænset. Imidlertid er genereringen og karakteriseringen af disse stabile iPSC / ESC-linjer meget tidskrævende og tager ofte flere måneder, indtil de første modificerede hjerneorganoider kan analyseres (gennemgået af f.eks. Fischer et al.17, Kyrousi et al.19 eller Teriyapirom et al.20).
I modsætning hertil defineres forbigående genetisk modifikation ved levering af genetisk last (f.eks. Et genekspressionsplasmid), der ikke integreres i værtsgenomet. Mens denne modifikation i princippet kan overføres til efterfølgende cellegenerationer, vil den leverede genetiske last gradvist blive fortyndet med hver celledeling. Derfor er denne type genetisk modifikation normalt tidsmæssigt og rumligt begrænset. Forbigående genetisk modifikation kan udføres i hjerneorganoider af adeno-associerede vira eller ved elektroporation (gennemgået af f.eks. Fischer et al.17, Kyrousi et al.19 og Teriyapirom et al.20), hvor sidstnævnte beskrives detaljeret i denne artikel. I modsætning til stabil genetisk modifikation er denne tilgang meget hurtig og omkostningseffektiv. Faktisk kan elektroporation udføres inden for få minutter, og afhængigt af målcellepopulationen (e) er elektroporerede organoider klar til analyse inden for få dage (gennemgået af f.eks. Fischer et al.17 og Kyrousi et al.19). Imidlertid kan grove morfologiske ændringer i hjerneorganoiden, såsom forskelle i størrelse, ikke påvises ved hjælp af denne metode, da denne type genetisk modifikation er tidsmæssigt og rumligt begrænset. Denne begrænsning kan også være en fordel, for eksempel i tilfælde af at studere individuelle cellepopulationer inden for organoiden eller virkningerne på hjerneorganoider på specifikke udviklingstidspunkter (gennemgået af f.eks. Fischer et al.17 og Kyrousi et al.19).
En klassisk tilgang til at studere genfunktion under hjernens udvikling og evolution er in utero elektroporation. In utero elektroporation er en velkendt og nyttig teknik til levering af genekspressionskonstruktioner i gnavere 21,22,23 og fritter 24,25 hjerner. For det første mikroinjiceres en opløsning, der indeholder ekspressionskonstruktionen (e) af interesse, gennem livmodervæggen ind i en bestemt ventrikel i den embryonale hjerne, afhængigt af den region, der skal målrettes. I det andet trin påføres elektriske impulser for at transfektere cellerne, der direkte forer den målrettede ventrikel. Denne tilgang er ikke kun begrænset til ektopisk ekspression eller overekspression af gener, da den også kan anvendes i KD- eller KO-undersøgelser ved mikroinjektion af kort hårnål (shRNA) eller CRISPR/Cas9 (i form af ekspressionsplasmider eller ribonukleoproteiner [RNP'er]), henholdsvis26,27. Imidlertid har in utero elektroporation af muse-, rotte- og fritteembryoner de samme begrænsninger som beskrevet ovenfor for disse dyremodeller.
Ideelt set vil man gerne udføre i utero elektroporation direkte i primater. Selv om dette i princippet er teknisk muligt, udføres elektroporation in utero ikke i primater på grund af etiske bekymringer, høje omkostninger til dyrevedligeholdelse og små kuldstørrelser. For visse primater, såsom menneskeaber (herunder mennesker), er dette slet ikke muligt. Imidlertid har disse primater det største potentiale for undersøgelsen af menneskelig (pato) fysiologisk neocortexudvikling og dens udvikling. En løsning på dette dilemma er at anvende elektroporationsteknikken på primathjerneorganoider28.
Dette papir præsenterer en protokol til elektroporation af en undertype af primathjerneorganoider, primathjerneorganoider. Denne tilgang muliggør hurtig og omkostningseffektiv genetisk modifikation af cellepopulationer inden for organoidernes ventrikellignende strukturer. Specifikt beskriver vi en samlet protokol til generering af primater cerebrale organoider fra mennesker (Homo sapiens), chimpanser (Pan troglodytes), rhesus makak (Macaca mulatta) og almindelige silkeaber (Callithrix jacchus) iPSC'er. Desuden beskriver vi mikroinjektions- og elektroporationsteknikken detaljeret og giver "go" og "no-go" kriterier for udførelse af primat cerebral organoid elektroporation. Denne tilgang er et effektivt redskab til at studere (pato)fysiologisk neocortex udvikling og dens udvikling i en model, der er særlig tæt på den menneskelige situation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 &#181;L Microloader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Merck</td>
			<td>8.05740.0005</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm cell culture dishes</td>
			<td>Sarstedt</td>
			<td>83.3900</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dishes</td>
			<td>CytoOne</td>
			<td>CC7682-3359</td>
			<td></td>
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		<tr>
			<td>Activin A</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3003</td>
			<td></td>
		</tr>
		<tr>
			<td>AOC1</td>
			<td>Selleckchem</td>
			<td>S7217</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Observer.Z1 Inverted Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>replacable by comparable fluorescent microscopes</td>
		</tr>
		<tr>
			<td>AZD0530</td>
			<td>Selleckchem&#160;</td>
			<td>S1006</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement with Vitamin A (retinoic acid, RA) (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement without Vitamin A (50x)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>BTX ECM 830 Square Wave Electroporation System</td>
			<td>BTX</td>
			<td>45-2052</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP77675</td>
			<td>Sigma-Aldrich</td>
			<td>SML0314</td>
			<td></td>
		</tr>
		<tr>
			<td>Chimpanzee induced pluripotent stem cell line Sandra A</td>
			<td></td>
			<td></td>
			<td>doi: 10.7554/elife.18683&#160;</td>
		</tr>
		<tr>
			<td>Common marmoset induced pluripotent stem cell line cj_160419_5</td>
			<td></td>
			<td></td>
			<td>doi: 10.3390/cells9112422</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>pH 7.0&#8722;7.3; warm to room temperature before use</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Selleckchem</td>
			<td>2449</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100x)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>glutamine substitute supplement</td>
		</tr>
		<tr>
			<td>Heparin (1 mg/mL stock)</td>
			<td>Sigma-Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Human induced pluripotent stem cell line iLonza2.2</td>
			<td></td>
			<td></td>
			<td>doi: 10.3390/cells9061349</td>
		</tr>
		<tr>
			<td>Human Neurotrophin-3 (NT-3)</td>
			<td>PeproTech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>19278</td>
			<td></td>
		</tr>
		<tr>
			<td>IWR1</td>
			<td>Sigma-Aldrich</td>
			<td>I0161</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MS5 stereomicroscope (MDG 17 transmitted-light base)</td>
			<td>Leica</td>
			<td>10473849</td>
			<td>replacable by comparable stereomicroscopes</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354277/354234</td>
			<td>basement membrane matrix; alternatively, Geltrex (ThermoFisher Scientific, A1413302) can be used</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Sigma-Aldrich</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>818715</td>
			<td>handle with causion due to cancerogenecity</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (10,000 U/mL)</td>
			<td>PanBiotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish electrode chamber</td>
			<td>self-produced (see Supplemental File 1)</td>
			<td></td>
			<td>also commertially available</td>
		</tr>
		<tr>
			<td>Pre-Pulled Glass Pipettes</td>
			<td>WPI</td>
			<td>TIP10LT</td>
			<td>borosilicate glass pipettes with long taper, 10 &#181;m tip diameter</td>
		</tr>
		<tr>
			<td>Pro-Survival Compound</td>
			<td>MerckMillipore</td>
			<td>529659</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Murine/RatBrain-Derived Neurotrophic Factor (BDNF)</td>
			<td>PeproTech</td>
			<td>AF-450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhesus macaque induced pluripotent stem cell line iRh33.1</td>
			<td></td>
			<td></td>
			<td>doi: 10.3390/cells9061349</td>
		</tr>
		<tr>
			<td>StemMACS iPS-Brew XF</td>
			<td>Miltenyi Biotech</td>
			<td>130-104-368</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>proteolytic and collagenolytic enzyme mixture</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12604-013</td>
			<td>recombinant trypsin substitute; warm to room temperature before use</td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 96-well plates</td>
			<td>Costar</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Stemcell Technologies</td>
			<td>72305</td>
			<td></td>
		</tr>
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</table>

targeted microinjection and electroporation of primate cerebral organoids for genetic modification

Hjernebarken er den yderste hjernestruktur og er ansvarlig for behandlingen af sensorisk input og motorudgang; Det ses som sæde for højere ordens kognitive evner hos pattedyr, især primater. At studere genfunktioner i primathjerner er udfordrende af tekniske og etiske årsager, men etableringen af hjerneorganoidteknologien har muliggjort studiet af hjernens udvikling i traditionelle primatmodeller (f.eks. rhesusmakak og almindelig silkeaber) såvel som i tidligere eksperimentelt utilgængelige primatarter (f.eks. Store aber) i et etisk forsvarligt og mindre teknisk krævende system. Desuden tillader menneskelige hjerneorganoider avanceret undersøgelse af neuroudviklingsmæssige og neurologiske lidelser.
Da hjerneorganoider rekapitulerer mange processer i hjernens udvikling, repræsenterer de også et kraftfuldt værktøj til at identificere forskelle i og funktionelt sammenligne de genetiske determinanter, der ligger til grund for hjernens udvikling af forskellige arter i en evolutionær sammenhæng. En stor fordel ved at bruge organoider er muligheden for at indføre genetiske modifikationer, som gør det muligt at teste genfunktioner. Indførelsen af sådanne ændringer er imidlertid besværlig og dyr. Dette papir beskriver en hurtig og omkostningseffektiv tilgang til genetisk modifikation af cellepopulationer inden for ventrikellignende strukturer af primater cerebrale organoider, en undertype af hjerneorganoider. Denne metode kombinerer en modificeret protokol til pålidelig generering af cerebrale organoider fra human-, chimpanse-, rhesusmakak- og almindelige marmosetafledte inducerede pluripotente stamceller (iPSC'er) med en mikroinjektions- og elektroporationstilgang. Dette giver et effektivt værktøj til undersøgelse af neurodevelopmental og evolutionære processer, der også kan anvendes til sygdomsmodellering.

Investigating the (patho)physiological development and evolution of the cerebral cortex is a formidable task that is hampered by the lack of suitable model systems. Previously, such studies were confined to two-dimensional cell culture models (such as primary neural progenitor or neuronal cell cultures) and evolutionarily distant animal models (such as rodents)1,2. While these models are useful for addressing certain questions, they are limited in modeling the complexity, cell type composition, cellular architecture, and gene expression patterns of the developing human neocortex in healthy and diseased states. These limitations lead, for example, to the poor translatability of mouse models of human diseases to the human situation, as described for certain cases of microcephaly (e.g., Zhang et al.3). Recently, transgenic non-human primates, which are an evolutionarily, functionally, and morphologically closer model of human neocortex development, have come into focus4,5,6,7,8 as they overcome many limitations of cell culture- and rodent-based models. However, the use of non-human primates in research is not only highly expensive and time-consuming but also raises ethical concerns. More recently, the development of brain organoid technology9,10 has emerged as a promising alternative that solves many of the limitations of previous models11,12,13,14,15,16.
Brain organoids are three-dimensional (3D), multicellular structures that emulate the main features of the cytoarchitecture and cell-type composition of one or multiple brain regions for a defined developmental time window11,12,13,14,17. These 3D structures are generated either from induced pluripotent stem cells (iPSCs) or, if available for the species of interest, from embryonic stem cells (ESCs). In general, two types of brain organoids can be distinguished based on the methodology used: unguided and regionalized (guided) brain organoids18. In generating the latter type of organoids, small molecules or factors are provided that guide the differentiation of the pluripotent stem cells to organoids of a particular brain region (e.g., forebrain organoids)18. By contrast, in unguided organoids, the differentiation is not guided by the addition of small molecules but rather relies exclusively on the spontaneous differentiation of the iPSCs/ESCs. The resulting brain organoids consist of cell types representing different brain regions (e.g., cerebral organoids)18. Brain organoids combine many key features of brain development with relatively cost- and time-efficient generation from any species of interest for which iPSCs or ESCs are available11,12,13,14. This makes brain organoids an excellent model for many kinds of neurobiological studies, ranging from evolutionary and developmental questions to disease modeling and drug testing15,16. However, addressing such questions using brain organoids strongly depends on the availability of different methods for genetic modification.
One key aspect of studying neocortex (patho)physiological development and its evolution is the functional analysis of genes and gene variants. This is usually achieved by (ectopic) expression and/or by knock-down (KD) or knock-out (KO) of those genes. Such genetic modifications can be classified into stable and transient genetic modification, as well as into the modifications being temporally and spatially restricted or not restricted. Stable genetic modification is defined by the introduction of a genetic alteration into the host genome that is passed on to all subsequent cell generations. Depending on the time point of genetic modification, it can affect all the cells of an organoid or can be restricted to certain cell populations. Most frequently, stable genetic modification is achieved in brain organoids at the iPSC/ESC level by applying lentiviruses, transposon-like systems, and the CRISPR/Cas9 technology (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20). This has the advantage that all cells of the brain organoid carry the genetic modification and that it is not temporally or spatially restricted. However, the generation and characterization of these stable iPSC/ESC lines are very time-consuming, often taking several months until the first modified brain organoids can be analyzed (reviewed by e.g., Fischer et al.17, Kyrousi et al.19, or Teriyapirom et al.20).
In contrast, transient genetic modification is defined by the delivery of genetic cargo (e.g., a gene expression plasmid) that does not integrate into the host genome. While this modification can, in principle, be passed on to subsequent cell generations, the delivered genetic cargo will be progressively diluted with each cell division. Therefore, this type of genetic modification is usually temporally and spatially restricted. Transient genetic modification can be carried out in brain organoids by adeno-associated viruses or by electroporation (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20), with the latter being described in detail in this article. In contrast to stable genetic modification, this approach is very fast and cost-efficient. Indeed, electroporation can be performed within minutes, and, depending on the target cell population(s), electroporated organoids are ready for analysis within days (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19). However, gross morphological changes of the brain organoid, such as differences in size, cannot be detected using this method, as this type of genetic modification is temporally and spatially restricted. This restriction can also be an advantage, for example, in the case of studying individual cell populations within the organoid or the effects on brain organoids at specific developmental time points (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19).
A classical approach to study gene function during brain development and evolution is in utero electroporation. In utero electroporation is a well-known and useful technique for the delivery of gene expression constructs into rodent21,22,23 and ferret24,25 brains. First, a solution containing the expression construct(s) of interest is microinjected through the uterine wall into a certain ventricle of the embryonic brain, depending on the region to be targeted. In the second step, electric pulses are applied to transfect the cells directly lining the targeted ventricle. This approach is not only limited to ectopic expression or the overexpression of genes, as it can also be applied in KD or KO studies by microinjecting short hairpin (shRNA) or CRISPR/Cas9 (in the form of expression plasmids or ribonucleoproteins [RNPs]), respectively26,27. However, the in utero electroporation of mouse, rat, and ferret embryos has the same limitations as described above for these animal models.
Ideally, one would like to perform in utero electroporation directly in primates. While this is, in principle, technically possible, in utero electroporation is not conducted in primates due to ethical concerns, high animal maintenance costs, and small litter sizes. For certain primates, such as great apes (including humans), this is not possible at all. However, these primates have the greatest potential for the study of human (patho)physiological neocortex development and its evolution. One solution to this dilemma is to apply the electroporation technique to primate brain organoids28.
This paper presents a protocol for the electroporation of a subtype of primate brain organoids, primate cerebral organoids. This approach allows the fast and cost-efficient genetic modification of cell populations within the ventricle-like structures of the organoids. Specifically, we describe a unified protocol for the generation of primate cerebral organoids from human (Homo sapiens), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and common marmoset (Callithrix jacchus) iPSCs. Moreover, we describe the microinjection and electroporation technique in detail and provide &#34;go&#34; and &#34;no-go&#34; criteria for performing primate cerebral organoid electroporation. This approach is an effective tool for studying (patho)physiological neocortex development and its evolution in a model especially close to the human situation.

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

Målrettet mikroinjektion og elektroporation af primathjerneorganoider til genetisk modifikation

Our field of research is primate brain development and evolution. We are trying to understand the factors underlying neocortical expansion in primates. To address this question in a practical and ethically justifiable way, we use brain organoids as our research model.Differences in brain development between modern humans, us, and neanderthals have recently emerged. These differences are due to a small number of amino acid changes in proteins with key roles in brain development. Cerebral organoids have been a crucial model system in these studies.The study of primate development from an evolutionary point of view conducting gain and loss of function studies is important. However, apes including humans naturally cannot be used for such experiments, so genetic modification of organoids plays a vital role in this field. While working as a postdoc in my lab, Michael Heide demonstrated that the human specific gene ARHGAP11B was a key player in increasing brain size during human evolution.As a group leader at the German Primate Center now, Michael contributed crucial insight by comparing human versus chimpanzee cerebral organoids. Our protocol offers a targeted approach for genetic modifications by specific micro injecting ventricle-like structures instead of commercial electroporation cuvettes. This approach uses a cost efficient setup including square wave electroporator and Petri dish electrode chamber instead of expensive nuclear effector solutions.While studying function and brain development in primates by genetic modification is technically possible, it is methodologically demanding, expensive, and not allowed for great apes. The electroporation of primate cerebral organoids provides a fast and cost efficient approach to introduce genetic modifications in a model close to primate brain development. In the future, we would like to focus on the characterization of differentially expressed genes in the cortex of different primate species.We will study these genes by the electroporation of primate cerebral organoids and analyze their potential effects on cortical progenitor cells.

Protokollen beskrevet her tillader effektiv generering af cerebrale organoider fra humane, chimpanser, rhesusmakak og almindelige silkeaber iPSC-linjer med minimale tidsændringer, der kræves mellem arter (figur 1A). Disse organoider kan elektroporeres i området fra 20 dps til 50 dps, afhængigt af tilgængeligheden af de ventrikellignende strukturer og overflod af cellepopulationen (e) af interesse. Før elektroporation er det imidlertid vigtigt at afgøre, om cerebrale organoider er af t...

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Elektroporation af primater cerebrale organoider giver en præcis og effektiv tilgang til at introducere forbigående genetisk modifikation (er) i forskellige stamfadertyper og neuroner i et modelsystem tæt på primats (pato)fysiologiske neocortexudvikling. Dette muliggør undersøgelse af neurodevelopmental og evolutionære processer og kan også anvendes til sygdomsmodellering.

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 ...

Vores forskningsfelt er primathjernens udvikling og evolution. Vi forsøger at forstå de faktorer, der ligger til grund for neokortikal ekspansion hos primater. For at løse dette spørgsmål på en praktisk og etisk forsvarlig måde bruger vi hjerneorganoider som vores forskningsmodel.Forskelle i hjernens udvikling mellem moderne mennesker, os og neandertalere er for nylig opstået. Disse forskelle skyldes et lille antal aminosyreændringer i proteiner med nøgleroller i hjernens udvikling. Cerebral organoider har været et afgørende modelsystem i disse undersøgelser.Undersøgelsen af primatudvikling fra et evolutionært synspunkt, der udfører gevinst- og tabsundersøgelser af funktion, er vigtig. Imidlertid kan aber, herunder mennesker, naturligvis ikke bruges til sådanne eksperimenter, så genetisk modifikation af organoider spiller en afgørende rolle på dette område. Mens han arbejdede som postdoc i mit laboratorium, demonstrerede Michael Heide, at det menneskespecifikke gen ARHGAP11B var en nøglespiller i at øge hjernens størrelse under menneskelig evolution.Som gruppeleder på German Primate Center bidrog Michael nu med afgørende indsigt ved at sammenligne menneskelige versus chimpansers cerebrale organoider. Vores protokol tilbyder en målrettet tilgang til genetiske modifikationer ved specifikke mikroinjicerende ventrikellignende strukturer i stedet for kommercielle elektroporationskuvetter. Denne tilgang bruger en omkostningseffektiv opsætning, herunder firkantet bølgeelektroporator og petriskålelektrodekammer i stedet for dyre nukleare effektorløsninger.Mens det er teknisk muligt at studere funktion og hjerneudvikling hos primater ved genetisk modifikation, er det metodisk krævende, dyrt og ikke tilladt for store aber. Elektroporation af primater cerebrale organoider giver en hurtig og omkostningseffektiv tilgang til at indføre genetiske modifikationer i en model tæt på primathjernens udvikling. I fremtiden vil vi gerne fokusere på karakteriseringen af differentielt udtrykte gener i cortex af forskellige primatarter.Vi vil studere disse gener ved elektroporation af primat cerebrale organoider og analysere deres potentielle virkninger på kortikale stamceller.