Name: Forfatter Spotlight: Optimering af den neurovaskulære udvikling af menneskelig hjerneorganoid i kyllingeembryo
Uploaded: 2024-02-16T13:00:00
Description: Watch this Scientific Journal Video about Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo at JoVE.com

Vi takker Dr. Alcántara og Dr. Ortega fra UB og resten af medlemmerne i Dr. Acostas laboratorium for de indsigtsfulde diskussioner. S.A. er Serra-Hunter adjunkt fra Generalitat de Catalunya ved Universitat de Barcelona.

De hvide leghorn kylling (Gallus gallus) embryoner blev behandlet ved at følge vejledningen for pleje og brug af forsøgsdyr fra Institute of Laboratory Animals Resources, Commission of Life Sciences, National Research Council, USA, og eksperimenterne blev godkendt af Rådet for pleje og brug af forsøgsdyr fra universitetet i Barcelona.
1. Ikke-guidet hjerneorganoid forberedelse
<ol><li>H9 humane embryonale stamceller (hESC) opbevares i mTESR1 forvarmet ved stuete...

Podning af menneskelige hjerneorganoider i den chorioallantoiske membran af kyllingeembryoet

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+of+human+brain+organoids.">Vascularization of human brain organoids.</a> Stem Cells. 39 (8), 1017-1024 (2021).</li><li>Apostolou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+challenges+in+stem+cell+biology.">Progress and challenges in stem cell biology.</a> Nat Cell Biol. 25 (2), 203-206 (2023).</li><li>Pham, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+vascularized+brain+organoids.">Generation of human vascularized brain organoids.</a> Neuroreport. 29 (7), 588-593 (2018).</li><li>Cakir, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+human+brain+organoids+with+a+functional+vascular-like+system.">Engineering of human brain organoids with a functional vascular-like system.</a> Nat Methods. 16 (11), 1169-1175 (2019).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+human+cortical+organoids+(vorganoids)+model+cortical+development+in+vivo.">Vascularized human cortical organoids (vorganoids) model cortical development in vivo.</a> PLoS Biol. 18 (5), e3000705 (2020).</li><li>Mansour, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+model+of+functional+and+vascularized+human+brain+organoids.">An in vivo model of functional and vascularized human brain organoids.</a> Nat Biotechnol. 36 (5), 432-441 (2018).</li><li>Revah, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+and+circuit+integration+of+transplanted+human+cortical+organoids.">Maturation and circuit integration of transplanted human cortical organoids.</a> Nature. 610 (7931), 319-326 (2022).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+chorioallantoic+membrane+angiogenesis+model.">Chicken chorioallantoic membrane angiogenesis model.</a> Methods Mol Biol. 843, 47-57 (2012).</li><li>Nowak-Sliwinska, P., Segura, T., Iruela-Arispe, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+chorioallantoic+membrane+model+in+biology,+medicine+and+bioengineering.">The chicken chorioallantoic membrane model in biology, medicine and bioengineering.</a> Angiogenesis. 17 (4), 779-804 (2014).</li><li>Kennedy, D. C., Coen, B., Wheatley, A. M., Mccullagh, K. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+experimentation+in+the+chick+chorioallantoic+membrane+as+a+model+for+screening+angiogenic+agents+including+from+gene-modified+cells.">Microvascular experimentation in the chick chorioallantoic membrane as a model for screening angiogenic agents including from gene-modified cells.</a> Int J Mol Sci. 23 (1), 452 (2021).</li><li>Garreta, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+tuning+the+extracellular+environment+accelerates+the+derivation+of+kidney+organoids+from+human+pluripotent+stem+cells.">Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells.</a> Nat Mater. 18 (4), 397-405 (2019).</li><li>Kaisto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+renal+organoid+and+organotypic+culture+for+vascularization,+extended+development,+and+improved+microscopy+imaging.">Optimization of renal organoid and organotypic culture for vascularization, extended development, and improved microscopy imaging.</a> J Vis Exp. (157), e60995 (2020).</li><li>Varzideh, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cardiomyocytes+undergo+enhanced+maturation+in+embryonic+stem+cell-derived+organoid+transplants.">Human cardiomyocytes undergo enhanced maturation in embryonic stem cell-derived organoid transplants.</a> Biomaterials. 192, 537-550 (2019).</li><li>Komatsu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cam+model+for+cic-dux4+sarcoma+and+its+potential+use+for+precision+medicine.">The cam model for cic-dux4 sarcoma and its potential use for precision medicine.</a> Cells. 10 (10), 2613 (2021).</li><li>Worsdorfer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+complex+human+organoid+models+including+vascular+networks+by+incorporation+of+mesodermal+progenitor+cells.">Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells.</a> Sci Rep. 9 (1), 15663 (2019).</li><li>Janzer, R. C., Jaff, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+induce+blood-brain+barrier+properties+in+endothelial+cells.">Astrocytes induce blood-brain barrier properties in endothelial cells.</a> Nature. 325 (6101), 253-257 (1987).</li><li>Janzer, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+Cellular+basis.">The blood-brain barrier: Cellular basis.</a> J Inherit Metab Dis. 16 (4), 639-647 (1993).</li><li>Lobrinus, J. A., Juillerat-Jeanneret, L., Darekar, P., Schlosshauer, B., Janzer, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+blood-brain+barrier+specific+ht7+and+neurothelin+epitopes+in+endothelial+cells+of+the+chick+chorioallantoic+vessels+by+a+soluble+factor+derived+from+astrocytes.">Induction of the blood-brain barrier specific ht7 and neurothelin epitopes in endothelial cells of the chick chorioallantoic vessels by a soluble factor derived from astrocytes.</a> Brain Res Dev Brain Res. 70 (2), 207-211 (1992).</li><li>Holash, J. A., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorioallantoic+membrane+(cam)+vessels+do+not+respond+to+blood-brain+barrier+(bbb)+induction.">Chorioallantoic membrane (cam) vessels do not respond to blood-brain barrier (bbb) induction.</a> Adv Exp Med Biol. 331, 223-228 (1993).</li><li>Holash, J. A., Noden, D. M., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluating+the+role+of+astrocytes+in+blood-brain+barrier+induction.">Re-evaluating the role of astrocytes in blood-brain barrier induction.</a> Dev Dyn. 197 (1), 14-25 (1993).</li><li>Giandomenico, S. L., Sutcliffe, M., Lancaster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+long-term+culture+of+advanced+cerebral+organoids+for+studying+later+stages+of+neural+development.">Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development.</a> Nat Protoc. 16 (2), 579-602 (2021).</li><li>Wagner-Amos, K., Seymour, R. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+tube+patterns+vessels+developmentally+using+the+vegf+signaling+pathway.">The neural tube patterns vessels developmentally using the vegf signaling pathway.</a> Development. 131 (7), 1503-1513 (2004).</li><li>Bozoyan, L., Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+control+the+development+of+the+migration-promoting+vasculature+scaffold+in+the+postnatal+brain+via+vegf+signaling.">Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via vegf signaling.</a> J Neurosci. 32 (5), 1687-1704 (2012).</li><li>Himmels, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neurons+control+blood+vessel+patterning+in+the+developing+spinal+cord.">Motor neurons control blood vessel patterning in the developing spinal cord.</a> Nat Commun. 8, 14583 (2017).</li><li>Di Lullo, E., Kriegstein, A. 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I denne undersøgelse beskriver vi en detaljeret protokol med adskillige vigtige trin, der giver gunstig vækst og udvikling af menneskelige hjerneorganoider ved podning uden at forstyrre kyllingembryonernes overlevelse. Vi anbefalede brug af sterile nåle til at punktere æggets luftkammer efter 24 timers inkubation (dag 1). Derudover forsøgte vi også at punktere på dag 4 (efter at have kontrolleret æggeskallen med lys for at teste udviklingen af vaskulaturen for at være sikker på, at vi kun arbejdede med sunde em...

Menneskelige hjerneorganoider er en ny teknik, der giver os mulighed for at rekapitulere den tidlige udvikling af den menneskelige hjerne in vitro 1,2,3. Ikke desto mindre er en af de største begrænsninger ved denne model manglen på vaskularisering, som spiller uundværlige roller ikke kun i hjernens homeostase, men også i hjernens udvikling4. Ud over levering af ilt og næringsstoffer tyder akkumulerende beviser på, at hjernens vaskulære system regulerer neural differentiering, migration og synaptogenese under udvikling 5,6. Derfor er der et presserende behov for at etablere pålidelige modeller, der kan give den manglende vaskulære signalering og struktur til hjerneorganoider, hvilket øger kompleksiteten af menneskelig hjerneorganoid generation7.
Blandt de foreslåede metoder til vaskularisering kan to hovedstrømlinjer overvejes: organoid indpodning i en levende organisme og rent in vitro teknologier co-kultivering af endotelceller og neurale celler 8,9,10,11,12. Intracerebral transplantation i mus er dyrt og tidskrævende, hvilket gør andre teknologier relevante for enklere modeller. Chick chorioallantoic membrane (CAM) assay er blevet brugt i vid udstrækning til at studere angiogenese 13,14,15. I det sidste årti har flere grupper med succes indpodet forskellige typer organoider, herunder nyre16,17, hjerte18 og tumororganoider19,20, i CAM'er. Ikke desto mindre er der lidt kendt om effektiviteten, toksicitet / afvisning, fysiologisk effekt og metoder til at indpode menneskelige hjerneorganoider i CAM. Et andet interessant og endnu uudforsket aspekt er dannelsen af en kimær blod-hjerne-barriere (BBB) mellem CAM og den organoide astrocytiske grænseflade. Tidligere banebrydende arbejde foreslog den formodede gennemførlighed af at generere en BBB i CAM ved at transplantere astrocytter og astrocytkonditioneret medium 21,22,23. Imidlertid synes modne astrocytter ikke at være i stand til at opnå disse24,25. Således forbliver den astrocyt-inducerede dannelse af BBB diskutabel, og transplantation af menneskelige hjerneorganoider ville give os mulighed for at kaste lys over denne kontrovers.
Denne videoartikel beskriver en protokol for en in ovo menneskelig hjerneorganoidtransplantation i CAM, der fremmer vækst, forbedring og vaskularisering, hvilket resulterer i organoider, der omfatter histologisk kompatible BBB-elementer. Her præsenterer vi en protokol, der sikrer kyllingembryoets overlevelse og rapporterer om CAM's tilladelse til at opretholde hjernens organoidvækst.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-TUBB3 [Tuj1], mouse&#160;</td>
			<td>BioLegend</td>
			<td>801201</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-GFAP, rabbit</td>
			<td>GeneTex</td>
			<td>GTX108711</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Anti-rabbit AlexaFluor 488, goat.</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-mouse AlexaFluor 594, goat</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-585-150</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Fertilized White Leghorn chicken (Gallus gallus) eggs</td>
			<td>Granja Gibert (Cambrils, Spain)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>1:10,000</td>
		</tr>
		<tr>
			<td>DPX</td>
			<td>Sigma</td>
			<td>100579</td>
			<td>xylene-based mounting medium&#160;</td>
		</tr>
		<tr>
			<td>Gentle Dissociation Solution</td>
			<td>CreativeBiolabs</td>
			<td>ITS-0622-YT187</td>
			<td>cell dissociation solution</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88 mounting media</td>
			<td>Merk</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel, lab-grade</td>
			<td>Sigma-Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y27632</td>
			<td>Millipore</td>
			<td>SCM075</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>Sharp-Point Surgical Scissors</td>
			<td>VWR</td>
			<td>470106-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Adhesion Microscope Slides</td>
			<td>Epredia</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

early unguided human brain organoid neurovascular niche modeling into the permissive chick embryo chorioallantoic membrane

Indpodning af organoider i vaskulariseret væv i modeldyr, såsom immundefekt mus eller kyllingembryo chorioallantoic membran (CAM), har vist sig effektiv til neovaskulariseringsmodellering. CAM er en rigt vaskulariseret ekstraembryonisk membran, som viser begrænset immunreaktivitet og dermed bliver en fremragende værtsmodel for celletransplantationer af human oprindelse.
Dette papir beskriver strategien til at indpode menneskelige hjerneorganoider, der er differentieret på flere modningsstadier, i CAM. Den cellulære sammensætning af hjerneorganoider ændrer sig med tiden, hvilket afspejler milepælene i den menneskelige hjerneudvikling. Vi podede hjerneorganoider på relevante modningsstadier: neuroepitelekspansion (18 DIV), tidlig neurogenese (60 DIV) og tidlig gliogenese (180 DIV) i CAM af embryonale dag (E) 7 kyllingeembryoner. Indpodede hjerneorganoider blev høstet 5 dage senere, og deres histologiske egenskaber blev analyseret.
Der blev ikke påvist histologiske tegn på neovaskularisering i de podede organoider eller unormale blodkar ved siden af podningerne. Desuden blev der observeret bemærkelsesværdige ændringer i den cellulære sammensætning af de podede organoider, nemlig en stigning i antallet af gliafibrillære sure protein-positive-reaktive astrocytter. De cytoarkitektoniske ændringer var imidlertid afhængige af organoidmodningsstadiet. Alt i alt tyder disse resultater på, at hjerneorganoider kan vokse i CAM, og de viser forskelle i cytoarkitekturen afhængigt af deres modningsstadium ved podning.

Human brain organoids are an emerging technique that allows us to recapitulate the early development of the human brain in vitro1,2,3. Nevertheless, one of the major limitations of this model is the lack of vascularization, which plays indispensable roles not only in brain homeostasis but also in brain development4. In addition to the delivery of oxygen and nutrients, accumulating evidence suggests that the vascular system of the brain regulates neural differentiation, migration, and synaptogenesis during development5,6. Therefore, there is an urgent need to establish reliable models that can provide the missing vascular signaling and structure to brain organoids, enhancing the complexity of human brain organoid generation7.
Among the proposed methods for vascularization, two main streamlines can be considered: organoid engrafting into a living organism and purely in vitro technologies co-culturing endothelial cells and neural cells8,9,10,11,12. Intracerebral transplantation in mice is costly and time-consuming, making other technologies relevant for simpler models. The chick chorioallantoic membrane (CAM) assay has been used extensively to study angiogenesis13,14,15. In the last decade, several groups have successfully engrafted different types of organoids, including kidney16,17, cardiac18, and tumor organoids19,20, into CAMs. Nevertheless, little is known about the efficacy, toxicity/rejection, physiological effect, and methods to engraft human brain organoids into the CAM. Another interesting and yet unexplored aspect is the formation of a chimeric blood-brain barrier (BBB) between the CAM and the organoid astrocytic interface. Previous pioneering work suggested the putative feasibility of generating a BBB in the CAM by transplanting astrocytes and astrocyte-conditioned medium21,22,23. However, mature astrocytes seem to be unable to achieve this24,25. Thus, the astrocyte-induced formation of the BBB remains debatable, and transplanting human brain organoids would allow us to shed light on this controversy.
This video article describes a protocol for an in ovo human brain organoid transplant into CAM that promotes growth, improvement, and vascularization, resulting in organoids that encompass histologically compatible BBB elements. Here, we present a protocol ensuring the survival of the chicken embryo and report on the permissivity of the CAM to sustain brain organoid growth.

Forfatter Spotlight: Optimering af den neurovaskulære udvikling af menneskelig hjerneorganoid i kyllingeembryo

Tidlig uguidet menneskelig hjerneorganoid neurovaskulær nichemodellering i den tilladende chick embryo chorioallantoic membran

The key questions I aim to address revolve around the mechanisms and processes involving human brain formation in the context of normal development, as well as various pathological states. This includes investigating the factors influencing brain development, such as genetics, environmental, and epigenetic influences. Obtaining brain organoids that closely resemble the adult human brain, including vascularization, blood brain barrier functionality, the presence of microglia and cellular diversity is challenging.Overcoming this is essential for the development of more physiologically relevant brain organoid models that can efficiently mimic the adult human brain to enhance clinical applications. Through our research, we provide optimal conditions for transplanting human brain organoids into chorioallantoic membrane to ensure good development of the microglia. This clinical improvement is central for forthcoming efforts in the field to generate synthetic vascularized environment for brain organoids.The advantage of our protocol compared to other techniques lies in its high effectiveness and cost efficiency. We have achieved a high success rate in transplanting viable organoids using fertile eggs from farm chickens. In the future, our laboratory will focus on conducting functional assessments of neo vasculature associated with organoids, exploring potential use of this vascular design model for application of intravenous nanomedicine.Additionally, we aim to evaluate the biodistribution of these nanomedicines in the human central nervous system.

Valg af embryomodningsplan for transplantationen Forsøget begynder ved D0, når befrugtede æg inkuberes ved 38 °C og 60% relativ luftfugtighed. Den chorioallantoiske membran (CAM) er en stærkt vaskulariseret ekstraembryonisk membran, der udvikler sig efter æginkubation. Det er dannet ved fusion af allantois og chorion. Ved D1, efter 24 timers inkubation, punkteres luftkammeret for at forhindre CAM i at binde sig til den indre skalmembran. Punktering af luftkammeret ved D1 forbedrer luftkammerets ...

Watch this Scientific Journal Video about Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo at JoVE.com

Her præsenterer vi en protokol til at indpode menneskelige hjerneorganoider på flere modningsstadier i kyllingens chorioallantoiske membran (CAM). Hjerneorganoider blev dyrket efter uguidede standardiserede protokoller.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has ...

De centrale spørgsmål, jeg sigter mod at behandle, drejer sig om de mekanismer og processer, der involverer menneskelig hjernedannelse i forbindelse med normal udvikling samt forskellige patologiske tilstande. Dette omfatter undersøgelse af de faktorer, der påvirker hjernens udvikling, såsom genetik, miljømæssige og epigenetiske påvirkninger. At opnå hjerneorganoider, der ligner den voksne menneskelige hjerne, herunder vaskularisering, blodhjernebarrierefunktionalitet, tilstedeværelsen af mikroglia og cellulær mangfoldighed er udfordrende.At overvinde dette er afgørende for udviklingen af mere fysiologisk relevante hjerneorganoidmodeller, der effektivt kan efterligne den voksne menneskelige hjerne for at forbedre kliniske anvendelser. Gennem vores forskning giver vi optimale betingelser for transplantation af menneskelige hjerneorganoider i chorioallantoic membran for at sikre god udvikling af microglia. Denne kliniske forbedring er central for kommende bestræbelser på området for at generere syntetisk vaskulariseret miljø for hjerneorganoider.Fordelen ved vores protokol sammenlignet med andre teknikker ligger i dens høje effektivitet og omkostningseffektivitet. Vi har opnået en høj succesrate med at transplantere levedygtige organoider ved hjælp af frugtbare æg fra gårdkyllinger. I fremtiden vil vores laboratorium fokusere på at udføre funktionelle vurderinger af neovaskulatur forbundet med organoider og udforske potentiel anvendelse af denne vaskulære designmodel til anvendelse af intravenøs nanomedicin.Derudover sigter vi mod at evaluere biodistributionen af disse nanolægemidler i det menneskelige centralnervesystem.

early-unguided-human-brain-organoid-neurovascular-niche-modeling-into

Research

JoVE Journal

Developmental Biology

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants.
This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed.
No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Here, we present a protocol to engraft human brain organoids at multiple maturation stages into the chick chorioallantoic membrane (CAM). Brain organoids were grown following unguided standardized protocols.