Name: Autor destacado: Optimización del desarrollo neurovascular del organoide del cerebro humano en embrión de pollo
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

Agradecemos al Dr. Alcántara y al Dr. Ortega de la UB y al resto de los miembros del laboratorio del Dr. Acosta por las esclarecedoras discusiones. S.A. es profesor ayudante doctor de la Generalitat de Catalunya en la Universitat de Barcelona.

Los embriones de pollo Leghorn blanco (Gallus gallus) fueron tratados siguiendo la Guía para el Cuidado y Uso de Animales de Laboratorio del Instituto de Recursos para Animales de Laboratorio, Comisión de Ciencias de la Vida, Consejo Nacional de Investigación, EE.UU., y los experimentos fueron aprobados por el Consejo para el Cuidado y Uso de Animales de Experimentación de la Universidad de Barcelona.
1. Preparación no guiada de organoides cerebrales
<ol><li>Ma...

Injerto de organoides cerebrales humanos en la membrana corioalantoidea del embrión de pollo

<ol>
	<li>Camp, J. G., 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+cerebral+organoids+recapitulate+gene+expression+programs+of+fetal+neocortex+development.">Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.</a> Proc Natl Acad Sci U S A. 112 (51), 15672-15677 (2015).</li><li>Lancaster, M. A., Knoblich, 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=Organogenesis+in+a+dish:+Modeling+development+and+disease+using+organoid+technologies.">Organogenesis in a dish: Modeling development and disease using organoid technologies.</a> Science. 345 (6194), 1247125 (2014).</li><li>Yang, Q., Hong, Y., Zhao, T., Song, H., Ming, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+makes+organoids+good+models+of+human+neurogenesis.">What makes organoids good models of human neurogenesis.</a> Front Neurosci. 16, 872794 (2022).</li><li>Sun, X. 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=Generation+of+vascularized+brain+organoids+to+study+neurovascular+interactions.">Generation of vascularized brain organoids to study neurovascular interactions.</a> Elife. 11, e76707 (2022).</li><li>Paredes, I., 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=Oligodendrocyte+precursor+cell+specification+is+regulated+by+bidirectional+neural+progenitor-endothelial+cell+crosstalk.">Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor-endothelial cell crosstalk.</a> Nat Neurosci. 24 (4), 478-488 (2021).</li><li>Matsui, T. K., Tsuru, Y., Hasegawa, K., Kuwako, K. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+local+shell+conductance+on+the+vascularisation+of+the+chicken+chorioallantoic+membrane.">Effect of local shell conductance on the vascularisation of the chicken chorioallantoic membrane.</a> Respir Physiol Neurobiol. 134 (2), 155-167 (2003).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev Dyn. 195 (4), 231-272 (1992).</li><li>Paredes, I., Himmels, P., Ruiz De Almodovar, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+communication+during+cns+development.">Neurovascular communication during cns development.</a> Dev Cell. 45 (1), 10-32 (2018).</li><li>Hogan, K. A., Ambler, C. A., Chapman, D. L., Bautch, V. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+brain+organoids+to+investigate+neural+development+and+disease.">The use of brain organoids to investigate neural development and disease.</a> Nat Rev Neurosci. 18 (10), 573-584 (2017).</li></ol>

En este estudio, describimos un protocolo detallado con numerosos pasos clave que proporcionan un crecimiento y desarrollo favorable de los organoides del cerebro humano tras el injerto sin perturbar la supervivencia de los embriones de pollo. Se recomendó el uso de agujas estériles para perforar la cámara de aire del huevo después de 24 h de incubación (día 1). Además, también intentamos hacer la punción en el día 4 (después de revisar la cáscara del huevo con luz para probar el desarrollo de la vasculatura ...

Los organoides cerebrales humanos son una técnica emergente que permite recapitular in vitro el desarrollo temprano del cerebro humano 1,2,3. Sin embargo, una de las principales limitaciones de este modelo es la falta de vascularización, que desempeña un papel indispensable no solo en la homeostasis cerebral, sino también en el desarrollo cerebral4. Además del suministro de oxígeno y nutrientes, la evidencia acumulada sugiere que el sistema vascular del cerebro regula la diferenciación neuronal, la migración y la sinaptogénesis durante el desarrollo 5,6. Por lo tanto, existe una necesidad urgente de establecer modelos confiables que puedan proporcionar la señalización vascular y la estructura faltantes a los organoides cerebrales, lo que aumenta la complejidad de la generación de organoides cerebrales humanos7.
Entre los métodos propuestos para la vascularización, se pueden considerar dos líneas principales: el injerto de organoides en un organismo vivo y las tecnologías puramente in vitro que cocultivan células endoteliales y células neurales 8,9,10,11,12. El trasplante intracerebral en ratones es costoso y requiere mucho tiempo, lo que hace que otras tecnologías sean relevantes para modelos más simples. El ensayo de membrana corioalantoidea de pollitos (CAM) se ha utilizado ampliamente para estudiar la angiogénesis 13,14,15. En la última década, varios grupos han injertado con éxito diferentes tipos de organoides, incluidos el riñón16,17, el cardíaco18 y los organoides tumorales19,20, en CAM. Sin embargo, se sabe poco sobre la eficacia, la toxicidad/rechazo, el efecto fisiológico y los métodos para injertar organoides cerebrales humanos en la MCA. Otro aspecto interesante y aún inexplorado es la formación de una barrera hematoencefálica quimérica (BHE) entre la MCA y la interfase astrocítica organoide. Trabajos pioneros previos sugirieron la viabilidad putativa de generar una BHE en la MCA mediante el trasplante de astrocitos y medio condicionado por astrocitos 21,22,23. Sin embargo, los astrocitos maduros parecen ser incapaces de lograrlo24,25. Por lo tanto, la formación de la BHE inducida por astrocitos sigue siendo discutible, y el trasplante de organoides cerebrales humanos nos permitiría arrojar luz sobre esta controversia.
Este artículo de video describe un protocolo para un trasplante in ovo de organoides cerebrales humanos en MCA que promueve el crecimiento, la mejora y la vascularización, lo que da como resultado organoides que abarcan elementos de BHE histológicamente compatibles. Aquí, presentamos un protocolo que garantiza la supervivencia del embrión de pollo e informamos sobre la permisividad de la MCA para mantener el crecimiento de organoides cerebrales.

<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

El injerto de organoides en tejidos vascularizados en animales modelo, como la membrana corioalantoidea (CAM) inmunodeficiente de ratones o embriones de pollo, ha demostrado ser eficaz para el modelado de la neovascularización. La MCA es una membrana extraembrionaria ricamente vascularizada, que muestra una inmunorreactividad limitada, convirtiéndose así en un excelente modelo de hospedaje para trasplantes de células de origen humano.
En este trabajo se describe la estrategia para injertar organoides cerebrales humanos diferenciados en múltiples etapas de maduración en la CAM. La composición celular de los organoides cerebrales cambia con el tiempo, reflejando los hitos del desarrollo del cerebro humano. Injertamos organoides cerebrales en etapas de maduración relevantes: expansión neuroepitelial (18 DIV), neurogénesis temprana (60 DIV) y gliogénesis temprana (180 DIV) en la MCA de embriones embrionarios de pollo del día (E)7. Los organoides cerebrales injertados se recolectaron 5 días después y se analizaron sus características histológicas.
No se detectaron signos histológicos de neovascularización en los organoides injertados ni vasos sanguíneos anormales adyacentes a los injertos. Además, se observaron cambios notables en la composición celular de los organoides injertados, a saber, un aumento en el número de astrocitos gliales fibrilares ácidos positivos reactivos a proteínas. Sin embargo, los cambios citoarquitectónicos dependieron de la etapa de maduración de los organoides. En conjunto, estos resultados sugieren que los organoides cerebrales pueden crecer en la MCA y muestran diferencias en la citoarquitectura dependiendo de su etapa de maduración en el momento del injerto.

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.

Autor destacado: Optimización del desarrollo neurovascular del organoide del cerebro humano en embrión de pollo

Modelado de nicho neurovascular de organoides cerebrales humanos tempranos no guiados en la membrana corioalantoidea permisiva del embrión de pollo

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.

Selección del programa de maduración embrionaria para el trasplante El experimento comienza en D0 cuando los huevos fertilizados se incuban a 38 °C y 60% de humedad relativa. La membrana corioalantoidea (MCA) es una membrana extraembrionaria altamente vascularizada que se desarrolla después de la incubación de los huevos. Está formado por la fusión de los alantoides y el corion. En D1, después de 24 h de incubación, la cámara de aire se perfora para evitar que el CAM se adhiera a la membrana...

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

Aquí, presentamos un protocolo para injertar organoides cerebrales humanos en múltiples etapas de maduración en la membrana corioalantoidea (CAM) del pollo. Los organoides cerebrales se cultivaron siguiendo protocolos estandarizados no guiados.

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

Las preguntas clave que pretendo abordar giran en torno a los mecanismos y procesos que implican la formación del cerebro humano en el contexto del desarrollo normal, así como diversos estados patológicos. Esto incluye investigar los factores que influyen en el desarrollo del cerebro, como la genética, el medio ambiente y las influencias epigenéticas. La obtención de organoides cerebrales que se asemejan mucho al cerebro humano adulto, incluida la vascularización, la funcionalidad de la barrera hematoencefálica, la presencia de microglía y la diversidad celular es un desafío.Superar esto es esencial para el desarrollo de modelos de organoides cerebrales más relevantes fisiológicamente que puedan imitar eficientemente el cerebro humano adulto para mejorar las aplicaciones clínicas. A través de nuestra investigación, proporcionamos las condiciones óptimas para el trasplante de organoides cerebrales humanos a la membrana corioalantoidea para garantizar un buen desarrollo de la microglía. Esta mejora clínica es fundamental para los próximos esfuerzos en el campo para generar un entorno vascularizado sintético para organoides cerebrales.La ventaja de nuestro protocolo frente a otras técnicas radica en su alta efectividad y rentabilidad. Hemos logrado una alta tasa de éxito en el trasplante de organoides viables utilizando huevos fértiles de pollos de granja. En el futuro, nuestro laboratorio se centrará en la realización de evaluaciones funcionales de la neovasculatura asociada a organoides, explorando el uso potencial de este modelo de diseño vascular para la aplicación de nanomedicina intravenosa.Además, nuestro objetivo es evaluar la biodistribución de estas nanomedicinas en el sistema nervioso central humano.

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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.