Name: early unguided human brain organoid neurovascular niche modeling into the permissive chick embryo chorioallantoic membrane
Uploaded: 2024-02-16T13:00:00.000+00:00
Duration: 4 min 8 s
Description: Watch this Scientific Journal Video about Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo at JoVE.com

We thank Dr. Alc&#225;ntara and Dr. Ortega from UB and the rest of the members in Dr. Acosta&#39;s lab for the insightful discussions. S.A. is Serra-Hunter fellow assistant professor from the Generalitat de Catalunya at Universitat de Barcelona.

The White Leghorn chicken (Gallus gallus) embryos were treated by following the Guide for the Care and Use of Laboratory Animals from the Institute of Laboratory Animals Resources, Commission of Life Sciences, National Research Council, USA, and the experiments were approved by the Council for Care and Use of Experimental Animals from the University of Barcelona.
1. Non-guided brain organoid preparation
<ol>
	<li>Maintain H9 human embryonic stem cells (hESCs) in mTESR1 prewarmed at room temperature (RT) under a confluence of 70% in 6-well plates coated with Matrigel dissolved in DMEM (1:40) in a 5% CO2 incubator at 37 &#176;C. 
	NOTE: Matrigel must be kept in ice until its use as a coating to prevent its polymerization.</li>
	<li>Generate brain organoids following the unguided brain protocol 2.
	<ol>
		<li>Dissociate H9 hESCs using 500 &#181;L of the Cell Dissociation Solution and incubate for 3-5 min at 37 &#176;C. Resuspend in mTESR1 containing a ROCK inhibitor Y27632 (final concentration 50 &#181;M) and seed 9,000 cells per well in a low adhesion pretreated 96-well plate.</li>
		<li>Once the organoids aggregate in mTESR1 containing a ROCK inhibitor Y27632 (final concentration 50 &#181;M), transfer them into induction medium for 5-6 days.</li>
		<li>Following unguided brain organoid protocol26, transfer the organoids to neural induction media at ~days 4-5.</li>
		<li>Once they acquire the size and the neuroepithelium ring, transfer them into an ice-cold Matrigel drop. Once the Matrigel is polymerized, transfer the organoids to a 5 cm, low-adhesion Petri dish with 5 mL of prewarmed maturation medium.</li>
		<li>After 4 days, put the brain organoids under orbital agitation.</li>
	</ol>
	</li>
	<li>Collect the organoids for harvesting in fresh DPBS immediately before grafting.</li>
</ol>
2. Egg maintenance, development activation, and eggshell puncture
<ol>
	<li>Store fertilized eggs (at day 0) in an incubator at 18 &#176;C until use. 
	NOTE: At this temperature, they do not develop, remaining at the same stage of maturation.</li>
	<li>When required for use, incubate them at 38 &#176;C and 60% relative humidity with soft rocking rotation at an angle of 45 &#176;C.</li>
	<li>On day 1, clean the eggshell surface with 70% ethanol and dry it with a paper towel to sterilize it.</li>
	<li>Place a surgical paper adhesive bandage, hereafter referred to as &#34;bandage,&#34; on top of the egg, covering the air chamber of the egg. 
	NOTE: This will prevent dust from the eggshell from falling into the CAM where it gets stuck.</li>
	<li>Perform the air chamber perforation puncture, hereafter referred to as air chamber hole (ACH), with a sterile needle 21 G x 11/2&#39;&#39;, softly puncturing the eggshell. Open the ACH on the upper tip of the egg to avoid disturbing the CAM, which at this point lays separated from the shell.</li>
	<li>To find the best position to puncture the ACH, use a lamp to determine the thinnest eggshell surface at the upper tip of the egg. The direct light beam transmission indicates the best position to drill.</li>
	<li>Cover the ACH with a new bandage to protect the embryo from the external environment and place the egg back into the incubator.</li>
	<li>From this moment on, cease rotation in the incubator and maintain the eggs in a vertical position with the window on top. 
	NOTE: This will facilitate the development of the embryo, and the CAM becomes available at the top with a chamber of air between the embryo and the eggshell, allowing for subsequent grafting.</li>
	<li>Open the ACH from day 1 to day 4. Viability increases on day 4 (3 days prior to grafting day).</li>
</ol>
3. Brain organoid grafting
<ol>
	<li>Perform grafting on day 7 when the CAM is well developed and vascularized, covering the egg all over the embryo.</li>
	<li>Remove the bandage, and wipe the eggshell surface with 70% ethanol to sterilize the eggshell, prior to carefully widening the ACH into a grafting window.</li>
	<li>Place a new, bigger bandage covering the extension beyond the final grafting window to avoid fragments of eggshell falling into the CAM while enlarging the grafting window. 
	NOTE: This bigger window is needed to select the best grafting position and for subsequent grafting.</li>
	<li>Enlarge the grafting window by softly puncturing the eggshell until the window reaches a diameter of approximately 2 cm. Softly remove the edges of the eggshell window with tweezers until the window is enlarged. Remove the remaining dust and eggshell pieces that will remain attached to the bandage by softly peeling off the bandage.</li>
	<li>Place the organoid with a P200 automatic pipet. Cut the tips with sterile and sharp scissors to enlarge the opening accordingly to the size of the organoid and prevent damage to the organoid or use wide-gauge transferring sterile tips. Collect the organoid in a maximum volume of 50 &#181;L of PBS and transfer it directly into the CAM, on a side opposite to the location of the embryo.</li>
	<li>Ensure that the position of the graft is distant from the yolk to avoid its rupture during harvesting, and preferably, place it near a blood vessel.</li>
	<li>Place a drop of 7 &#181;L of Matrigel surrounding the organoid after placing the organoid onto the CAM. 
	NOTE: This will prevent the organoid from moving around the membrane.</li>
	<li>Close the window using a small piece of parafilm fitting the exact size of the window and attach it to the shell with an adhesive bandage. 
	NOTE: It is important to fully cover the window with the parafilm to avoid the embryo from drying out while allowing gas exchange. Delimit properly the extension of the window coverage, avoiding exceeding the window, as it could hinder the necessary gas exchange for correct embryo development27.</li>
</ol>
4. Transplanted organoid harvesting
<ol>
	<li>Harvest the grafted organoids and the surrounding CAM on day 12 of chicken development, corresponding to 38 Hamburger and Hamilton stage of development (HH38)28, 5 days after grafting. At this stage, the CAM is fully differentiated with mature vasculature while the feather germs coverts the wings as well as the upper eyelid of the embryo.</li>
	<li>Upon removing the adhesive bandage, further enlarge the window to facilitate sample harvest using scissors and fine tweezers.</li>
	<li>Visualize the organoid with the help of a binocular dissection microscopy if needed, followed by a 2 min prefixation step with 4% paraformaldehyde (PFA) to help organoid and CAM harvesting.</li>
	<li>Collect a 1.5 cm2 section of the CAM containing the organoid.</li>
	<li>Sample fixation
	<ol>
		<li>Wash the samples with 1x PBS and fix them with 4% PFA (in PBS, pH 7.2) for 30 min at 4 &#176;C. Then, wash the samples for 3 x 5 min in PBS.</li>
		<li>Store the samples at 4 &#176;C in PBS with sodium azide or directly cryoprotect them in 30% sucrose-PBS overnight at 4 &#176;C.</li>
		<li>Upon cryoprotection, embed the samples in optimal cutting temperature compound (OCT) and store them at -20 &#176;C until sectioning. Cut 20 &#181;m thick tissue slices using a cryostat and collect them on xylanized slides. Store the slides at -20 &#176;C until they are used for staining.</li>
	</ol>
	</li>
</ol>
5. Immunofluorescence
<ol>
	<li>Remove OCT. from the sections with a PBS solution with 0.1% Triton X-100 (PBS-T) for 10 min at room temperature (RT).</li>
	<li>Treat the sections with a blocking solution (BS) with a 2% BSA, 3% donkey serum PBS-T 0.01% solution for 1 h at RT.</li>
	<li>Dilute the selected primary antibody (see the Table of Materials) in BS and incubate samples overnight at 4 &#176;C.</li>
	<li>The next day, wash the samples 3 x 10 min in 1x PBS at RT.</li>
	<li>Dilute the secondary antibody in BS and incubate for 2 h at RT (see the Table of Materials).</li>
	<li>Incubate the samples for 10 min with 4&#39;,6-diamidino-2-phenylindole (DAPI) to mark the cell nuclei at RT, diluted in PBS-T at a 1:1,000 dilution.</li>
	<li>Mount the slides with mounting medium and cover with glass coverslips.</li>
	<li>Take all images using a widefield fluorescence microscope at 2.5, 10, 20, and 40x.</li>
	<li>Store the slices at 4 &#176;C.</li>
</ol>
6. Hematoxylin and eosin (H&#38;E) staining
NOTE: To prove that the grafting worked, perform H&#38;E staining.
<ol>
	<li>Remove OCT with PBS-T 0.1% for 10 min at RT.</li>
	<li>Perform serial dehydration in a hood as follows: 10 min in distilled water (DW), 45 s in hematoxylin, 1 min in DW, 20 s in PBS, 2 x 30 s in 70% EtOH, 2 x 30 s in 80% EtOH, 2 x 30 s in 96% EtOH, 30 s in eosin, 2 x 15 s in 96% EtOH, 2 x 15 s in 100% EtOH, 1 min in xylol-100% ETOH, 1 min in xylol, 1 min in xylol (fresh).</li>
	<li>Mount the glass coverslips with a xylene-based mounting medium.</li>
</ol>

Grafting Human Brain Organoids into the Chorioallantoic Membrane of the Chick Embryo

<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>

The authors do not report any conflicts of interest.

In this study, we describe a detailed protocol with numerous key steps that provide favorable growth and development of human brain organoids upon grafting without perturbing the survival of the chicken embryos. We recommended the use of sterile needles to puncture the air chamber of the egg after 24 h of incubation (day 1). Additionally, we also tried to make the puncture at day 4 (after checking through the eggshell by light to test the development of the vasculature to be sure that we were working only with healthy em...

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.

<table><tbody><tr><td>Anti-TUBB3 [Tuj1], mouse&amp;nbsp;</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&amp;nbsp;</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

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.

Selecting the embryo maturation schedule for the transplant 
The experiment begins at D0 when fertilized eggs are incubated at 38 &#176;C and 60% relative humidity. The chorioallantoic membrane (CAM) is a highly vascularized extraembryonic membrane that develops after egg incubation. It is formed by the fusion of the allantois and chorion. At D1, after 24 h of incubation, the air chamber is punctured to prevent the CAM from attaching to the inner shell membrane. Puncturing the air chamber at D1 impr...

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

Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo

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.

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 ...

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

Research

JoVE Journal

Developmental Biology

1.3K Views.  CONICET - Universidad de Buenos Aires. De belangrijkste vragen die ik wil beantwoorden, draaien om de mechanismen en processen die te maken hebben met de vorming van menselijke hersenen in de context van normale ontwikkeling, evenals verschillende pathologische toestanden. Dit omvat het onderzoeken van de factoren die de ontwikkeling van de hersenen beïnvloeden, zoals genetica, omgevings- en epigenetische invloeden. Het verkrijgen van hersenorganoïden die sterk lijken op het volwassen menselijke brein, inclusief vascularisatie, ...

Video: Auteur in de schijnwerpers: optimalisatie van de neurovasculaire ontwikkeling van menselijke hersenorganoïde in kuikenembryo

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biology

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

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 ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Medicine

Using Chick Embryo Brain as a Model to Study Human Glioblastoma Cell Behavior

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. 
Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 &#181;m) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 &#181;m) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. 
Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...

Neuroscience

Chick Chorioallantoic Membrane or CAM Tumor Model: An In Ovo Method to Simulate Perineural Invasion of Tumor Cells

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
In this video, we describe the method to co-culture dorsal root ganglia and cancer cells on chick chorioallantoic membrane or CAM to study perineural invasion.

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

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

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