Funds for this work were provided through the IRIC Philanthropic funds from the Marcelle and Jean Coutu foundation and from the Fonds de recherche du Qu&#233;bec - Sant&#233; (FRQS #295647). D.J.H.F.K. has salary support from FRQS in the form of a Chercheurs-boursiers Junior 1 fellowship (#283502). M.I.I.R. was supported by an IRIC Doctoral Award from the Institut of Research in Immunology and Cancer, a Bourse de passage acc&#233;l&#232;re de la maitrise au doctorat from the University of Montreal, and Bourse de M&#233;rite aux cycles sup&#233;rieurs.

<ol>
	<li>Spurlock, M. 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=Amelioration+of+penetrating+ballistic-like+brain+injury+induced+cognitive+deficits+after+neuronal+differentiation+of+transplanted+human+neural+stem+cells.">Amelioration of penetrating ballistic-like brain injury induced cognitive deficits after neuronal differentiation of transplanted human neural stem cells.</a> Journal of Neurotrauma. 34 (11), 1981 (2017).</li><li>Zhou, Y., Shao, A., Xu, W., Wu, H., Deng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advance+of+stem+cell+treatment+for+traumatic+brain+injury.">Advance of stem cell treatment for traumatic brain injury.</a> Frontiers in Cellular Neuroscience. 13, 301 (2019).</li><li>Hayashi, Y., Lin, H. -. T., Lee, C. -. C., Tsai, K. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neural+stem+cell+transplantation+in+Alzheimer's+disease+models.">Effects of neural stem cell transplantation in Alzheimer's disease models.</a> Journal of Biomedical Science. 27 (1), 29 (2020).</li><li>Kefalopoulou, Z., 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=Long-term+clinical+outcome+of+fetal+cell+transplantation+for+Parkinson+disease:+Two+case+reports.">Long-term clinical outcome of fetal cell transplantation for Parkinson disease: Two case reports.</a> JAMA Neurology. 71 (1), 83-87 (2014).</li><li>Li, W., 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=Extensive+graft-derived+dopaminergic+innervation+is+maintained+24+years+after+transplantation+in+the+degenerating+parkinsonian+brain.">Extensive graft-derived dopaminergic innervation is maintained 24 years after transplantation in the degenerating parkinsonian brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (23), 6544-6549 (2016).</li><li>Parmar, M., Grealish, S., Henchcliffe, 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+future+of+stem+cell+therapies+for+Parkinson+disease.">The future of stem cell therapies for Parkinson disease.</a> Nature Reviews Neuroscience. 21 (2), 103-115 (2020).</li><li>Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cell-based+therapy+for+Parkinson's+disease:+A+Kyoto+trial.">iPS cell-based therapy for Parkinson's disease: A Kyoto trial.</a> Regenerative Therapy. 13, 18-22 (2020).</li><li>Krause, M., Phan, T. G., Ma, H., Sobey, C. G., Lim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapies+for+stroke:+Are+we+there+yet.">Cell-based therapies for stroke: Are we there yet.</a> Frontiers in Neurology. 10, 656 (2019).</li><li>Coles-Takabe, B. L. K., 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=Don't+look:+Growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don't look: Growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li><li>Duval, K., 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=Modeling+physiological+events+in+2D+vs.+3D+cell+culture.">Modeling physiological events in 2D vs. 3D cell culture.</a> Physiology. 32 (4), 266-277 (2017).</li><li>Jakel, R. J., Schneider, B. L., Svendsen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+human+neural+stem+cells+to+model+neurological+disease.">Using human neural stem cells to model neurological disease.</a> Nature Reviews Genetics. 5 (2), 136-144 (2004).</li><li>Hodge, R. D., 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=Conserved+cell+types+with+divergent+features+in+human+versus+mouse+cortex.">Conserved cell types with divergent features in human versus mouse cortex.</a> Nature. 573 (7772), 61-68 (2019).</li><li>Li, J., 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=Conservation+and+divergence+of+vulnerability+and+responses+to+stressors+between+human+and+mouse+astrocytes.">Conservation and divergence of vulnerability and responses to stressors between human and mouse astrocytes.</a> Nature Communications. 12 (1), 3958 (2021).</li><li>Morizane, 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=MHC+matching+improves+engraftment+of+iPSC-derived+neurons+in+non-human+primates.">MHC matching improves engraftment of iPSC-derived neurons in non-human primates.</a> Nature Communications. 8 (1), 385 (2017).</li><li>Khrameeva, 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=Single-cell-resolution+transcriptome+map+of+human,+chimpanzee,+bonobo,+and+macaque+brains.">Single-cell-resolution transcriptome map of human, chimpanzee, bonobo, and macaque brains.</a> Genome Research. 30 (5), 776-789 (2020).</li><li>Powell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+brains:+The+ethics+of+transplanting+human+neurons+into+animals.">Hybrid brains: The ethics of transplanting human neurons into animals.</a> Nature. 608 (7921), 22-25 (2022).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Birey, 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=Assembly+of+functionally+integrated+human+forebrain+spheroids.">Assembly of functionally integrated human forebrain spheroids.</a> Nature. 545 (7652), 54-59 (2017).</li><li>Lancaster, M. 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Garc&#237;a-Delgado, A. 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=Brain+organoids+to+evaluate+cellular+therapies.">Brain organoids to evaluate cellular therapies.</a> Animals. 12 (22), (2022).</li><li>Lin, J. -. R., Fallahi-Sichani, M., Sorger, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+multiplexed+imaging+of+single+cells+using+a+high-throughput+cyclic+immunofluorescence+method.">Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.</a> Nature Communications. 6 (1), 8390 (2015).</li><li>Knapp, D. J. H. 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=Single-cell+analysis+identifies+a+CD33++subset+of+human+cord+blood+cells+with+high+regenerative+potential.">Single-cell analysis identifies a CD33+ subset of human cord blood cells with high regenerative potential.</a> Nature Cell Biology. 20 (6), 710-720 (2018).</li><li>Georgala, P. A., Carr, C. B., Price, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Pax6+in+forebrain+development.">The role of Pax6 in forebrain development.</a> Developmental Neurobiology. 71 (8), 690-709 (2011).</li><li>Britanova, 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=Satb2+is+a+postmitotic+determinant+for+upper-layer+neuron+specification+in+the+neocortex.">Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex.</a> Neuron. 57 (3), 378-392 (2008).</li><li>Kim, H., 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=Modeling+G2019S-LRRK2+sporadic+Parkinson's+disease+in+3D+midbrain+organoids.">Modeling G2019S-LRRK2 sporadic Parkinson's disease in 3D midbrain organoids.</a> Stem Cell Reports. 12 (3), 518-531 (2019).</li><li>Smits, L. M., 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=Modeling+Parkinson's+disease+in+midbrain-like+organoids.">Modeling Parkinson's disease in midbrain-like organoids.</a> NPJ Parkinson's Disease. 5, 5 (2019).</li><li>Jin, M., 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=Type-I-interferon+signaling+drives+microglial+dysfunction+and+senescence+in+human+iPSC+models+of+Down+syndrome+and+Alzheimer's+disease.">Type-I-interferon signaling drives microglial dysfunction and senescence in human iPSC models of Down syndrome and Alzheimer's disease.</a> Cell Stem Cell. 29 (7), 1135.e8-1153.e8 (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>Popova, 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+microglia+states+are+conserved+across+experimental+models+and+regulate+neural+stem+cell+responses+in+chimeric+organoids.">Human microglia states are conserved across experimental models and regulate neural stem cell responses in chimeric organoids.</a> Cell Stem Cell. 28 (12), 2153.e6-2166.e6 (2021).</li><li>Wang, S., Li, B., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+fluorophores+for+deep-tissue+bioimaging.">Molecular fluorophores for deep-tissue bioimaging.</a> ACS Central Science. 6 (8), 1302-1316 (2020).</li></ol>

The authors have nothing to disclose.

Given the significant interest in cell therapeutic approaches for the treatment of CNS injuries/neurodegenerative disorders1,2,3,4,5,6,7,8, models of cell function in a transplant setting are gaining importance. This paper presents a method for the transplantation of labeled, human NPCs into human cerebral organoids, along with their live-cell tracking and end-point assessment by histology and immunofluorescent staining. Importantly, we showed that the transplanted cells were capable of migration, differentiation, and long-term (4 month) persistence in the organoid setting. Such long-term persistence is a marked increase over the maintainability of brain slice cultures17. This system is, thus, appropriate for examining many of the behaviors one would need to assess in a potential therapeutic setting, such as survival, proliferation, and differentiation. Indeed, an orthogonal study recently demonstrated that transplanted NPCs behaved similarly in cerebral organoids compared to NPCs transplanted into NSG mouse brains20, thus confirming the utility of organoids as a transplant recipient. As this is an in vitro system, it is also straightforward to add cytokines or drugs of interest. This could be used to better understand the effects of specific environments such as inflammation and immunosuppressants on the transplanted cells to further mimic what they might encounter in a therapeutic setting. The cyclic immunofluorescence protocol we demonstrated (based on previous research21) further extends the power of this approach, allowing a wide array of lineage- and, potentially, disease-specific markers to be simultaneously assessed in a single section, and, thus, allowing accurate tracking of the transplanted cells and their impact on the tissue. Of course, other endpoint assessment methods could be used instead depending on the goals of the analysis. For example, tissue clearing with 3D reconstruction could be used if cell morphology is of primary interest, or dissociation followed by flow cytometry could be used if the quantification of specific cell types is the end goal. We expect this method to be easily extensible to other cell types such as CNS tumors, potentially allowing their study in a microenvironmentally relevant context. Similarly, the organoids used as recipients could be exchanged for disease-model organoids25,26,27, potentially allowing for the modeling of transplantation approaches for these conditions.
As with all models, the one presented here also has its own limitations. For one, iPSC-derived organoids are developmentally immature19 and, thus, have important differences compared to the aging brain, in which many neurodegenerative diseases manifest. Cerebral organoids are also non-uniform in development19, thus precluding consistent injection into the same exact physiological niche. Moreover, while they contain the cell types of the relevant brain regions18, 19, they lack the endothelial, microglial, and immune components, which are also important in the in vivo setting14. This limits the study of how the host will react to the cell transplantation. Techniques are currently coming online to add vascular28 and microglial29 cells, as well as to increase the organoid consistency and regionalization18, thus improving the modeling power of the organoid transplantation system. They would, however, require further testing and optimization beyond what is presented here. While this protocol is inexpensive and does not require specialized equipment, there remain a number of important technical considerations-the injection depth, for example. This is both due to the fact that organoids are not perfused and, thus, often have a necrotic center if they grow too large19 and that light cannot penetrate through the organoid core for live-cell tracking. Thus, the cells that have been injected too deep and colonies that have migrated inside may be missed. While this can be ameliorated by the use of longer-wavelength fluorophores with better tissue penetrance30, depending on the organoid size and detection apparatus, this will likely remain a consideration. Finally, as brain organoids are in a state of development, the transplantation timing is another key consideration, as the environment will likely differ depending on the developmental stage of the organoid into which it is injected. While this can be controlled to some extent by ensuring a consistent organoid age at time of injection, it is, without doubt, a factor that needs consideration.
This protocol is inexpensive, simple, animal-free, and does not require specialized equipment, thus making transplantation modeling accessible to a wider variety of labs. With the rapid pace of advancement both in neural cell therapeutics and organoid model systems, we anticipate that the organoid transplantation protocol presented here will be a useful model for a range of diseases and therapeutic approaches.

The human brain is a complex organ composed of multiple cell types of the neural and glial lineages. Together, these form a sophisticated network that gives rise to cognition. There is significant interest in the transplantation of cells into this system as a treatment for a wide variety of neurological disorders, including traumatic brain injury (TBI)1,2, neurodegenerative disorders3,4,5,6,7, and stroke8. One major limitation in the advancement of such strategies, however, is the relative paucity of available preclinical models to determine the expected transplantation outcomes. The most used models currently are in vitro culture methods to determine cell potential and xenotransplantation into mice. While cell culture methods can assess differentiation and self-renewal potential9, these are performed under optimal growth conditions that do not mimic the microenvironment the cells would encounter in a transplant context. Moreover, the way in which the cells are grown can influence their behavior10.
Mouse brains contain all the cells of the microenvironment and are, thus, extremely powerful model systems for transplantation11. There are, however, important differences between the mouse and human cortex12,13, and not all growth factors cross-react between species. Primate models are a closer alternative that better mimic the human system and have also yielded important preclinical results14. Even these more closely related relatives, however, retain important differences in their cellular makeup15. While both of these model systems provide valuable insights into cell behavior during transplant and incorporate the surgical elements of an eventual therapy, they remain imperfect. They are also costly and technically challenging (i.e., one must perform brain surgery on the animals), thus limiting the possible throughput. Moreover, there are a plethora of ethical issues associated with transplanting human brain cells into animals16. Brain slice cultures allow one brain to be cut and used for multiple treatments, thus removing some of the limitations of animal transplants; however, these have limited lifespans (weeks), are still animal-derived, and (being a thin slice) do not have sufficient volume/surface integrity to mimic the injection of cells17. Thus, there remains an important gap between strictly cell culture/potential models and in vivo transplantation.
Cerebral organoids are an in vitro model containing the main neural cell types present in the brain and can be generated in high numbers from human induced pluripotent stem cells (iPSCs)18,19. Such organoids thus provide a cellular context, which could allow the assessment of the functional capacity of a test cell of interest in the transplant setting. Indeed, a recent study demonstrated that neural progenitor cells (NPCs) transplanted into human cerebral organoids survive, proliferate, and differentiate similarly to NPCs transplanted into the brain of a non-obese diabetic severe combined immunodeficient gamma (NSG) mouse20. Cerebral organoids thus represent a cruelty-free, long-lived (&#62;6 months), cost-effective system that captures the cell types of the human brain. As such, they could represent an ideal transplant recipient for the early-stage testing of the regenerative capacity of neural cells.
This paper presents a protocol for the transplantation and subsequent tracking of labeled human NPCs into human cerebral organoids (Figure 1). This begins with the injection of GFP-labeled NPCs into mature (2-4 months old) cerebral organoids18. The transplanted cells are then followed by live-cell fluorescence microscopy over a 4 month period. During this time, we show both the persistence of cells at the injection site but also migration to distal regions of the organoid. At the endpoint, we demonstrate the antigen retrieval, staining, and imaging of histological sections derived from these organoids, including a protocol for the quenching of existing AlexaFluor-based dyes to allow additional staining and imaging rounds, based on previous work21. This protocol could, thus, be useful in the measurement of the differentiation capacity of cells in a transplant setting, graft durability, cell expansion in situ, and cell migration from the site of the transplant. We anticipate that this will be useful both for regenerative medicine/cell therapy applications, as well as tumor modeling by engrafting tumor cells into relevant region-specific organoids.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>StemCell Technologies</td>
			<td>7920</td>
			<td>proteolytic-collagenolytic enzyme mix</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-GFP Antibody</td>
			<td>BioLegend</td>
			<td>338008</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-MAP2 (clone SMI 52)</td>
			<td>BioLegend</td>
			<td>801804</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 anti-GFAP Antibody (clone SMI 25)</td>
			<td>BioLegend</td>
			<td>837510</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 anti-Nestin (clone 10C2)</td>
			<td>BioLegend</td>
			<td>656804</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-Tubulin &#946; 3 (TUBB3) (clone TUJ1)</td>
			<td>BioLegend</td>
			<td>801209</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid Monohydrate</td>
			<td>Fisher Chemical</td>
			<td>A104-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 Cell Imaging Multimode Reader</td>
			<td>Biotek</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Denaturated Ethyl Alcohol (Anhydrous)</td>
			<td>ChapTec</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12/Glutamax</td>
			<td>Thermo</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dymethil Sulfoxide (DMSO), Sterile</td>
			<td>BioShop</td>
			<td>DMS666.100</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI 1.53c</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Sigma</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5&#160;</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoCore Arcadia H</td>
			<td>Leica Biosystems</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Phenol Red-free, LDEV-free</td>
		</tr>
		<tr>
			<td>MX35 microtome blade&#160;</td>
			<td>Epredia&#160;</td>
			<td>3053835</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (-Ca -Mg)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin Dihydrochloride</td>
			<td>Thermo</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>Abcam</td>
			<td>ab120129</td>
			<td></td>
		</tr>
		<tr>
			<td>Simport Scientific&#160;Stainless-Steel Base Molds</td>
			<td>Fisher Scientific</td>
			<td>22-038-209</td>
			<td></td>
		</tr>
		<tr>
			<td>Simport Scientific&#160;UNISETTE Biopsy Processing/Embedding Cassette</td>
			<td>Fisher Scientific</td>
			<td>36-101-9255</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Forebrain Neuron Differentiation Kit</td>
			<td>StemCell Technologies</td>
			<td>8600</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Medium</td>
			<td>StemCell Technologies</td>
			<td>5833</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff SMADi Neural Induction Kit</td>
			<td>StemCell Technologies</td>
			<td>8581</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Shandon Finesse ME Microtome</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Prep</td>
			<td>Fisher Scientific</td>
			<td>T555</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek VIP 6&#160; AI Tissue Processor</td>
			<td>Sakura Finetek</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene (histological)</td>
			<td>ChapTec</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue; 0.4% (wt/vol)</td>
			<td>Thermo</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BioShop</td>
			<td>TWN510.100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a human cerebral organoid model of neural cell transplantation

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. 
We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.

As a validation of the cerebral organoid identity, histological sections of a mature (2 month old) cerebral organoid were stained for PAX6 (a marker of dorsal NPCs23) and SATB2 (a marker of mature, postmitotic, upper-layer neurons24). As expected, PAX6+ cells were present at the interior of the organoid, and SATB2+ cells were present in the upper layers (Figure 2). These results support that the cerebral organoids used were indeed dorsal forebrain as specified in the differentiation kit. To establish the dose-dependence of the cerebral organoid transplant system, 2 month old cerebral organoids were injected with increasing numbers of EGFP+ iPSC-derived NPCs. A clear dose-dependence of GFP fluorescence on input cell number was present, with consistent EGFP+ cell patch detection at 10,000 cells and above (Figure 3). The persistence and migration of the transplanted NPCs were next assessed by following the transplanted organoids over time. For this, 50,000 iPSC-derived EGFP+ NPCs were transplanted into 2-3 month old cerebral organoids generated from the same iPSC line. The injected organoids and controls were imaged for EGFP positivity at indicated timepoints over the next 3-4 months. In this transplantation series, we observed persistence of the injected site throughout the 4 month tracking period (Figure 4A). Additional EGFP+ cell patches appeared by 9 days post transplantation and persisted until the study endpoint (3-4 months depending on the organoid), indicating the migration of the cells and integration at their new sites (Figure 4A). At a higher magnification, clear neural morphology was observable with long projections into the organoid (Figure 4B), confirming the integration of the injected cells.
To determine the differentiation status of the injected cells late post injection, the 4 month tracked organoid and its control were fixed, paraffin-embedded, cut to 15 &#181;m thick slices, and mounted onto glass slides. The slices were then processed and stained in either a single round of fluorescent staining (EGFP, TUJ1, NESTIN) or for two consecutive staining cycles to add additional markers (MAP2, GFAP). The initial single-round staining confirmed the presence of EGFP+ cells at the injection site, including a mixture of cells retaining NPC status (NESTIN+TUJ1&#8722;) and those that had differentiated towards a neural fate (NESTIN&#8722;TUJ1+) (Figure 5). For both the control and injected organoids, very few NESTIN+ NPCs were observed (most, though not all being EGFP+ transplanted NPCs at the site of injection), with a majority of TUJ1+ immature-mature neurons (Figure 5). The two-round staining gave more detail, revealing mature neurons (NESTIN&#8722;TUJ1+MAP2+GFAP&#8722;) around most of the outer region of the organoid, with areas of immature (NESTIN&#8722;TUJ1+MAP2&#8722;GFAP&#8722;) neurons toward the middle (Figure 6A,B). Astrocytes (NESTIN&#8722;TUJ1&#8722;MAP2&#8722;GFAP+) were present in both the injected and control organoids and were interspersed around the outer edges (Figure 6A,B). The slice for which the two-round staining was performed in the injected organoid showed a small satellite colony of EGFP+ cells far from the injection site that had adopted the phenotype of mature neurons (Figure 6B,C). Some of these appeared to be in close proximity to astrocytes; however, there were no EGFP+ cells with complete overlap to the GFAP staining, suggesting they were adjacent rather than generating the astrocytes themselves (Figure 6B,C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64770/64770fig01.jpg" /> 
Figure 1: Transplantation model of labeled cells into cerebral organoids. Schematic overview of the generation of labeled cells by lentiviral transduction, their transplantation into cerebral organoids, and tracking by live-cell imaging and immunofluorescence. Abbreviation: GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64770/64770fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64770/64770fig02.jpg" /> 
Figure 2: Immunofluorescence of histological sections showing the architecture of early and late organoids. A 2 month old cerebral organoid was fixed, paraffin-embedded, sliced, and stained with PAX6, SATB2, and DAPI. An unstained section was used to set the exposure and integration time to avoid a false-positive signal from autofluorescence. PAX6+ cells were present at the interior of the organoid, while SATB2+ cells were present in the upper layers. Z-stack images were taken every 4.2 &#181;m through the whole 15 &#181;m tissue section. Optical sections were combined using the focus stacking option in the Gen5 software with default options. Scale bars = 100 &#181;m. Abbreviations: PAX6 = paired box 6 protein; SATB2 = special AT-rich sequence-binding protein 2; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www.jove.com/files/ftp_upload/64770/64770fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64770/64770fig3v2.jpg" /> 
Figure 3: Dose-dependent engraftment of NPCs into cerebral organoids. The organoids were transplanted with 0 (negative control), 1,000, 5,000, 10,000, or 50,000 GFP+ iPSC-derived NPCs. At 1 week post transplantation, the organoids were imaged on a Cytation 5 with a GFP filter cube. The negative control was used to set the exposure and integration time to minimize autofluorescence. Darker colors indicate more EGFP fluorescence relative to the negative control. Scale bars = 100 &#181;m. These are 4x images of the whole organoids. After imaging, rolling-ball background subtraction with a pixel radius of 50 was performed prior to display to correct for the variable background intensity across the organoids. Injection sites identified as the regions of highest engraftment are indicated with a red arrow where engraftment was present. Abbreviations: NPCs = neural progenitor cells; GFP = green fluorescent protein; EGFP = enhanced GFP; iPSC = induced pluripotent stem cell. <a href="https://www.jove.com/files/ftp_upload/64770/64770fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64770/64770fig4v2.jpg" /> 
Figure 4: Tracking of transplanted cell growth, migration, and persistence with fluorescence live-cell imaging. (A)&#160;Control and transplanted (50,000 GFP+ iPSC-derived NPCs) organoids were followed by fluorescence live-cell imaging over the course of 2-4 months from two independent transplant sets. Negative control organoids were used to set the exposure and integration time to minimize the autofluorescence at each time point. EGFP images were captured using a GFP filter cube on the Cytation 5 at indicated times post transplant. Darker colors indicate more EGFP fluorescence relative to the negative control for that time point. Rolling-ball background subtraction with a pixel radius of 50 was performed prior to display to correct for the variable background intensity across the organoids. The organoids were placed in approximately the same orientation at each time point, and the images were rotated for consistency of the display and to clearly show the transplanted cell growth. The injection sites identified as the regions of highest engraftment at the earliest time point are indicated with a red arrow. An example negative control organoid is shown in the bottom of the figure. (B) Example 20x images are shown from engrafted organoids at week 1 and week 15 post transplantation. The local contrast was enhanced prior to display using FIJI to ensure neurite visibility. Scale bars = 100 &#181;m. Abbreviations: NPCs = neural progenitor cells; GFP = green fluorescent protein; EGFP = enhanced GFP; iPSC = induced pluripotent stem cell. <a href="https://www.jove.com/files/ftp_upload/64770/64770fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64770/64770fig05.jpg" /> 
Figure 5: Immunofluorescence of the histological sections revealing the persistence of the transplanted NPCs at the injection site along with migration and neural differentiation. Single-channel fluorescence images from (A) non-injected and (B) transplanted organoids. The overlayed image on the right shows the three channels of interest (NESTIN, TUJ1, and EGFP), but does not include DAPI. The display minimums were set to just exclude the signal from the negative cells (determined for EGFP from the non-injected control and for other channels based on known marker combinations). The display maximums were based on the highest signal observed for that antibody in any cell. The display ranges were kept constant between the non-injected and transplanted organoids to allow for direct comparison. Scale bars = 100 &#181;m. Abbreviations: NPCs = neural progenitor cells; DAPI = 4&#39;,6-diamidino-2-phenylindole; TUJ1 = beta-III tubulin; EGFP = enhanced green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64770/64770fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64770/64770fig6v2.jpg" /> 
Figure 6: Assessment of engrafted cell differentiation state and localization&#160;using cyclic immunofluorescence of the histological sections. Single-channel fluorescence images from the non-injected, (A) age-matched, and (B) transplanted organoids are shown for the first and second round of staining as indicated. The display minimums were set to just exclude the signal from the negative cells (determined for EGFP from the non-injected control and for other channels based on known marker combinations). The display maximums were based on the highest signal observed for that antibody in any cell. The display ranges (and of course, the imaging parameters) were kept constant between the control and injected organoids to allow for direct comparison. Scale bars = 100 &#181;m. An overlayed image (excluding DAPI) is shown for each staining round for each organoid on the right. (B) For the injected organoid where image registration was performed, all images are cropped to the region observed in both the staining rounds. (B,C) For the injected organoid, the EGFP+ cell regions are outlined in blue. A diagram of how DAPI is used to match the features during image registration is shown in (C), followed by an overall merge of the registered image. Abbreviations: DAPI = 4&#39;,6-diamidino-2-phenylindole; TUJ1 = beta-III tubulin; EGFP = enhanced green fluorescent protein; MAP2 = microtubule-associated protein 2; GFAP = glial fibrillary acidic protein. <a href="https://www.jove.com/files/ftp_upload/64770/64770fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Clone</td>
			<td>Fluorophore</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>Anti-NESTIN</td>
			<td>10C2</td>
			<td>AlexaFluor 594</td>
			<td>1 in 2,000</td>
		</tr>
		<tr>
			<td>Anti-TUBB3</td>
			<td>TUJ1</td>
			<td>AlexaFluor 647</td>
			<td>1 in 2,000</td>
		</tr>
		<tr>
			<td>Anti-GFP</td>
			<td>FM264G</td>
			<td>AlexaFluor 488</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Anti-GFAP</td>
			<td>SMI 25</td>
			<td>AlexaFluor 594</td>
			<td>1 in 500</td>
		</tr>
		<tr>
			<td>Anti-MAP2</td>
			<td>SMI 52</td>
			<td>AlexaFluor 488</td>
			<td>1 in 1,000</td>
		</tr>
		<tr>
			<td>Anti-PAX6</td>
			<td>O18-1330</td>
			<td>AlexaFluor 647</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>Anti-SATB2</td>
			<td>EPNCIR130A</td>
			<td>AlexaFluor 594</td>
			<td>1 in 500</td>
		</tr>
	</tbody>
</table>
Table 1: Antibody concentrations for staining. Abbreviations: TUBB3 = beta-tubulin III; GFP = green fluorescent protein; GFAP = glial fibrillary acidic protein; MAP2 = microtubule-associated protein 2; PAX6 = paired box 6 protein; SATB2 = special AT-rich sequence-binding protein 2.

Watch this Scientific Journal Video about A Human Cerebral Organoid Model of Neural Cell Transplantation at JoVE.com

A Human Cerebral Organoid Model of Neural Cell Transplantation

Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For ...

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Neuroscience

982 Views.  Université de Montréal. Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

Video: A Human Cerebral Organoid Model of Neural Cell Transplantation

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...