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>
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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><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 &amp;beta; 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&amp;nbsp;</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&amp;nbsp;</td><td>Epredia&amp;nbsp;</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&amp;nbsp;Stainless-Steel Base Molds</td><td>Fisher Scientific</td><td>22-038-209</td><td></td></tr><tr><td>Simport Scientific&amp;nbsp;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&amp;nbsp; 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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

1.1K 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

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 ...

Developmental Biology

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

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 ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

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 ...

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

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.

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.

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

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Bioengineering

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

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Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

A Human Cerebral Organoid Model of Neural Cell Transplantation

Summary

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