Name: A Human Cerebral Organoid Model of Neural Cell Transplantation (Video) | JoVE Journal
Uploaded: 2023-07-21T12:50:00.000+00:00
Duration: 8 min 58 s
Description: Learn neural cell transplantation in human cerebral organoids using iPSC-derived progenitors and fluorescence tracking. This protocol enables long-term assessment of engraftment, migration, and integration for cell therapy research.

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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.

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</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.

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a human cerebral organoid model of neural cell transplantation

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 µm 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−) and those that had differentiated towards a neural fate (NESTIN−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−TUJ1+MAP2+GFAP−) around most of the outer region of the organoid, with areas of immature (NESTIN−TUJ1+MAP2−GFAP−) neurons toward the middle (Figure 6A,B). Astrocytes (NESTIN−TUJ1−MAP2−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="Dorsal forebrain organoids: GFP-cells injection, microscopy, live-cell imaging, immunofluorescence." class="xfigimg" src="/files/ftp_upload/64770/64770fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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="Fluorescence microscopy image; DNA, PAX6, SATB2 staining; cell marker colocalization analysis." class="xfigimg" src="/files/ftp_upload/64770/64770fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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 µm through the whole 15 µm tissue section. Optical sections were combined using the focus stacking option in the Gen5 software with default options. Scale bars = 100 µm. Abbreviations: PAX6 = paired box 6 protein; SATB2 = special AT-rich sequence-binding protein 2; DAPI = 4',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="Cell count effect on stain uptake; microscopic images showing varying cell densities, experimental results." class="xfigimg" src="/files/ftp_upload/64770/64770fig3v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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 µm. 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="Cell transplantation results showing transplanted cell growth over weeks; microscopy images, 100 µm scale." class="xfigimg" src="/files/ftp_upload/64770/64770fig4v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Tracking of transplanted cell growth, migration, and persistence with fluorescence live-cell imaging. (A) 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 µm. 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="Fluorescence microscopy of organoid injection; DNA, GFP, NESTIN, TUJ1 markers; comparative analysis." class="xfigimg" src="/files/ftp_upload/64770/64770fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
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 µm. Abbreviations: NPCs = neural progenitor cells; DAPI = 4',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="Immunofluorescence microscopy DNA, GFP, MAP2, GFAP; image registration diagram; cellular analysis." class="xfigimg" src="/files/ftp_upload/64770/64770fig6v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Assessment of engrafted cell differentiation state and localization 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 µm. 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',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.

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

Source: Palma-Tortosa, S., et.al. Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation. J. Vis. Exp. (2022)This video demonstrates the transplantation of stem cell-derived neuronal progenitor cells into human cortical tissue slices. The procedure involves isolating and resuspending progenitor cells in a basement membrane matrix, followed by their injection into a cortical slice placed on a transwell assembly. After incubation to allow...

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Injection of Fluorescent Neural Progenitor Cells into Human Cerebral Organoids for Transplantation Modeling

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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.

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Learn neural cell transplantation in human cerebral organoids using iPSC-derived progenitors and fluorescence tracking. This protocol enables long-term assessment of engraftment, migration, and integration for cell therapy research.

A Human Cerebral Organoid Model of Neural Cell Transplantation

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