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 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.
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 (>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.
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 authors have nothing to disclose.
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 Québec – Santé (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 accélère de la maitrise au doctorat from the University of Montreal, and Bourse de Mérite aux cycles supérieurs.
Accutase | StemCell Technologies | 7920 | proteolytic-collagenolytic enzyme mix |
Alexa Fluor 488 anti-GFP Antibody | BioLegend | 338008 | |
Alexa Fluor 488 anti-MAP2 (clone SMI 52) | BioLegend | 801804 | |
Alexa Fluor 594 anti-GFAP Antibody (clone SMI 25) | BioLegend | 837510 | |
Alexa Fluor 594 anti-Nestin (clone 10C2) | BioLegend | 656804 | |
Alexa Fluor 647 anti-Tubulin β 3 (TUBB3) (clone TUJ1) | BioLegend | 801209 | |
Citric Acid Monohydrate | Fisher Chemical | A104-500 | |
Cytation 5 Cell Imaging Multimode Reader | Biotek | – | |
Denaturated Ethyl Alcohol (Anhydrous) | ChapTec | – | |
DMEM F12/Glutamax | Thermo | 10565018 | |
Dymethil Sulfoxide (DMSO), Sterile | BioShop | DMS666.100 | |
FIJI 1.53c | – | – | |
Formalin solution, neutral buffered, 10% | Sigma | HT501128-4L | |
Gen5 | – | – | |
HistoCore Arcadia H | Leica Biosystems | – | |
Matrigel Growth Factor Reduced (GFR) | Corning | 356231 | Phenol Red-free, LDEV-free |
MX35 microtome blade | Epredia | 3053835 | |
NaOH | Sigma | 655104 | |
PBS (-Ca -Mg) | Sigma | D8537 | |
Puromycin Dihydrochloride | Thermo | A1113803 | |
ROCK inhibitor Y-27632 | Abcam | ab120129 | |
Simport Scientific Stainless-Steel Base Molds | Fisher Scientific | 22-038-209 | |
Simport Scientific UNISETTE Biopsy Processing/Embedding Cassette | Fisher Scientific | 36-101-9255 | |
STEMdiff Forebrain Neuron Differentiation Kit | StemCell Technologies | 8600 | |
STEMdiff Neural Progenitor Medium | StemCell Technologies | 5833 | |
STEMdiff SMADi Neural Induction Kit | StemCell Technologies | 8581 | |
Thermo Scientific Shandon Finesse ME Microtome | Thermo Scientific | – | |
Tissue Prep | Fisher Scientific | T555 | |
Tissue-Tek VIP 6 AI Tissue Processor | Sakura Finetek | – | |
Toluene (histological) | ChapTec | – | |
Trypan blue; 0.4% (wt/vol) | Thermo | 15250061 | |
Tween 20 | BioShop | TWN510.100 |