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 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.
Our current knowledge of the human brain has largely been based on animal models and post-mortem brain specimens. Stem cell biology is a rapidly advancing field that provides new insights into the basic biology of human brain development and the pathological drivers of human brain disorders. Human pluripotent stem cells (hPSCs) are an invaluable tool for modeling the human brain via the generation of organoids, organ-like three-dimensional (3D) tissue that typically recapitulates the developmental trajectories, cellular make-up, and architecture of the developing human brain. Brain organoids are self-assembled and composed of neural stem cells, specified neural progenitors, mature neurons, and glial cell types. Organoids, therefore, provide a unique opportunity to study the early human brain, which is often inaccessible for direct experimentation but also has intrinsic limitations such as the absence of vasculature and an immune system.
Methodologies to generate brain organoids have been pursued in two different ways: unguided and guided differentiation. Unguided brain organoid methods rely on the spontaneous intrinsic differentiation capacities of the stem cells that drive tissue morphogenesis1,2 and allow for the emergence of a variety of cell lineage identities ranging from forebrain, midbrain, and hindbrain, to choroid plexus, retina, and mesoderm. In contrast, guided brain organoid methods require substantial use of external factors to drive hPSCs toward the desired patterning of neuronal lineages representing one brain region type, such as medial ganglionic eminence3, forebrain4, midbrain5, hypothalamus6, cerebellum7, and choroid plexus8. This ability to generate different brain regions with different cell lineages, and the potential to fuse these at will, makes brain organoids an excellent model for investigating human brain development and deciphering the underlying mechanisms of brain-related diseases. Although these methods for generating brain organoids offer a breakthrough in modeling human brain regions, the variability and heterogeneity between organoids remain a significant limitation for systematic and quantitative studies, such as drug screening.
The current protocol is based on a method developed in our recent paper9 and involves the selective differentiation of hPSC colonies toward neuroectoderm (NEct) identity with dual SMAD inhibitors (SB-431542 and LDN 193189), which then have the ability to self-organize within 4 days into 3D neuroepithelium spheroids under the influence of FGF2 signaling. These neuroepithelium spheroids reliably generate homogenous cortical organoids with an in vivo-like cellular composition within 4 weeks of differentiation. The protocol described here is built on our previous findings showing that inhibition of dual SMAD (Suppressor of Mothers Against Decapentaplegic) signaling promotes the differentiation of hPSCs toward rostral neural stem cells derived from neuroectodermal progenitors10 by, among others, inhibiting endodermal, mesodermal, and trophectoderm cell fate choice11. Furthermore, the embedding of the neuroepithelium spheroids in the hESC-qualified basement membrane matrix triggers significant budding of the neuroepithelia, forming ventricles with apicobasal polarity. Large-scale culture showed reproducibility and homogeneity of cortical organoids independent of cell lines, clones, or batches, and thus represents a reliable and stable stem cell system to mimic early human cortical development in health and disease in vitro. We further outline a protocol for detecting senescent neuronal cell markers in hPSCs-derived cortical brain organoids that have been cultured for prolonged periods of time.
After plating hPSCs at a seeding density of 20%-30%, the cells are treated with dual SMAD inhibitors for 3 days to differentiate hPSC colonies toward neuroectodermal colonies. These colonies are then gently lifted with dispase and seeded into ultra-low attachment 6-well plates supplemented with FGF2. The floating 2D colonies self-organize into 3D neuroectodermal spheroids overnight and are maintained for 4 days in N2 medium supplemented daily with FGF2. Once the spheroids have established the neuroepithelial layer, they can be embedded in the basement membrane matrix. By routinely adding fresh terminal differentiation medium, the researchers will observe progressive expansion and budding of neuroepithelia in cortical organoids. Researchers may wish to dissociate these organoids to conduct transcriptional and proteomic profiling. Additionally, brightfield imaging is recommended for monitoring the quality of the cortical organoids. Analysis can be performed by fixation, cryosection, and immunostaining. Descriptions and methods for these techniques have been previously described12. Ultimately, this protocol allows researchers to rapidly and robustly generate homogeneous cortical brain organoids for modeling the developing human cortical brain, with low cost and limited equipment, and for studying aspects of cellular neuronal senescence, as outlined in this paper.
To enable the use of hPSC-derived brain organoids in drug screening and disease modeling, it is crucial to make organoids following a replicable and reliable protocol15. Brain organoids are commonly generated from embryoid bodies derived from hPSCs, which are then embedded in an extracellular matrix that promotes tissue expansion and neural differentiation. When compared to such protocols as Lancaster's1,16,17 and Velasco18, which begin from embryoid bodies and allow for a default differentiation pathway to be followed by the developing organoids, we have found that commencing cortical brain organoid creation with human NEct cells rather than with embryoid bodies improves the consistency of cortical brain organoid formation. This consequently also allows for the scaling required for drug and phenotypic screening. Since human NEct cells can not only be expanded into considerable quantities but can also be readily cryopreserved, this approach also improves replicability between experiments. It should also be noted that, compared to other protocols that have adopted the use of Bioreactors and similar technologies, no specialized equipment is required for this protocol, making it suitable for any lab6. Finally, the time required to generate mature organoids that are positive for cortical layer markers such as SATB2 is reduced compared to both Lancaster1 and bioreactor protocols6,19 making it more suitable for studying the developmental trajectory of human cortical development in health and diseases1,6,16.
Furthermore, given the continuously growing global health care impact of aging-related diseases such as dementia, which are associated with an increase in senescent cell types in the brain that contributes to pathogenesis, the ability to identify and test compounds that can ameliorate brain aging are of enormous interest. Despite hPSCs being known to be epigenetically rejuvenated during the reprogramming process20, we find robust increases in senescent cells in cortical brain organoids cultured for prolonged periods of time. This is a promising development that now enables the screening of drugs that eliminate such senescent cells from the brain (senolytics) or that slow this process down (senostatics)21. Since human NEct-derived cortical brain organoids are of human origin, this approach will likely shorten the traditional path to market such novel therapeutics.
There are two critical steps in this protocol. The first is the correct level of confluency of the hPSC colonies at the time of differentiation. hPSC colonies must be at most 30% confluent to ensure that generated NEct colonies do not fuse with neighboring colonies and that individual organoids are clonally driven. The second critical step involves the correct use of dispase to lift the NEct colonies and produce the neural spheroids. The timing of incubation with dispase is critical to the eventual quality of the neural spheroids generated. This is because over-exposure of colonies with dispase is toxic to the cells22 and eventually affects the quality of generated organoids. The limitation of this protocol is that it is difficult to control the size of the neural spheroids because it is dependent on the size of the initial colonies that are lifted with dispase. However, this issue can be overcome by selecting neural spheroids that are of a similar size when proceeding to the embedding stage.
Finally, future applications could extend to the use of these reproducible cortical organoids in robotic analysis and biopharmaceutical screening approaches typically used in that industry. This is supported by preliminary data from our laboratory indicating that the generation of cortical brain organoids from human NEct cells can be readily automated, making it compatible with these approaches.
The authors have nothing to disclose.
This work is supported by the Medical Research Future Fund-Accelerated Research, Leukodystrophy flagship Massimo's Mission (EPCD000034), Medical Research Future Fund-Stem Cell Mission (APP2007653). Authors would like to thank Dr. Ju-Hyun Lee (Korea University) for generating data in Supplementary Video 1.
16% Formaldehyde (W/V) Methanol-free | Thermo Fisher Scientific | 28908 | 4% of PFA are diluted in 1x PBS |
2-Mercaptoethanol 50 mL(1000x) | Life Technologies Australia (TFS) | 21985023 | Used in NM and DM media |
B 27 Supplement 10 mL | Life Technologies Australia (TFS) | 17504044 | Used in NM and DM media |
CKX53 microscope with SC50 camera | Olympus | ||
Corning Costar 6 well cell culture plates | Sigma Aldrich Pty Ltd | CLS3516-50EA | |
Dispase II powder | Thermo Fisher Scientific | 17105041 | Powder is dissolve in HBSS, filtered through 0.22 µm filter, aliquote at 10 mL and store at -20 °C |
DMEM Nutrient Mix F12 10x 500 mL (DMEM/F-12) | Thermofisher | 11320082 | Used in NM and DM media |
DMSO Dimethyl Sulfoxide | Sigma Aldrich Pty Ltd | D2650-100ML | |
Dulbecco's Phosphate Buffered Saline | Sigma Aldrich Pty Ltd | D1408-500ML | |
Falcon Matrigel hESC-qualified Matrix | In Vitro Technologies Pty Ltd | FAL354277 | Make aliquotes of 100 µL and stored at -20 °C |
GlutaMAX Supplement 100x | Thermo Fisher Scientific | 35050061 | Used in NM and DM media |
Hanks Balanced Salt Solution | Sigma Aldrich Pty Ltd | H8264 | |
Human induced pluripotent stem cells (EU79) | In-house reporogrammed from skin fibroblast | ||
Human induced pluripotent stem cells (G22) | Genea Biocells | Obtained from Genea Biocells (San Diego, United States) | |
Human induced pluripotent stem cells (WTC) | Gift from Professor Bruce Conklin | ||
InSolution TGF-Β RI Kinase Inhibitor VI, SB431542 | Merck | US1616464-5MG | |
Insulin Solution Human Recombinant | Sigma Aldrich Pty Ltd | I9278 | Used in NM and DM media |
LDN193189 Dihydrochloride | Sigma Aldrich Pty Ltd | SML0559-5MG | Used during differentiation |
MEM Non-Essential Amino Acids Solution (100x) | Thermo Fisher Scientific | 11140050 | Used in NM and DM media |
mTeSR Plus | STEMCELL TECHNOLOGIES | 100-0276 | Used to maintain hiPSC colonies prior to differentiation with NM media |
N2 Supplement 5 mL (100x) | Life Technologies Australia Pty Ltd | 17502048 | Used in NM and DM media |
Neurobasal Medium | Thermo Fisher Scientific | 21103049 | Used in DM media |
OCT Embedding Compound Sakura Clear (118 mL/Bottle) | Tissue Tek | 4583 | |
Parafilm M Roll Size 4 in. x 125 Ft | Sigma Aldrich Pty Ltd | P7793 | |
Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | Used in NM and DM media |
Potassium Hexacyanoferrate (II) Trihydrate | Sigma Aldrich Pty Ltd | CP1087 | |
Potassium hexacyanoferrate(III) | Sigma Aldrich Pty Ltd | 455946 | |
Prolong Glass Antifade Mountant | Life Technologies Australia (TFS) | P36980 | |
Recombinant Human FGF basic | R&D Systems | 233-FB-01M | Aliquotes are made at 20 µg/mL and stored at -20 °C |
SB431542 | Tocris | 1614 | Used during differentiation |
Sucrose | Sigma Aldrich Pty Ltd | PHR1001-1G | 30% of sucrose are diluted in 1x PBS |
Ultra-Low attachment multiwell plates , 24 well plate, polystyrene | Sigma Aldrich Pty Ltd | CLS3473-24EA | |
X-GAL EA | Life Technologies Australia (TFS) | R0404 | Make aliquotes of 20 mg/mL and storde at -80 °C |