This study introduces and describes protocols to derive two specific human neural organoids 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 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.
The World Health Organization (WHO) classifies astrocytomas as low grade (grade I to II) or high grade (grade III and IV). Glioblastoma multiforme (GBM) is an astrocytoma grade IV, the most lethal of primary brain tumors, that is resistant to all current forms of treatments1. Despite standard-of-care therapy including neurosurgery, chemotherapy, and radiotherapy, GBM remains fatal and the 15-month overall survival rate has not dramatically changed over the past 15 years2. To make significant progress in understanding GBM pathogenesis, the use of relevant models is key. So far, the study of GBM has relied on cell lines, rodent organotypic slices, and xenotransplantation of patient-derived cells into mice or transgenic mice developing spontaneous tumors3,4. Although these models have been useful to study brain metastasis and tumor aggressiveness, they are restricted by differences among species, and resulting conclusions may be incorrectly translated to human tissues. Moreover, existing models with human cells are also limited by the absence of host tissue/tumor interactions3,4. Experimental models are critical for the translation from basic science to therapeutic targets. Therefore, describing a protocol to produce in vitro human neural organoids co-cultured with GBM-initiating cells (GICs) can provide a relevant system that mimics morphological and functional features of GBM development. This system reproduces some in vivo features of GBM developmentsuch as diffuse migration of invading cells and necrosis areas, and it highlights gene expression relevant to tumor biology. As previously revealed, some critical microRNAs are induced during GIC development within 3D nervous tissue5,6.
PD is a major neurodegenerative disorder and associated with the degeneration of multiple neuronal subtypes7. Even if a progressive onset of symptoms (e.g., bradykinesia, asymmetric rest tremor, rigidity and posture instability) characterizes the disease, its exact etiology is not clearly established. Indeed, many studies have highlighted evidence that major risk factors can result from a combination of genetic and environmental factors. Parkinsonian symptoms are associated with the bilateral degeneration of dopaminergic neurons in the substancia nigra (SN), leading to the disappearance of dopaminergic (DA) axons projecting to the striatum8,9. Therefore, the reduction of striatal dopamine levels is correlated with progression of motor dysfunction in PD patients. Dopaminergic neurons contain tyrosine hydroxylase (TH), a key enzyme in the synthesis of catecholaminergic neurotransmitters that converts the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA, a dopamine precursor) to dopamine10. Early loss of TH activity followed by a decline in TH protein expression is a hallmark of PD.
This study describes two protocols using human neural organoids, with one specifically oriented towards a midbrain-like phenotype enriched with TH-positive cells.
This protocol follows the guidelines of University of Geneva's human research ethics committee.
1. Maintenance and culture of undifferentiated human embryonic stem cells (hESCs)
2. hESC-derived neural organoids for GBM studies
3. Isolation and cultivation of glioblastoma-initiating cells (GICs)
4. hESC-derived dopaminergic organoids for PD studies
5. Quantification of TH and Nurr1 gene expression for validation of dopaminergic differentiation
6. High pressure liquid chromatography (HPLC) detection
7. Raw data recording with microelectode array (MEA) platform
The critical steps of this protocol must be well-identified and handled properly. Therefore, a diagram of culture conditions indicating the time-lapse for each step as well as the compounds used for the differentiation protocol are illustrated in Figure 1A and Figure 3A for NO plus GBM and DA neural organoids, respectively. Figure 1B,C,D,E,F illustrates the cells, spheres, and NO and show the typical morphology for each step. Figure 1G,H,I illustrates immunofluorescence staining with some neural markers.
Figure 1: Human neural organoid (NO) differentiation protocol. (A) Standardized protocol for the generation of NO derived from human embryonic stem cells (hESC). (B) hESCs are maintained on extracellular matrix in hESC medium. (C) Microwell plates were used to generate calibrated neurospheres. At 2 weeks, neurospheres were plated onto the insert containing a PTFE membrane (scale bar = 50 µm). (D) Macroscopic view of NO into the insert in one well of a 6 well plate. During the first days, rosettes were observed (black arrow) (E). (F) Macroscopic view of a NO plus GIC sphere on the top. (G–I) Immunofluorescence analysis of NO plus GIC sphere (EGFR-positive; scale bar = 50 µm) (G) and NO alone, which showed immune reactivity for the neuronal marker βIII-tubulin and slightly positive for nestin; however, synapsin 1 showed a weak signal (H,I) (scale bars = 100 µm and 50 µm, respectively). Please click here to view a larger version of this figure.
Figure 2: Illustration of necrotic spheres and immature NO. The neurospheres (A) and NO (B) can undergo necrosis when they are too numerous in the well or oversized (C) (scale bar = 10 µm). (D) One GIC infected with a tomato reporter help to track tumor cell invasion in NO, scale bar, 10 µm. Example of immature NO with neural tubes (E) and no neural tubes (F) (scale bar = 50 µm). Please click here to view a larger version of this figure.
Figure 3: Standardized protocol for generation of DA neural organoid and electrophysiological and morphological analysis. (A) Standardized protocol for the generation of DA neural organoids. (B) Immunofluorescence analysis of DA neural organoid; TH-immunoreactive cells co-expressing Nurr1, a midbrain specific marker (scale bar = 50 µm). Data are represented as mean ± SEM (n = 3). (C) Graphs represent kinetics of TH and Nurr1 gene expression evaluated by qRT-PCR. (D) Representative HPLC: dopamine peak (arrow) was detected by HPLC from DA neural organoid lysate. (E) Example of raw data recorded with MEA platform. Each spike is displayed by a vertical line (time stamps), whereas the remaining trace is noise. (F) Picture representing a neurosphere deposited on the MEA. (G) Superposition of typical spikes (blue and red curves) detected from the raw data. The black bold curve indicates the average of the corresponding red curves. (H) Raster plot showing the time stamps associated with each spike detected. The different colors highlight the different electrodes. Please click here to view a larger version of this figure.
Gene | Foward | Reverse |
Nurr1 | GGCTGAAGCCATGCCTTGT | GTGAGGTCCATGCTAAACTTGACA |
TH | GCACCTTCGCGCAGTTCT | CCCGAACTCCACCGTGAA |
EEF1 | AGCAAAAATGACCCACCAATG | GCCTGGATGGTTCAGGATA |
GAPDH | GCACAAGAGGAAAGAGAGAGAACC | AGGGGAGATTCAGTGTGGGT |
Table 1: Primers used in this protocol.
One of the most critical aspects of this protocol includes the maintenance of hESC pluripotency during cell culturing and close monitoring of the spheres and neural organoid morphology. hESCs are very sensitive, and every manipulation can lead to early uncontrolled differentiation as well as cell death. In order to increase experimental reproducibility and avoid the occurrence of abnormal karyotype events, it is advised to cryopreserve several batches of hESCs at the lowest passage after validation of their chromosome stability. Moreover, it is recommended to thaw a new vial for each experiment and check the behavior of the cells every day. If the spheres are less refractive with abnormal higher size, they will likely start to aggregate and die.
One improvement upon this system is either perfusion or implementing a vascularized system (by adding endothelial cells or within a 3D fluidic microchip)12,13. However, controlling the thickness of the neural organoid (≤300 µm) allows efficient passive perfusion of oxygen and nutriments and prevents necrosis. Another improvement is the introduction of immune cells (microglia). With these limitations in mind, neural organoids plus a GIC system may be a relevant tool for several reasons. First, this system allows drug screening to monitor how a therapeutic compound may affect an organoid or tumor cell. Second, cell-to-cell interactions can be studied, and micro-environmental determinants underlying individual and collective invasions can be visualized and explored5,6,13.
In the context of Parkinson's disease, a neural organoid enriched in DA neurons can represent a relevant and accurate 3D model to study disease development. In previous studies, Parkinson's patient-derived induced pluripotent stem cells differentiated towards DA neurons have been used to study the affected neuronal subtypes. Of note, some disease-related phenotypes such as the accumulation of α-synuclein and sensitivity to oxidative stress have been observed14,15. Moreover, the neural organoid may be used as a tool to screen therapeutic molecules. However, specific and relevant readouts should be set up to evaluate DA neuron survival and functionality, such as dopamine production and electrophysiological activity. Altogether, this protocol provides two standardized and accurate stem cell-based approaches to generate neural organoids.
The authors have nothing to disclose.
The authors thank la Ligue Genevoise Contre le Cancer (Geneva, Switzerland), ISREC Foundation (Lausanne, Switzerland), and the Clayton Foundation for Research (Houston, TX, USA) for financial support. Moreover, the authors thank HES-HO and the Wyss Center for financial support. We thank the Krause's lab for helpful discussions and support and Dr. Halah Kutaish for proofreading.
6-well plate (6-well plate) | Falcon / Corning | 07-201-588 | |
ABI Prism 7900 HT detection system (Real-Time PCR detection systems) | Applied Biosystems | Discontinued | |
Aggrewell 400 (Microwell culture plates ) | StemCell Technologies | 34421 | |
Amplifier (W2100-HS32) (Amplifier) | Multi Channel Systems | ||
Anti-EGFR (phospho Y1101) antibody | Abcam | ab76195 | 1/100 dilution |
Anti-GFAP Antibody | Dako | Z334 | 1/1000 dilution |
Anti-Nestin, Human Antibody | Millipore | ABD69 | 1/400 dilution |
Anti-Synapsin I Antibody | Chemicon | AB1543P | 1/500 dilution |
B27 supplements (B27) | Life Technologies / Invitrogen | 1238 | For both protocol, stock solution 100x, final solution 1x |
Brain-derived neurotrophic factor (BDNF) | Cell Guidance | GFH1-2 | For both protocol, stock solution 100 µg/mL in pure H2O, final solution 20 ng/mL |
CHIR-99021 (GSK-3β inhibitor ) | Axon Medchem | ct99021 | For Dopaminergic protocol, stock solution 7.5 mM in DMSO, final solution 3 µM |
Compound E a γ–secretase inhibitor (γ–secretase inhibitor) | Calbiochem | CAS 209986-17-4 | For both protocol (gamma-secretase inhibitor XXI), stock solution 5 mM in DMSO, final solution 1 µM |
Coulochem III (Coulometric detector parameters) | Thermo scientific | ||
Dibutyryl cyclic-AMP (Dibutyryl cAMP) | Sigma | D0627 | For Dopaminergic protocol, stock solution 0.5 M in DMSO, final solution 0.5 mM |
Dimethyl Sulfoxide Pure (DMSO) | Sigma-Aldrich | C6164 | Compounds solvent, ready to use |
Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 12491-015 | For cell culture, ready to use |
Dulbecco's Modified Eagle Medium Mixture F-12 (DMEM-F12) | Gibco | 11320033 | For cell culture, ready to use |
EDTA 0.1 mM (EDTA) | Life Technologies | AM9912 | For cell culture, ready to use |
Epidermal Growth Factor (EGF) | Gibco | PHG0313 | For GIC culture, stock solution 100 µg/mL in pure H2O, final solution 10 ng/mL |
Fibroblast Growth Factor 20 (FGF20) | Peprotech | 100-41 | For Dopaminergic protocol, stock solution 100 µg/mL in pure H2O, final solution 5 ng/mL |
fibroblast growth factor 8 (FGF8) | Peprotech | GFH176-5 | For Dopaminergic protocol, stock solution 100 µg/mL in pure H2O, final solution 100 ng/mL |
Fibroblast growth factor-basic (bFGF) | Gibco | PHG0024 | For GIC culture, stock solution 100 µg/mL in pure H2O, final solution 10 ng/mL |
G5 supplements (G5) | Invitrogen | 17503012 | For GIC culture, stock solution 100x, final solution 1x |
Glial cell-derived neurotrophic factor (GDNF) | Cell Guidance | GFH2-2 | For both protocol, stock solution 100 µg/mL in pure H2O, final solution 20 ng/mL |
Hydrophilic polytetrafluoroethylene membrane (PTFE membrane) | BioCell-Interface | Discontinued | |
LDN-193189 (BMP inhibitor) | Axon Medchem /Stemgen | 04-0072-02 /1509 | Dual/Smad, stock solution 5 mM in DMSO, final solution 0.5 µM |
L-glutamine (L-glutamine) | Gibco | 25030081 | L-Glutamine (200 mM), stock solution 200 mM, final solution 2 mM |
Matrigel (extracellular matrix) | BD Biosciences | 354277 | hESC-qualified Matrix, stock solution 18-22 mg/mL, final solution 180-220 µg/mL |
Millicell-CM Culture plate insert (0.4 µm) (Culture plate insert) | Millipore | PICM03050 | |
Monoclonal Anti-β-Tubulin III antibody | Sigma | T8660 | 1/1000 dilution |
MS Orbital Shaker, MS-NOR-30 (Orbital shaker) | Major Science | MS-NRC-30 | |
N2 supplements (N2) | Invitrogen | 17502-048 | For GIC culture, stock solution 100x, final solution 1x |
Nanodrop (Nanodrop) | Thermo Fisher Scientific | Discontinued | |
Neurobasal (Neurobasal) | Life Technologies / Gibco | 21103049 | Maintenance and maturation embryonic neuronal cell populations , ready to use |
Non-Essential Amino Acids (NEAA) | Gibco | 11140 | Non-essential Amino Acids 100X, stock solution 100x, final solution 1x |
Nurr1 Antibody (M-196) | Santa Cruz | Sc-5568 | 1/100 dilution |
Nutristem (hESC medium ) | Biological Industries | 05-100-1A | Stem cell media, ready to use |
Penicilin / Streptomycin (Penicilin / Streptomycin ) | Life Technologies / Gibco | 15140122 | For cell culture, stock solution 5 mg/mL, final solution 50 µg/mL |
Perchloric acid 0.1N (HCLO4) | Merck | 100519 | For HPLC, ready to use |
Phosphate Buffered Saline without Ca2+/Mg2+ (PBS without Ca2+/Mg2+ ) | Life Technologies | 14190250 | For cell culture, ready to use |
PrimeScript RT-PCR Kit (Reverse transcription kit) | Takara | RR014A | |
Purmorphamin (smoothened agonist) | Calbiochem | SML0868 | For Dopaminergic protocol, stock solution 10 mM in DMSO, final solution 2 µM |
Rho-associated Kinase Y-27632 (ROCK) | Abcam Biochemicals | ab120129-1 | Rock Inhibitor, stock solution 50 mM in DMSO, final solution 10 µM |
RNeasy mini kit (RNA extraction kit ) | Qiagen | 74104 | |
SB-431542 (TGFβ/Activin/Nodal inhibitor ) | Ascent | Asc- 163 | Dual-Smad, stock solution 50 mM in DMSO, final solution 10 µM |
Sonic Hedgehog (SHH) | Cell Guidance | GFH168-5 | For Dopaminergic protocol, stock solution 100 µg/mL in pure H2O, final solution 100 ng/mL |
StemPro Accutase (hESC enzymatic solution) | Gibco | A11105-01 | hESC enzymatic solution, ready to use |
Symmetry C-18,5 mm (4.6 150mm2) (Reversed-phase column) | Waters Corporation | ||
T150 flask (T150 flask) | Falcon | 08-772-1F | |
TH Antibody (F-11) | Santa Cruz | Sc-25269 | 1/200 dilution |
Transforming Growth Factors beta 3 (TGFβ3) | Cell Guidance | GFH109-2 | For Dopaminergic protocol, stock solution 100 µg/mL in pure ethanol , final solution 1 ng/mL |
Trichostatine A (inhibitor of histone deacetylase ) | Sigma | T8552 | For Dopaminergic protocol, stock solution 100 µM in DMSO, final solution 20 nM |
TrypLE (recombinant enzymatic solution) | Invitrogen | 12604021 | recombinant enzymatic solution, ready to use |
Trypsin 0.25% (enzymatic solution) | Life Technologies | 15050065 | enzymatic solution, ready to use |
W2100, Multi Channel Systems (Data acquisition system ) | WAT045905 | ||
X-vivo (serum free medium) | Lonza | BE04-743Q | serum free medium, ready to use |