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 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'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.
Due to myriad practical and ethical limitations, there has been a great deal of difficulty in studying the human brain. While studies utilizing rodents have been critical to our understanding of the human brain, the mouse brain has many dissimilarities1,2. Interestingly, mice have a neuronal density that is at least 7 times less than the primate brain3,4. Although primates are closer to humans than rodents from an evolutionary standpoint, it is not practical for most researchers to work with them. The purpose of this protocol was to recapitulate many important features of the human brain using a simplified and less expensive method without the need for a heterologous basement membrane matrix or exogenous growth factors while maintaining brain cell diversity and cellular organization.
Formative work from the Sasai lab used the serum free culture of embryoid bodies (SFEBq) method to generate two- and three- dimensional neuronal cell types from signalized embryonic stem cells (ESCs)5,6. Many human brain organoid methods have followed a relatively similar path from signalized ESCs7,8. In contrast, this protocol starts with clusters of detached human ESCs (hESCs), similar to the initial steps of seminal work of the Thomson and Zhang laboratories prior to the plating steps9,10 as well as the initial step of the brain organoid protocol of the Pasca laboratory before the addition of exogenous growth factors11. Basement membrane matrices (e.g., matrigel) have been utilized in many brain organoid protocols and it has been shown to be an effective scaffold8. However, most commonly used basement membrane matrices do not come without complications as they co-purify with unknown quantities of growth factors with batch to batch variability during production12. In addition, these matrices can complicate imaging, and increase the risk of contamination and cost.
While human brain organoids can be used to answer many questions, there are certain limitations to bear in mind. For one, starting from embryonic stem cells, organoids more closely resemble immature brains than aged brains and as such may not be ideal models for diseases that occur in old age, like Alzheimer's disease. Second, while our protocol found markers of forebrain, midbrain and hindbrain development which are useful to study the effect of a treatment or disease on cells from multiple brain regions in concert, other protocols can be followed to concentrate on specific brain regions13,14. Finally, another limitation of organoid models is that of size, while the average length of a human brain approximately 167 mm, brain organoids made with the use of agitation grow up to 4 mm8 and the organoids formed by this protocol grow to 1-2 mm by 10 weeks. Nonetheless, this protocol provides an important tool to study human brain tissue and the interaction of multiple cell types.
1. Stem Cell Maintenance
2. Dissociation of the hESCs for Organoid Culture
3. Generation of Organoids
4. RNA Extraction and Preparation
5. Immunohistochemistry
Figure 1 shows representative brightfield images of several time points to demonstrate what the cells/organoids look like throughout the different stages of the protocol. The hESCs were removed from the tissue culture plate, broken into small pieces, and placed in a T75 ultra-low attachment flask where they formed spheres. It is important to note that the cells look bright and similar in size, without dark, dying cells in the centers of these clusters. The cells were gradually weaned off bFGF. On day 5, they were placed into neural induction media and they remained in this media throughout the culture period. Although the organoids get larger and thus darker over time, it is important to take note of the neural rosette-like structures (black arrows) that are present throughout the brain organoid development and expand. The rosettes indicate the initiation of neural differentiation and contain features of the embryonic neural tube, displaying epithelial characteristics and surrounding an apical lumen15.
Staining of the organoids with hematoxylin and eosin at 5 months in culture indicated that there were not vast amounts of necrosis even in the centers, which was of initial concern given the stagnant culture system (Figure 2A). These organoids demonstrated a histologic morphology similar to the human cortex based on light microscopic evaluation by an experienced neuropathologist (Figure 2B). By histology, many unique cell morphologies were observed resembling glia (blue arrow head), neurons (red arrowhead), cells with Cajal-Retzius morphology (black arrows), and neuropil (orange arrow head) (Figure 2B,C).
To take a more in depth look at gene expression within the cells, qRT-PCR was performed. For the results shown in Figure 3, each bar represents 3 separate batches of cells grown independently and harvested at the specified time point. These samples were then run in triplicate with a primer pair to the indicated gene in addition to the housekeeping gene, GAPDH. The glutamate transporter, Vglut1 (Figure 3A), was expressed at 2.5 weeks, increased at 5 weeks, and remained consistent through 5 months in culture. A forebrain marker, Foxg1 (Figure 3B), was expressed at low levels until 5 weeks in culture. The deep layer marker, Tbr1 (Figure 3C), peaked around 5 weeks and decreased subsequently, whereas the upper layer marker, Satb2 (Figure 3D), increased over time.
The expression of the ventral marker Engrailed1 (Eng1) (Figure 3E), the hindbrain/spinal cord marker Hoxb4 (Figure 3F), as well as the oligodendrocyte marker, Olig2 (Figure 3G), all increased over time. In contrast, the stem cell marker, Sox2 (Figure 3H), decreased over time. The glial marker, FAP (Figure 3I), peaked at 5 weeks and remained relatively constant subsequently. In addition, immunofluorescence data was consistent with the qRT-PCR data. At 10 weeks there was a robust expression of Foxg1 (Figure 4A). Sox2 expression was more confined to areas resembling the subventricular zone (SVZ) (Figure 4B,C). Interestingly, there was also some expression of the outer radial glial cell marker, HopX (Figure 4D).
Figure 1: Overview of organoid growth conditions and morphology. (A) Schematic of media changes. (B-M) Representative images of organoids as they matured. (B-M) H9 hESCs (B) were utilized to form the brain organoids. Organoids on (C) day 2 in 20 ng/mL bFGF media, and (D) day 3 and (E) day 4 in 10 ng/mL bFGF media. (F-M) Organoids in neural induction media (NIM) on days 5 (F), 8 (G), 10 (H), 17 (I) 35 (J,K), and 70 (L,M). Arrows point to neural rosettes. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 2: Organoids shared histologic similarities to human brain tissue. H&E staining of the organoids at 5 months (A) with some layering resembling the human cortex (B). At higher magnification many cell morphologies were observed including glia (blue arrow head), neurons (red arrowhead), neuropil (orange arrow head), and cells with Cajal-Retzius morphology (black arrows) (B,C). Please click here to view a larger version of this figure.
Figure 3: Expression of neurodevelopmental genes within the brain organoids over time. Quantitative RT-PCR data using SYBR green evaluating the expression of Vglut1 (A), Foxg1 (B), Tbr1 (C), Satb2 (D), En1 (E), Hoxb4 (F), Olig2 (G), Sox2 (H), and GFAP (I). Error bars = mean ± standard deviation (n ≥ 3). This figure has been modified from16. See Table 1 for primer information. Please click here to view a larger version of this figure.
Figure 4: Expression of neurodevelopmental proteins within the brain organoids at 10 weeks. Immunofluorescence revealed a robust expression of Foxg1 (A), localized expression of Sox2 (B,C) and the presence of HopX (D). Please click here to view a larger version of this figure.
Gene | Sequence (5' to 3') | Amplicon | Exon | |
En1 | F | GGACAATGACGTTGAAA CGCAGCA |
149 | 2 |
R | AAGGTCGTAAGCGGTTT GGCTAGA |
2 | ||
Foxg1 | F | AGAAGAACGGCAAGTAC GAGA |
188 | 1 |
R | TGTTGAGGGACAGATTG TGGC |
1 | ||
GAPDH | F | ACCACAGTCCATGCCAT CAC |
449 | 8 |
R | CACCACCCTGTTGCTGT AGCC |
9 | ||
GFAP | F | AGAGATCCGCACGCAGT ATG |
80 | 4 |
R | TCTGCAAACTTGGAGCG GTA |
5-Apr | ||
Hoxb4 | F | AAAGCACCCTCTGACTG CCAGATA |
80 | 2 |
R | ATGGGCACGAAAGATGA GGGAGA |
2 | ||
Olig2 | F | CCCTGAGGCTTTTCGGA GCG |
451 | 1 |
R | GCGGCTGTTGATCTTGA GACGC |
2 | ||
Satb2 | F | TAGCCAAAGAATGCCCT CTC |
94 | 6 |
R | AAACTCCTGGCACTTGG TTG |
7 | ||
Sox2 | F | CCCAGCAGACTTCACAT GT |
150 | 1 |
R | CCTCCCATTTCCCTCGT TTT |
1 | ||
Tbr1 | F | GTCACCGCCTACCAGAA CAC |
101 | 4 |
R | ACAGCCGGTGTAGATCG TG |
6 | ||
Vglut1 | F | CAGAGTTTTCGGCTTTG CTATTG |
183 | 5-Apr |
R | GCGACTCCGTTCTAAGG GTG |
6 |
Table 1: Primer sequences used for quantitative RT-PCR in Figure 3.
Similar to other organoid models, this is an artificial system that comes with several caveats. Although there was little batch to batch variation in terms of overall expression levels, individual organoids did exhibit differences. For example, the location of Sox-2 positive areas were not identical in every organoid (Figure 3). While qPCR is suitable to look for overall changes in batches of cells, additional techniques such as single cell RNAseq will be utilized in future studies to gather more information on a cell-by-cell basis. Another limitation of this system, is that it does not integrate vasculature within the organoids as has been done in some of the more recent studies17,18,19. However, transitioning the hESCs from a low to high oxygen environment may more closely resemble the anaerobic to aerobic transitioning in a developing embryo.
The critical steps within this protocol are the formation of the neurospheres as well as the appropriate maintenance including media changes with the proper culture media to ensure healthy cells and adequate nutrients to the growing organoids without overcrowding. To troubleshoot inadequate cell proliferation or differentiation, we recommend starting with a fresh batch of low passage ESCs and freshly prepared media including supplements. Occasionally there can be batch variation of reagents and materials. Thus, we recommend purchasing multiple bottles of reagents such as N2 and ultra-low attachment flasks which are from the same lot as long as they can be utilized in a reasonable amount of time.
Unlike many other brain organoid protocols, this method does not use a bioreactor; instead the cells stay relatively stagnant aside from media changes. This is similar to previous work with neurospheres, which were eventually broken apart to make 2D neuronal cultures20. In this model the cells are kept in a 3D format and allowed to grow for up to 6 months in culture. It was found that when utilizing small clusters instead of aggregating single cells that the organoids looked brighter, which we interpreted as less necrotic. As previously reported, when the brain organoid clusters were evaluated by histology and immunofluorescence at 5 months, there were no obvious areas of necrosis16. Although starting from small clusters of cells introduces a little variety in the size of organoids formed, the majority of organoids were of a roughly similar size.
The use of a heterologous basement membrane matrix and a bioreactor have both advantages and disadvantages. Certain cell types, or larger brain organoids might prefer growth under one condition or another. Basement membrane matrices or other hydrogels might be beneficial to selectively add growth factors to particular regions or create specific molds. Although basement membrane matrices have been shown to support three-dimensional organization and differentiation15, it is worth emphasizing that some of these products have a poorly defined and variable composition that includes quantities of growth factors12. In addition to simplifying the workflow while culturing the brain organoids, the absence of a basement membrane matrix might also improve three-dimensional imaging techniques.
The development of this brain organoid model system offers a new approach for many potential applications. For example, toxic insults like hypoxia, hyperglycemia, hypercapnia, and infection among others, may be tested with this system. In addition, neurodevelopmental disorders may be studied with this system by starting with either genetically modified stem cells or patient-specific human induced pluripotent stem cells (hPSCs). The ability to add different cell types during organoid culture also offers the possibility to study tumor-brain interactions. Given the simplicity of the protocol and lack of expensive, specialty materials we hope that this approach may be considered by laboratories both within and outside of the field as one potential method with its own unique benefits to further advance this rapidly progressing and exciting discipline.
The authors have nothing to disclose.
We thank the Yale Stem Cell Core (YSCC), and the Yale Cancer Center (YCC) for assistance. We thank Dr. Jung Kim for his neuropathology review. This work was supported by Connecticut Regenerative Medicine Research Fund, March of Dimes, and NHLBI R01HL131793 (S.G.K.), the Yale Cancer Center and the Yale Cancer Biology Training Program NCI CA193200 (E.B.) and a generous unrestricted gift from Joseph and Lucille Madri.
Alexa Fluor 488 goat anti-mouse | Thermo Fisher Scientific, Waltham, MA, USA | A11029 | |
Alexa Fluor 546 goat anti-rabbit | Thermo Fisher Scientific, Waltham, MA, USA | A11035 | |
B27 Supplement | Gibco, Waltham, MA, USA | 17504-044 | |
bFGF | Life Technologies, Carlsbad, CA, USA | PHG0263 | |
BSA | Sigma-Aldrich, St. Louis, MO, USA | A9647 | |
BX43 microscope | Olympus, Shinjuku, Tokyo, Japan | ||
DAPI stain | Thermo Fisher Scientific, Waltham, MA, USA | D1306 | |
Dispase | STEMCELL Technologies, Vancouver, Canada | 07913 | |
DMEM/F12 | Thermo Fisher Scientific, Waltham, MA, USA | 11330-032 | |
DPBS | Gibco, Waltham, MA, USA | 10010023 | |
FluroSave | MilliporeSigma, Burlington, MA | 345789 | |
GFAP antibody | NeuroMab, Davis, CA | N206A/8 | |
Growth Factor Reduced Matrigel (Matrix) | Corning, Corning, NY, USA | 356231 | |
H9 hESCs | WiCell, Madison, WI, USA | WA09 | |
Heparin | Sigma-Aldrich, St. Louis, MO, USA | 9041-08-1 | |
iQ SYBR Green Supermix | Bio-Rad, Hercules, CA, USA | 1708880 | |
iScript cDNA Synthesis Kit | Bio-Rad, Hercules, CA, USA | 1708891 | |
L-glutamine | Gibco, Waltham, MA, USA | 25030-081 | |
Monothioglycerol | Sigma-Aldrich, St. Louis, MO, USA | M6145 | |
mTESR media | STEMCELL Technologies, Vancouver, Canada | 85850 | |
N2 NeuroPlex | Gemini Bio Products, West Sacramento, CA, USA | 400-163 | |
Nanodrop | Thermo Fisher Scientific, Waltham, MA, USA | ND-2000 | |
NEAA | Gibco, Waltham, MA, USA | 11140-050 | |
Normal Donkey Serum (NDS) | ImmunoResearch Laboratories Inc., West Grove, PA, USA | 017-000-121 | |
OCT | Sakura Finetek, Torrance, CA, USA | 25608-930 | |
PFA | Electron Microscopy Sciences, Hatfield, PA | RT15710 | |
qPCR machine | Bio-Rad, CFX96, Hercules, CA, USA | 1855196 | |
RNeasy kit | Qiagen, Hilden, Germany | 74104 | |
Sox2 | MilliporeSigma, Burlington, MA | AB5603 | |
TMS-F microscope | Nikon, Melville, NY, USA | ||
Triton X-100 | Sigma-Aldrich, St. Louis, MO, USA | T8787-100ML | |
Ultra-low attachment T75 flasks | Corning, Corning, NY, USA | 3814 |