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Generation of embryonic organoids using NDiff 227 neural differentiation medium

Data kindly provided by Susanne van den Brink, PhD student at Alexander van Oudenaarden laboratory, Hubrecht Institute, Utrecht, Netherlands

Learn how NDiff 227 neural differentiation medium supports: 

  • A simple method to generate a large number of mouse gastruloids (embryonic organoids) from mouse embryonic stem cells
  • The high-throughput in vitro study of mouse embryonic development
  • An 80–90% success rate in the formation of elongating aggregates that resemble post-implantation mouse embryos
  • The induction of somite-like structures in up to 50% of the aggregates
Introduction Results Conclusions Methods

Introduction  

During early embryonic development, cellular interactions coordinate the transformation of a small, uniform cluster of cells into a complex three-dimensional multicellular organism. Errors during early embryonic development can result in developmental abnormalities, but it is largely unknown how and when such abnormalities arise. A majority of studies investigating the mechanisms underlying embryonic development rely on animal models, such as mice. However, it is technically challenging to maintain and image mouse embryos in long-term cultures. Additionally, the generation of genetically modified embryos is time-consuming and inefficient, and mouse embryos cannot be easily obtained in large numbers, limiting their use in large-scale genetic or drug screening procedures.

The discovery that mouse embryonic stem (ES) cells derived from mouse embryos can be propagated in a pluripotent state in vitro circumvented some of the abovementioned challenges, allowing scientists to study the processes that regulate embryonic development in vitro at a larger scale. Following this discovery, early two-dimensional (2D) and three-dimensional (3D) ES cell culture systems were developed, but they did not accurately capture the complex 3D morphology of the embryo. Therefore, these culture systems could not be used to understand, among other topics, the 3D interactions between the embryonic and extra-embryonic lineages, coordinated 3D cellular morphogenetic rearrangements (associated with embryonic processes such as gastrulation), or processes that ensure the appearance of embryonic organs in the correct position along the embryo's body axes.

To overcome these obstacles, Susanne van den Brink and her colleagues at the van Oudenaarden lab (Hubrecht Institute, Netherlands) recently developed a mouse ES cell-based protocol with which 3D embryo-like organoid structures can be generated. These structures (termed gastruloids) recapitulate many of the key events of mouse embryonic post-implantation development, including germ layer and body axis formation (van den Brink et al. 2014; Turner et al. 2017; Beccari et al. 2018; van den Brink et al. 2020). To generate these structures, the scientists used Takara Bio's NDiff 277 medium to efficiently and reproducibly differentiate mouse ES cell aggregates towards an early embryonic state. Upon subsequent application of a Wnt agonist, these aggregates elongate, resulting in the formation of a structure containing most embryonic cell types present in the correct location along the anterior-posterior (head-tail) body axis. As these processes normally take place during the embryonic development phase known as gastrulation, these embryo-like organoid structures are termed gastruloids, and they can be used to study mouse embryonic development in vitro in a high-throughput manner.

Results  

NDiff 227 medium is a defined, serum-free medium that was developed initially to differentiate mouse embryonic stem (ES) cells in adherent monoculture towards a neural lineage (Ying et al. 2003). However, the researchers discovered that NDiff 277 medium could also be used to efficiently and reproducibly generate 3D embryo-like organoid structures (gastruloids) from mouse ES cells for high-throughput studies of embryonic development in vitro. The development of gastruloids is a new and previously undescribed application of this medium.

In this protocol, mouse ES cells are aggregated by plating ~300 cells in each well of a low-attachment U-bottomed 96-well plate in NDiff 277 medium (Figure 1). During the two days following plating, the cells sink to the bottom of the well and attach to form one small, spherical aggregate per well. Upon treatment with the Wnt-agonist Chiron (CHIR99021) for 24 hr on Day 3, these aggregates break their symmetry and form an elongated structure at 4–5 days after aggregation. These structures elongate towards the posterior end over time (van den Brink et al. 2014) and generate most of the cell types that are present in mouse embryos, all of which are positioned in the correct location along the anterior-posterior axis of the elongated aggregates (van den Brink et al. 2020). Analysis of the resulting elongated structures revealed that they develop three germ layers and the three body axes.

Figure 1. Workflow for generating gastruloids from mouse ES cells. Gastruloids are produced by plating 300 mouse ES cells per well in NDiff 227 medium in a U-bottomed 96-well plate. After seeding, the plates are placed in the incubator for 48 hr. During the incubation, the cells sink to the bottom of the well and stick together to form a round aggregate. At 48 hr, NDiff 227 medium is supplemented with the Wnt-agonist Chiron (Chi), which induces a symmetry-breaking event. At 72 hr, the Chiron-supplemented NDiff 227 medium is replaced with plain NDiff 227 medium. Between 96 and 120 hr after aggregation, the aggregates elongate and form an embryonic organoid that contains all three germ layers and all three body axes. Optional: adding 10% Matrigel to gastruloids at 96 hr after aggregation induces the formation of somite-like structures and results in gastruloids that resemble mouse embryos more closely. Approximate hands-on time per 96-well plate for each step is indicated within brackets.

The first version of the gastruloid-development protocol (van den Brink et al. 2014) resulted in elongated structures that recapitulated many, but not all, aspects of mouse embryonic development. An important limitation of the first version of this system was that the elongated aggregates were, in contrast to mouse embryos, unable to generate somites (blocks of tissue that line the back of the embryo and give rise to the vertebral column, ribs, and skeletal muscles of the embryo). In 2020, however, the researchers discovered that the formation of such somite-like structures could be induced in gastruloids by adding a low percentage of Matrigel to the aggregates at 96 hr post culture (van den Brink et al. 2020). The addition of Matrigel resulted in gastruloids that resembled mouse embryos more accurately (Figure 2). This recent discovery revealed that it is possible to efficiently generate a large number of complex gastruloids in vitro using NDiff 227 medium.

Figure 2. Multi-photon microscopy image of a gastruloid that generates somite-like structures in vitro. A mouse gastruloid was embedded in 10% Matrigel at 96 hr, later stained for Uncx4.1 (which marks the posterior half of somites), and then visualized at 120 hr using hybridization chain reaction (HCR) imaging. Pink arrowheads point at the boundaries between the individual somites. A: anterior (head); P: posterior (tail). Scale bar = 100 µm. Image credit: Vincent van Batenburg.

Conclusions  

Researchers at the van Oudenaarden lab demonstrated that Takara Bio's NDiff 227 neural differentiation medium can help facilitate the study of embryonic development in vitro in a high-throughput manner by efficiently and reproducibly generating embryonic organoids (gastruloids) from mouse ES cells. The production of gastruloids has some critical advantages over traditionally used methods: they are easier to modify genetically than mouse embryos, and they can be used to study complex processes, such as body axis formation, currently not possible with other in vitro culture systems. Additionally, the gastruloids can be easily generated in large numbers, making them very suitable for large-scale drug screening procedures. The protocol for generating such gastruloids is simple and fast, uses only standard tissue culture equipment, and requires minimal prior experience with cell culture. Importantly, the NDiff 227 medium used in this protocol is defined and serum-free, which reduces batch effects between experiments and ensures high reproducibility between the different laboratories that are working with this in vitro system.

Methods  

Mouse ES cells were cultured in serum + leukemia inhibitory factor (LIF) conditions in a CO2- and temperature-controlled cell culture incubator (37ºC; 5% CO2). Before aggregation, cells were trypsinized, washed in PBS, and resuspended in Takara Bio's NDiff 277 medium (Cat. # Y40002). Subsequently, 300 cells were seeded in each well of a low-adherent 96-well U-bottomed plate in 40 µl of NDiff 227 medium per well and placed in the cell culture incubator for 48 hr. During this time, the cells sank to the bottom of the well, resulting in the formation of one small, spherical aggregate per well. After 48 hr, 150 µl of NDiff medium supplemented with 3 µM Chiron (CHIR99021) was added to the wells using a multichannel pipette, after which the plate was returned to the incubator for another 24 hr. Next, the Chiron-supplemented NDiff 227 medium was removed from the wells and replaced with 150 µl of fresh NDiff 227 medium (without Chiron), and the plate was returned to the incubator. Finally, 24 hr later (at 96 hr after aggregation), the medium was changed one last time by removing 150 µl of the NDiff 227 medium and replacing it with 150 µl of fresh NDiff 227 medium. After five days (120 hr after aggregation), ~80–90% of the aggregates elongated and displayed an embryo-like morphology.

Optional: embedding the aggregates in 10% Matrigel (in NDiff 227 medium) at 96 hr after aggregation induces the formation of somite-like structures in up to 50% of the aggregates. A detailed protocol for the generation of gastruloids without and with somite-like structures is available in this paper. An older version of this protocol is also available in video format.

It was recently shown that similar aggregates could also be generated when mouse induced pluripotent stem (iPS) cells are aggregated in NDiff 227 medium. A detailed protocol explaining how to generate such iPS cell-based mouse gastruloids is available here.

References

Beccari, L. et al. Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids. Nat. Lett. 562, 272–276 (2018).

van den Brink, S. C. et al. Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. Nature 582, 405–409 (2020).

van den Brink, S. C. et al. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development 141, 4231–42 (2014).

Turner, D. A. et al. Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids. Development 144, 3894–3906 (2017).

Ying, Q. et al. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotech. 21, 183–86 (2003).

Related Products

Cat. # Product Size License Quantity Details
Y40002 NDiff® 227 500 mL USD $378.00

NDiff 227 is a proprietary, defined, serum-free medium for the neural differentiation of mouse ES cells in adherent monolayer culture conditions. NDiff 227 has also been shown to support the serum-free, feeder-free culture of mouse ES cells. More recently, 227-like medium supplemented with growth factors has also been used to successfully maintain feeder-free human ES cells in culture. When supplemented with FGF, serum-free 227 also generates Anterior Definitive Endoderm (ADE) precursors from mouse ES cells, which can then be differentiated into liver and pancreas with enhanced efficiency.

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Neural differentiation of mouse embryonic stem cells cultured in adherent monoculture conditions with NDiff 227 medium

Neural differentiation of mouse embryonic stem cells cultured in adherent monoculture conditions with NDiff 227 medium
Neural differentiation of mouse embryonic stem cells cultured in adherent monoculture conditions with NDiff 227 medium.

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Y40002: NDiff 227

Y40002: NDiff 227

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