小鼠多能干细胞衍生干细胞样结构的生成：植入术后胚胎发生的体外模型

Adriano Bolondi; Leah Haut; Seher Ipek Gassaloglu; Polly  Burton; Helene  Kretzmer; René  Buschow; Alexander Meissner; Bernhard G. Herrmann; Jesse V. Veenvliet

doi:10.21769/BioProtoc.4042

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Peer-reviewed

Generation of Mouse Pluripotent Stem Cell-derived Trunk-like Structures: An in vitro Model of Post-implantation Embryogenesis

小鼠多能干细胞衍生干细胞样结构的生成：植入术后胚胎发生的体外模型

AB Adriano Bolondi

LH Leah Haut

SG Seher Ipek Gassaloglu

PB Polly Burton

HK Helene Kretzmer

RB René Buschow

AM Alexander Meissner email

BH Bernhard G. Herrmann email

JV Jesse V. Veenvliet email

发布: 2021年06月05日第11卷第11期 DOI: 10.21769/BioProtoc.4042 浏览次数: 4794

评审: Giusy TornilloDavid TurnerStefano Vianello

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参见作者原研究论文

The authors used this protocol in:

Cover of Science, featuring study using the protocol.

Dec 2020

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Abstract

Post-implantation mammalian embryogenesis involves profound molecular, cellular, and morphogenetic changes. The study of these highly dynamic processes is complicated by the limited accessibility of in utero development. In recent years, several complementary in vitro systems comprising self-organized assemblies of mouse embryonic stem cells, such as gastruloids, have been reported. We recently demonstrated that the morphogenetic potential of gastruloids can be further unlocked by the addition of a low percentage of Matrigel as an extracellular matrix surrogate. This resulted in the formation of highly organized trunk-like structures (TLSs) with a neural tube that is frequently flanked by bilateral somites. Notably, development at the molecular and morphogenetic levels is highly reminiscent of the natural embryo. To facilitate access to this powerful model, here we provide a detailed step-by-step protocol that should allow any lab with access to standard cell culture techniques to implement the culture system. This will provide the user with a means to investigate early mid-gestational mouse embryogenesis at an unprecedented spatiotemporal resolution.

Keywords: Trunk-like structures (像树干的结构)

Gastrulation (原肠胚形成)

Somites (体节)

Self-organization (自我组织)

In vitro models (体外模型)

Stem cells (干细胞)

Background

Gastrulation and early organogenesis represent developmental events that are crucial for the successful generation of a functional body plan. In mammals, these processes start just after the embryo implants in utero and within few days, a variety of morphologically and functionally diverse tissues emerge. It is currently difficult to study these highly dynamic changes in vivo, and ex vivo culture of post-implantation mouse embryos is laborious, costly, and requires rigorous training, which often renders it impractical for most laboratories. These impediments have led to extensive efforts to model post-implantation and early mid-gestational development in vitro using embryonic stem cells (reviewed in Shahbazi and Zernicka-Goetz 2018; Shahbazi et al., 2019; Baillie-Benson et al., 2020; Veenvliet and Herrmann, 2021). In particular, post-implantation development can be modeled with gastruloids, mouse or human embryonic stem cell (mESC/hESC) aggregates that self-organize (van den Brink et al., 2014 and 2020; Moris et al., 2020). The original mouse gastruloid culture protocol resulted in elongated structures with embryo-like expression domains similar to the post-occipital mouse embryo and with correct positioning of the three body axes, but limited morphogenesis (van den Brink et al., 2014; Baillie-Johnson et al., 2015; Beccari et al., 2018a and 2018b; Turner et al., 2017). More recent efforts have managed to introduce embryo-like morphological features by changing the cellular environment, such as the formation of somite-like structures or a heart tube (van den Brink et al., 2020; Rossi et al., 2021). Further advances have demonstrated that the addition of an extracellular matrix (ECM) surrogate to gastruloids can trigger a more embryo-like architecture with a gut tube as well as somites flanking a neural tube (Veenvliet et al., 2020). We dubbed these embryonic organoids trunk-like structures (TLSs), since they resemble the core part of the trunk of an early mid-gestational embryo (~embryonic stage (E) 8.5-9). Importantly, during the timeframe of TLS induction (96-120 h post-aggregation), the gene regulatory programs are highly similar to the developing embryo. Moreover, the segmentation clock, an oscillator driving the rhythmic deposition of somites in vivo, is active at an embryo-like pace in the TLS (Pourquié, 2003; Veenvliet et al., 2020).

The TLS model is easy to access, track, manipulate, and scale, which makes it a powerful tool to study post-implantation and early mid-gestational mammalian development in a dish. Here, we provide a comprehensive step-by-step procedure to facilitate the generation of trunk-like structures. We also describe how to process TLSs for downstream analysis, including whole-mount immunofluorescent staining and (single cell) RNA sequencing.

Materials and Reagents

Pipet tips, variable volumes (Biozym, SafeSeal SurPhob VT)
1.5 ml tubes (Sarstedt, catalog number: 72.706)
15 ml Falcon tubes (Sarstedt, catalog number: 62.554.502)
50 ml Falcon tubes (Sarstedt, catalog number: 62.547.254)
6 cm cell culture plates (Sarstedt, catalog number: 83.3901.300)
Ultra-low attachment 96-well plates (Corning, Costar, catalog number: CLS7007)
6-well cell culture plates (Corning, catalog number: 3516)
10 cm cell culture plates (Corning, catalog number: 430167)
Luna cell counting slides (Logos Biosystems, catalog number: L12001)
µ-Slide 8-well glass bottom (Ibidi, catalog number: 80827)
Flowmi cell strainers 40 µm (Merck, catalog number: BAH136800040)
Bottle top vacuum filter unit (Corning, catalog number: CLS431096)
KnockOut DMEM (Gibco, catalog number: 10829018)
100× Penicillin (5000 U/ml)-Streptomycin (5,000 µg/ml) (Lonza, catalog number: DE17-603E)
100× Glutamine, 200 mM (Lonza, catalog number: BE17-605E)
100× Nucleosides (Sigma, catalog number: ES-008D)
Gibco 2-Mercaptoethanol, 55 mM solution in DPBS (Gibco, catalog number: 21985023)
Fetal Calf Serum (FCS), both regular (Pan Biotech, catalog number: P30-3306) and qualified and embryonic stem cell culture tested (Pan Biotech, catalog number: P30-2602)
TrypLE (Gibco, catalog number: 12604013) OR 0.05% Trypsin-EDTA (1x) (Gibco, catalog number: 25300-054)
NDiff 227 medium (Takara, catalog number: Y40002)
CHIR99021 InSolution (Sigma, catalog number: 361571) OR 10 mM in dimethyl sulfoxide (DMSO) (Tocris Biosciences, catalog number: 4423)
LDN193189 (Reprocell, catalog number: 04-0074-10)
DMSO (Sigma, catalog number: D2650)
Matrigel Growth Factor Reduced (GFR), Phenol Red-free (Corning, catalog number: 356231) – multiple lots/batches have been tested yielding similar results in terms of trunk-like-structure generation efficiency
Gelatin 2% solution (Sigma, catalog number: G1393)
DPBS, w/o MgCl₂/CaCl₂ (Gibco, catalog number: 14190144)
PBS with MgCl₂/CaCl₂ (Sigma, catalog number: D8662)
Murine Leukemia Inhibitory Factor (LIF) ESGRO^TM (10⁷U/ml) (Millipore, catalog number: ESG1107)
Trypan Blue (Bio-Rad, catalog number: 1450021)
UltraPure Dnase/Rnase-Free Distilled Water (Invitrogen, catalog number: 10977049)
Reagent Reservoirs 60 ml (Merck, catalog number: BR703411)
Bovine Serum Albumin powder (BSA) (Sigma, catalog number: A2153)
Dulbecco’s Modified Eagle's Medium (DMEM) 4,500 mg/ml glucose, without sodium pyruvate (Lonza, catalog number: BE12-733F)
Cell culture grade water (Lonza, catalog number: BE17-724Q)
0.1% Gelatin solution (see Recipes)
Mouse embryonic fibroblast (MEF) medium (see Recipes)
Mouse embryonic stem cell (mESC) medium (see Recipes)
PBS/0.5% BSA solution (see Recipes)

Equipment

Biological safety cabinet (Thermo Fisher Scientific, model: Herasafe KS12)
Clean horizontal laminar flow hood (Thermo Fisher Scientific, model: HeraGuard ECO)
Cell culture incubator (Thermo Fisher Scientific, model: Heracell Vios 160i)
Cell culture centrifuge (Eppendorf, model: Centrifuge 5804R)
Variable volume pipets and multichannel pipets (Eppendorf, model: Research^® plus pipette)
Horizontal light source, Light ring (Nikon, P-DF LED Darkfield Unit) or other stereomicroscope stand
Automated cell counter (Logos biosystems, Luna automated cell counter, L10001)
Cell culture water bath (LAUDA Aqualine, catalog number: AL18)
Tissue culture vacuum pump (Vacuubrand, catalog number: 20727200)
Microcentrifuge (Eppendorf, model: 5424R)

Equipment set up:

Cell culture incubators are set to 37°C, 5% CO₂.
NOTE: We have also successfully generated TLSs at 7.5% CO₂, but routinely use 5%.
Cell culture water bath (set to 37°C).
All centrifugation steps are performed at room temperature, unless otherwise indicated.

Procedure

English

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Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Bolondi, A., Haut, L., Gassaloglu, S. I., Burton, P., Kretzmer, H., Buschow, R., Meissner, A., Herrmann, B. G. and Veenvliet, J. V. (2021). Generation of Mouse Pluripotent Stem Cell-derived Trunk-like Structures: An in vitro Model of Post-implantation Embryogenesis. Bio-protocol 11(11): e4042. DOI: 10.21769/BioProtoc.4042.
Veenvliet, J. V., Bolondi, A., Kretzmer, H., Haut, L., Scholze-Wittler, M., Schifferl, D., Koch, F., Guignard, L., Kumar, A. S., Pustet, M., Heimann, S., Buschow, R., Wittler, L., Timmermann, B., Meissner, A. and Herrmann, B. G. (2020). Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. Science 370(6522).