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Dec 2020

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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   

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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), Morphogenesis (形态发生), Gastruloids (类原肠胚), Embryoids (胚状体), Organoids (类器官), 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

  1. Pipet tips, variable volumes (Biozym, SafeSeal SurPhob VT)

  2. 1.5 ml tubes (Sarstedt, catalog number: 72.706)

  3. 15 ml Falcon tubes (Sarstedt, catalog number: 62.554.502)

  4. 50 ml Falcon tubes (Sarstedt, catalog number: 62.547.254)

  5. 6 cm cell culture plates (Sarstedt, catalog number: 83.3901.300)

  6. Ultra-low attachment 96-well plates (Corning, Costar, catalog number: CLS7007)

  7. 6-well cell culture plates (Corning, catalog number: 3516)

  8. 10 cm cell culture plates (Corning, catalog number: 430167)

  9. Luna cell counting slides (Logos Biosystems, catalog number: L12001)

  10. µ-Slide 8-well glass bottom (Ibidi, catalog number: 80827)

  11. Flowmi cell strainers 40 µm (Merck, catalog number: BAH136800040)

  12. Bottle top vacuum filter unit (Corning, catalog number: CLS431096)

  13. KnockOut DMEM (Gibco, catalog number: 10829018)

  14. 100× Penicillin (5000 U/ml)-Streptomycin (5,000 µg/ml) (Lonza, catalog number: DE17-603E)

  15. 100× Glutamine, 200 mM (Lonza, catalog number: BE17-605E)

  16. 100× Nucleosides (Sigma, catalog number: ES-008D)

  17. Gibco 2-Mercaptoethanol, 55 mM solution in DPBS (Gibco, catalog number: 21985023)

  18. 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)

  19. TrypLE (Gibco, catalog number: 12604013) OR 0.05% Trypsin-EDTA (1x) (Gibco, catalog number: 25300-054)

  20. NDiff 227 medium (Takara, catalog number: Y40002)

  21. CHIR99021 InSolution (Sigma, catalog number: 361571) OR 10 mM in dimethyl sulfoxide (DMSO) (Tocris Biosciences, catalog number: 4423)

  22. LDN193189 (Reprocell, catalog number: 04-0074-10)

  23. DMSO (Sigma, catalog number: D2650)

  24. 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

  25. Gelatin 2% solution (Sigma, catalog number: G1393)

  26. DPBS, w/o MgCl2/CaCl2 (Gibco, catalog number: 14190144)

  27. PBS with MgCl2/CaCl2 (Sigma, catalog number: D8662)

  28. Murine Leukemia Inhibitory Factor (LIF) ESGROTM (107U/ml) (Millipore, catalog number: ESG1107)

  29. Trypan Blue (Bio-Rad, catalog number: 1450021)

  30. UltraPure Dnase/Rnase-Free Distilled Water (Invitrogen, catalog number: 10977049)

  31. Reagent Reservoirs 60 ml (Merck, catalog number: BR703411)

  32. Bovine Serum Albumin powder (BSA) (Sigma, catalog number: A2153)

  33. Dulbecco’s Modified Eagle's Medium (DMEM) 4,500 mg/ml glucose, without sodium pyruvate (Lonza, catalog number: BE12-733F)

  34. Cell culture grade water (Lonza, catalog number: BE17-724Q)

  35. 0.1% Gelatin solution (see Recipes)

  36. Mouse embryonic fibroblast (MEF) medium (see Recipes)

  37. Mouse embryonic stem cell (mESC) medium (see Recipes)

  38. PBS/0.5% BSA solution (see Recipes)

Equipment

  1. Biological safety cabinet (Thermo Fisher Scientific, model: Herasafe KS12)

  2. Clean horizontal laminar flow hood (Thermo Fisher Scientific, model: HeraGuard ECO)

  3. Cell culture incubator (Thermo Fisher Scientific, model: Heracell Vios 160i)

  4. Cell culture centrifuge (Eppendorf, model: Centrifuge 5804R)

  5. Variable volume pipets and multichannel pipets (Eppendorf, model: Research® plus pipette)

  6. Horizontal light source, Light ring (Nikon, P-DF LED Darkfield Unit) or other stereomicroscope stand

  7. Automated cell counter (Logos biosystems, Luna automated cell counter, L10001)

  8. Cell culture water bath (LAUDA Aqualine, catalog number: AL18)

  9. Tissue culture vacuum pump (Vacuubrand, catalog number: 20727200)

  10. Microcentrifuge (Eppendorf, model: 5424R)


Equipment set up:

  1. 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%.

  2. Cell culture water bath (set to 37°C).

  3. All centrifugation steps are performed at room temperature, unless otherwise indicated.

Procedure

  1. Seeding mouse embryonic fibroblasts (MEFs)
    NOTES:

    1. Seed MEFs at least one day prior to seeding the mESCs.
    2. Pre-warm MEF medium in the water bath for at least 20 min before starting.
    3. MEF plates should be used within one week of seeding.


    1. Coat a 6 cm cell culture plate with 3 ml 0.1% gelatin solution.

      NOTE: Gelatin-coated culture plates have to be prepared fresh on the day of seeding MEFs and cannot be stored.

    2. Leave the plate at room temperature for 15 min.

      Next, thaw a vial of mitotically inactive MEFs at 37°C in the water bath.

      NOTES:

      1. Inactive MEFs are mitotically inactivated in-house using mitomycin C treatment (3 h at 37°C).

      2. You need 1.0 × 106 MEFs to coat a 6 cm cell culture plate. Thaw the appropriate number depending on the number of 6 cm cell culture plates needed.

    3. Add the MEFs to a 15 ml Falcon tube containing 5 ml pre-warmed MEF medium.

    4. Centrifuge the cells at 200 × g for 5 min.

    5. While centrifuging, aspirate gelatin from each 6 cm plate and add 2 ml MEF medium.

    6. Aspirate the supernatant from the 15 ml tube containing MEFs and resuspend the cell pellet at a concentration of 1.0 × 106 cells per ml.

      NOTE: Viable MEFs are counted at the time of freezing and there is no need to count them again after thawing.

    7. Add 1 ml cell suspension to each prepared 6 cm plate.

    8. Place the plate in the incubator and swirl the plate to ensure even distribution of cells.


  1. Seeding mouse embryonic stem cells (mESCs)
    NOTES:

    1. Pre-warm mESC medium in the water bath for at least 20 min before starting.

    2. We routinely use mESCs with an F1G4 genetic background for TLS protocol generation (George et al., 2007).


    1. Thaw a vial of mESCs at 37°C in the water bath immediately before plating.

      NOTE: You need 3.5 × 105 mESCs for a 6 cm MEF-coated plate. Thaw the appropriate number depending on the number of 6 cm cell culture plates needed.

    2. Add the mESCs to a 15 ml Falcon tube containing 5 ml pre-warmed mESC medium.

    3. Centrifuge the cells at 200 × g for 5 min.

    4. While centrifuging, aspirate MEF medium from each 6 cm plate containing MEFs and add 2 ml mESC medium.

    5. Aspirate the supernatant from the 15 ml tube containing mESCs and resuspend the cell pellet at a concentration of 3.5 × 105 cells per ml.

      NOTE: Viable mESCs are counted at the time of freezing and there is no need to count them again after thawing.

    6. Add 1 ml cell suspension to each MEF-coated 6 cm plate.

    7. Place the plate in the incubator and swirl the plate to ensure even distribution of cells.

    8. Replace the medium daily with 3 ml fresh mESC medium.


  1. Passaging mESCs
    NOTES:

    1. Passage mESCs every 48 h at a splitting ratio of 1:8-1:10. Colony density and morphology should look similar to that shown in Figure 1A. Do not let your culture overgrow (Figure 1B).

    2. The splitting time and ratios detailed here are optimized for the mESC lines used in Veenvliet et al. (2020). Based on the proliferation rate of the mESC line used, splitting times and ratios may need to be adjusted. This may be especially true if transgenic lines and/or mESCs with different genetic backgrounds are used.

    3. Pre-warm mESC medium and TrypLE in the water bath for at least 20 min before starting.

    4. Prepare MEF-coated plates one day prior to passaging mESCs.

    5. Instead of TrypLE, 0.05% Trypsin-EDTA can be used.



    Figure 1. Optimal embryonic stem cell culture densities for successful TLS generation. A. mESC culture densities suitable for TLS generation (24 h and 48 h after seeding). B. mESC culture density unsuitable for TLS generation (96 h after seeding). Scale bars for all panels, 50 μm.

    1. Aspirate the medium from the mESC plate and wash with 3 ml DPBS.

    2. Aspirate the DPBS and add 1 ml TrypLE.

    3. Ensure that the plate surface is evenly covered with TrypLE and place it in the incubator at 37°C for 5 min.

    4. After 5 min, dislodge the colonies with a P1000 pipet set to 800 μl by pipetting up and down in the plate 20 times.

    5. Inactivate the TrypLE by adding 1 ml mESC medium and pipet further to obtain a single cell suspension.

    6. Transfer the cell suspension to a 15 ml Falcon tube and wash the plate with an additional 3 ml mESC medium to recover all cells. Transfer these cells to the same 15 ml tube.

    7. Centrifuge the cells at 200 × g for 5 min.

    8. While centrifuging, aspirate the MEF medium from the previously prepared 6 cm plate containing MEFs and add 2 ml mESC medium.

    9. Aspirate the supernatant from the 15 ml tube containing mESCs and resuspend the cell pellet in 2 ml mESCs medium.

    10. Add the appropriate amount of cell suspension to each MEF-coated 6 cm plate (ratio 1:8-1:10 → 200-250 μl). Adjust the final volume to 3ml.

    11. Place the plate in the incubator and swirl the plate to ensure even distribution of cells.

    12. Replace the medium daily with 3 ml fresh pre-warmed mESC medium.


  1. Generation of trunk-like structures (TLSs)

    NOTES:
    1. The input cell number for each well detailed here is optimized for the mESC lines used in Veenvliet et al. (2020). Based on the proliferation rate of the mESC line (especially for transgenic lines and/or mESCs with a different genetic background), the cell amount may need to be adjusted to reach the same efficiency reported in Veenvliet et al. (2020).

    2. We recommend first optimizing the standard gastruloid protocol for new cell lines, using gastruloid elongation efficiency as a fast experimental readout (Cermola et al., 2021). In our experience, good gastruloid elongation efficiency (>95%) is essential to achieve a similar TLS efficiency to that reported in Veenvliet et al. (2020). A routine optimization procedure involves the seeding of 100-600 mESCs per well, with a stepwise increase of 50 cells.

    3. mESCs must be in culture for at least one passage before starting.

    4. Pre-warm mESC medium and TrypLE in the water bath for at least 20 min before starting.

    5. Here, we use commercially available, quality controlled NDiff 227 medium (N2B27). We and others have successfully generated gastruloids with home-made N2B27 (Beccari et al., 2018b); however, in our hands, more robust results of the gastruloid and TLS protocols are obtained with the NDiff 227 medium.

    6. Instead of TrypLE, 0.05% Trypsin-EDTA can be used.

    7. A schematic overview of the TLS generation protocol indicating critical timepoints is provided in Figure 2.



      Figure 2. Schematic overview of the TLS generation protocol. Workflow for the generation of trunk-like-structures (TLS) from seeding of MEFs to downstream analysis. MG, Matrigel; CL, CHIR+LDN; MEFs, mouse embryonic fibroblasts; mESCs, mouse embryonic stem cells.


    D1. Prepare 6-well plate for MEF depletion
    1. Coat three wells of a 6-well plate with 2 ml 0.1% gelatin solution for each 6 cm plate that will be used for TLS generation.

    2. Incubate the 6-well plate at room temperature for 15 min.

    3. Aspirate gelatin solution and add 1 ml mESC medium to each well.

    4. Store plate in the incubator until use.


    D2. Prepare a single cell suspension
    1. Aspirate the medium from the mESC plate and wash with 3 ml DPBS.

    2. Aspirate the DPBS and add 1 ml TrypLE.

    3. Ensure that the plate surface is evenly covered with TrypLE and place it in the incubator at 37°C for 5 min.

    4. After 5 min, dislodge the colonies with a P1000 pipet set to 800 μl by pipetting up and down in the plate 20 times.

    5. Inactivate the TrypLE by adding 1 ml mESC medium and pipet-mix.

    6. Transfer the cell suspension to a 15 ml Falcon tube and wash the plate with an additional 3 ml mESC medium to recover all cells. Transfer these cells to the same 15 ml tube.

    7. Centrifuge the cells at 200 × g for 5 min.

    8. Resuspend the cell pellet in 1 ml mESC medium and pipet up and down 50 times.

      NOTE: Here, it is critical to achieve a proper single cell suspension to avoid losing mESCs (or retaining MEFs) during MEF depletion and to ensure the best protocol performance. We recommend checking for a proper single cell suspension under a microscope.


    D3. MEF depletion

    NOTE: An example how the wells with cells attached to the bottom should look like after each step of MEF depletion is provided in Figure 3.



    Figure 3. MEF depletion prior to mESC aggregation. MEFs attach to the 0.1% gelatin-coated wells. Scale bars, 50 μm.
    NOTE: With consecutive transfers, the amount of attached cells decreases. After the third incubation, MEF depletion is completed and mESCs are ready to be used for aggregation.


    1. Transfer the obtained cell suspension to a well of the prepared 6-well plate (see D1).

      NOTE: Transfer the amount of mESCs present in one 6 cm plate into one well of the prepared 6-well plate. The presence of too many cells could result in decreased depletion efficiency.

    2. Pipet-mix 10 times.

    3. Place the plate in the incubator and swirl the plate to ensure even distribution of cells. Leave untouched for 25 min.

    4. Next use a P1000 pipet set to 1 ml to carefully transfer all the cells in suspension to another well.

      NOTE: It is critical not to dislodge the MEFs, which are attached to the bottom of the wells.

    5. Pipet up and down 10 times in the new well to ensure a single cell suspension.

      NOTE: Cells may clump during the incubation; therefore, it is critical to pipet once the cells are transferred to the new well. We recommend confirming under the microscope that you have obtained a proper single cell suspension.

    6. Place the plate in the incubator and swirl the plate to ensure even distribution of cells. Leave untouched for 20 min.

    7. Next, use a P1000 pipet set to 1 ml to carefully transfer all the cells in suspension to another well.

      NOTE: It is critical not to dislodge the MEFs, which are attached to the bottom of the wells.

    8. Pipet up and down 10 times in the new well to ensure a single cell suspension.

      NOTE: Cells may clump during the incubation; therefore, it is critical to pipet once the cells are transferred in the new well. We recommend confirming under the microscope that you have obtained a proper single cell suspension.

    9. Place the plate in the incubator and swirl the plate to ensure even distribution of cells. Leave untouched for 15 min.

    10. During this last 15-min step, equilibrate the required amount of NDiff 227 in a 10 cm dish in the incubator for at least 20 min. Longer incubation is also possible (e.g., NDiff 227 can be placed in the incubator after step 6. See Table 1 for the volume needed as a function of the number of 96-well plates to seed).

      NOTE: NDiff 227 is light-sensitive and should be protected from (direct) light as much as possible.

    11. Carefully transfer all MEF-depleted mESCs to a 15 ml Falcon tube with a P1000 pipet.

      NOTE: It is critical not to dislodge the MEFs, which are attached to the bottom of the wells.


    D4. mESC aggregation (0 h)

    NOTE: The first 96 h of the TLS protocol are similar to the gastruloids protocol (Baillie-Johnson et al., 2015; van den Brink et al., 2014; Beccari et al., 2018b; Anlas et al., 2021). Detailed protocols for gastruloid formation, including troubleshooting, are provided elsewhere (Baillie-Johnson et al., 2015; Beccari et al., 2018b; Anlas et al., 2021).

    1. Centrifuge the cells at 200 × g for 5 min.

    2. Resuspend the cell pellet in 5 ml PBS with MgCl2/CaCl2 and pipet up and down 20 times (Wash 1).

    3. Centrifuge the cells at 200 × g for 5 min.

      NOTE: In case of low starting cell numbers, steps 2 and 3 can be omitted. This may however slightly compromise protocol efficiency.

    4. Resuspend the cell pellet in 5 ml pre-equilibrated NDiff 227 and pipet up and down 20 times (Wash 2).

    5. Centrifuge the cells at 200 × g for 5 min.

    6. Resuspend the cell pellet in 500 μl pre-equilibrated NDiff 227 and pipet up and down 30 times. NOTE: It is critical to obtain a single cell suspension prior to counting and plating.

    7. For counting, prepare a 1:2 dilution of the cell suspension by adding 10 μl cell suspension to 10 μl Trypan Blue.

    8. Count using the Luna automated cell counter with the following set up: Dilution factor → 2; Noise reduction → 5; Live detection sensitivity → 5; Roundness → 85%; Min cell size → 10 μm; Max cell size → 20 μm; Declustering level → High.

    9. Transfer the amount of cells needed for the experiment to a new Falcon tube (see Table 1 for the number of cells needed as a function of the number of 96-well plates to seed).

    10. Add the pre-incubated NDiff 227 volume required to bring the cell suspension to a concentration of 5.7 × 103 cells/ml (see Table 1 for the volume to add as a function of the number of 96-well plates to seed).

      NOTE: This cell concentration is optimized for an input of 200 cells/well, which has been shown to give high TLS generation efficiency for all cell lines tested (Veenvliet et al., 2020). Similar results were obtained for inputs ranging from 200 to 250 cells/well.

    11. Mix the new cell suspension vigorously and transfer it to a reservoir.

    12. Use a multichannel pipet to transfer 35 μl to each well of an ultra-low attachment 96-well plate. Pipet gently up and down in the reservoir between each transfer.

    13. Gently tap the plate 5 times on a clean bench, transfer to the incubator, and allow undisturbed aggregation for 48 h.

      NOTE: Keeping NDiff 227 outside the incubator for longer periods of time (more than 5 min) will lead to disequilibration of the medium. Therefore, try to avoid keeping NDiff 227 or plates with freshly seeded cells in NDiff 227 out the incubator for too long. In the case of handling multiple plates, we recommend putting each plate into the incubator directly after pipetting.


    Table 1. Cell numbers and volumes required for mESC aggregation. The amounts are calculated for an input of 200 cells/well in 35 μl. Volumes and cell numbers in column 4 are calculated for 100 samples (instead of 96) per plate plus a 10% excess dead volume. In column 2, the volume of NDiff 227 to equilibrate is calculated based on the amount needed for washing and counting the cells (5.5 ml per experiment, independent of the number of plates, see step D4.4 and D4.6), plus the amount indicated in column 4, plus an extra volume to account for dead space in the dish and evaporation during medium equilibration.


    D5. CHIR pulse (48 h)

    NOTES:

    1. Start this procedure at least one hour before the end of the 48 h.

    2. Cells at 48 h should have formed one single round aggregate with a diameter measuring 214 ± 13 μm (seeFigure 4A).

    3. CHIR99021 is light-sensitive and should be protected from (direct) light as much as possible.

    4. CHIR99021 should be aliquoted in single-use aliquots in brown (light-protected) sterile tubes upon arrival and not subjected to repeated freeze-thaw cycles.


    1. Equilibrate the needed amount of NDiff 227 in a 10-cm dish in the incubator for at least 20 min. (see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment).

    2. Transfer the required amount of NDiff 227 to a 50-ml Falcon tube (see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment).


      Table 2. Volumes of NDiff 227, CHIR99021, and Matrigel required during the last three steps (protocol steps D5, D6, D7) of the TLS generation protocol. In column 2, the volume of NDiff 227 to equilibrate is calculated including an excess volume (2 ml for each dish that is used for medium equilibration) to account for dead space in the dish(es) and medium evaporation during equilibration.

      *For volumes higher than 30 ml, use more than one 10-cm plate to equilibrate NDiff 227 in the incubator.

      **Use two 50-ml tubes if the volume exceeds 50 ml.

      ***Note that we calculate 5% v/v Matrigel as a function of the final volume in each well (35 μl + 150 μl = 185 μl) (as opposed to CHIR99021). This means that in the NDiff 227, the concentration of Matrigel is slightly higher than 5% (6.17%). For instance, for 1 plate of TLSs, the volume of Matrigel added is calculated as (0.05*16)*(185/150) = 0.987 ml.


    1. Add CHIR99021 to the NDiff 227 medium to obtain a final concentration of 3 μM.

    2. Mix the medium vigorously and transfer it to a reservoir.

    3. Use a multichannel pipet to add 150 μl CHIR99021-supplemented medium to each well of the plates containing the aggregates.

    4. Gently tap the plate 10 times on a clean bench, transfer to the incubator, and allow further undisturbed development for 24 h.

      NOTES:

      1. The tapping is critical to prevent cell aggregates from attaching to the culture plates. Ensure that the aggregates are freely moving immediately after tapping (this can be checked under the microscope).

      2. Take caution to avoid splashing medium on the lid while tapping the plates.



      Figure 4. Examples of expected morphology of mESC-derived aggregates at several timepoints during TLS generation. A, B. mESC-derived aggregates at 48 h and 72 h after aggregation are round without clear signs of symmetry breaking. C. At 96 h after aggregation, the structures have clearly broken symmetry and are teardrop shaped. The white arrowheads indicate the posterior pole, where localized expression of Brachyury is expected. Note that, depending on the cell line used, aggregates may establish the teardrop-like morphology prior to 96 h. In that case, structures should be embedded in Matrigel earlier (as soon as the teardrop-like morphology is observed) to achieve optimal TLS efficiency (see main text for details). D. Upon addition of 5% Matrigel, the aggregates will establish an architecture reminiscent of the embryonic trunk, with somites (magenta arrowheads) flanking a neural tube (green arrowheads). Chemical modulation with a WNT agonist (5 μM CHIR99021) and BMP inhibitor (600 nM LDN193189) results in compromised neural tube development and formation of excess somites arranged like a “bunch-of-grapes” (TLSCL). Scale bars for all panels, 50 μm. A, Anterior; P, Posterior.


    D6. Media change (72 h)

    NOTES:

    1. Start this procedure at least one hour before the end of the 72 h.

    2. Aggregates at 72 h should look like the example given in Figure 4B and measure 244 ± 15 μm in diameter.

    3. If available, perform this step on a clean bench containing a stereoscope or a light ring to help visualize the structures and avoid loss of structures while pipetting off the old medium.


    1. Equilibrate the required amount of NDiff 227 in a 10-cm dish in the incubator for at least 20 min (see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment).
    2. Transfer the required amount of NDiff 227 to a 50-ml tube (see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment).
    3. Use a multichannel pipet to remove 150 μl from each well without disturbing the structure.
      NOTE: CRITICAL STEP. Avoid losing structures while pipetting off the medium. This is best achieved by keeping the plate under a 30-40° angle and putting the pipet tips against the side opposite to that where the aggregate should be located. As a visual aid, a stereoscope or light ring could be used as stated above (see Figure 5 for a schematic of how to position the plate and tips).




      Figure 5. Schematic representation of plate positioning for media changes during the TLS generation procedure. The plate is tilted at a 30-40° angle on a clean bench and the media is carefully aspirated with a multichannel pipet, avoiding disturbance of the aggregates.


    4. Pour the pre-equilibrated medium in a reservoir.

    5. Use a multichannel pipet to add 150 μl medium to each well of the plates containing the aggregates.

    6. Gently tap the plate 10 times on a clean bench, transfer to the incubator, and allow further undisturbed development for 16-24 h.

      NOTES:

      1. The tapping is critical to prevent cell aggregates from attaching to the culture plates. Ensure that the aggregates are freely moving immediately after tapping (this can be checked under the microscope).

      2. Take caution to avoid splashing medium on the lid while tapping the plates.

      3. Thaw overnight at 4 °C on ice the amount of Matrigel needed the following day (see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment).

      4. We have used multiple Matrigel batches with comparable results.


    D7. TLS generation (88-96 h)

    NOTES:

    1. Start monitoring the TLSs around 88 h after aggregation for the appearance of a “teardrop-like” shape (see Figure 4C). Structures should present a longer axis (421 ± 33 μm) and a shorter axis (337 ± 30 μm), with an axis ratio of 0.8 ± 0.07.

    2. Start this procedure immediately when a “teardrop-like” shape is observed in the majority of the TLSs (or latest 96 h after aggregation) to achieve optimal TLS formation efficiency.

    3. If available, perform this step on a clean bench containing a stereoscope or a light ring to help visualize the structures and avoid losing structures while pipetting off the old medium.

    4. If performing chemical modulation at this step, follow the “Variant protocol: chemical modulation during TLS generation.”


    1. Equilibrate the required amount of NDiff 227 in a 10-cm dish in the incubator for at least 20 min (see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment).

    2. Transfer the required amount of NDiff 227 to a 50-ml Falcon tube and place it on ice (see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment).

    3. Once the medium has cooled down, supplement it with the correct volume of Matrigel on ice and mix vigorously.

      NOTE: It is critical, while handling Matrigel, that every step is performed on ice to avoid clumping. It is also recommended to pre-cool the pipet tips used for the handling of 100% Matrigel by placing the box in the fridge until use; see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment.

    4. Move the Falcon tube with Matrigel-supplemented medium to room temperature.

    5. Use a multichannel pipet to remove 150 μl from each well without disturbing the structure.

      NOTES:

      1. CRITICAL STEP. Avoid losing structures while pipetting out the medium. This is best achieved by keeping the plate under a 30-40° angle and putting the pipet tips against the side opposite to that where the aggregate should be located. As a visual aid, a stereoscope or light ring could be used as stated above (see Figure 5 for a schematic of how to position the plate and tips).

      2. This step should not take more than 5 min after Matrigel-supplemented medium has been placed at room temperature. If there are multiple plates, it is advisable to remove the medium from all plates before equilibrating the Matrigel-supplemented medium at room temperature and keep the structures in the incubator.

    6. Pour the Matrigel-supplemented medium into a reservoir.

    7. Use a multichannel pipet to add 150 μl Matrigel-supplemented medium in each well of the plates containing the aggregates.

    8. Gently tap the plate 10 times on a clean bench and transfer to the incubator.

      NOTES:

      1. The tapping is critical to prevent cell aggregates from attaching to the culture plates. Ensure that aggregates are freely moving immediately after tapping (this can be checked under the microscope).

      2. Take caution to avoid splashing medium on the lid while tapping the plates.

      3. If performing Live Cell Imaging after embedding, allow the TLSs to settle for 1 h in the incubator before starting imaging.


    Variant protocol: chemical modulation during TLS embedding

    Follow “D7. TLS generation (88-96 h)” for all passages except for point 3.

    3’. Once the medium has cooled down, supplement it with the correct volume of Matrigel and appropriate chemical compounds for your modulations on ice and mix vigorously.

    NOTES:

    1. It is critical, while handling Matrigel, that every step is performed on ice to avoid clumping; see Table 2 for the volume needed as a function of the number of 96-well plates used in the experiment.

    2. Add an equal volume of diluent to the control sample TLSs when performing chemical modulations.

    3. In Veenvliet et al. (2020), we induced excess somite production and compromised neural tube development by supplementing the medium with 5 μM CHIR99021, alone (TLSC) or in combination with 600 nM LDN193189 (TLSCL).


EXPECTED OUTCOME:

TLSs at 120 h should look elongated with a clear anterior and posterior domain (Figure 4D upper panel; Figure 6A). Moreover, they should show clear segmentation (somites) on one or both sides of a tubular structure (neural tube). TLSs subjected to chemical modulation (TLSCL) should display compromised neural tube development and formation of excess somites arranged like a “bunch-of-grapes” (Figure 4D lower panel; Figure 6B).



Figure 6. Expected outcome of the TLS protocol. A. Examples of trunk-like structures (TLSs) 24 h after addition of 5% Matrigel (total culture time 120 h). The left upper structure is immunostained with a SOX2 antibody and counterstained with DAPI, labeling the neural tube and nuclei, respectively. Somites are indicated with magenta arrowheads or with S1, S2, etc. (in magnifications); S1-S8, Somite 1-Somite 8. Neural tube (NT) is indicated with a green arrowhead. A, Anterior; P, Posterior. B. Expected outcome of TLSs subjected to chemical modulation (TLSCL) is compromised neural tube development and formation of excess somites arranged like a “bunch-of-grapes.” Somites are indicated with magenta arrowheads. Scale bars, 100 μm (whole structure) or 20 μm (magnifications). A, Anterior; P, Posterior.


D8. TLS analysis (108-120 h)

NOTES:

  1. Depending on the specific biological question, the exact time of analysis may vary (see Veenvliet et al., 2020 for time-resolved expression dynamics).

  2. If available, perform this step on a clean bench containing a stereoscope or a light ring to help visualize the structures and avoid losing structures while processing them.

  3. The following protocol variants are performed (D8’, D8’’, D8’’’) depending on the downstream applications.

  4. Prepare a P200 tip box with the tip cut-off at the 50-μl mark for TLS picking (if using unmarked tips, cut approximately 9 mm off the tip).

D8’. Staining and imaging
  1. Use a P200 pipet set to 50 μl and the cut-off tips to manually pick each individual TLS that needs to be analyzed in a well of an Ibidi 8-chamber plate.

  2. Add 200 μl cold PBS/0.5% BSA solution to each well containing a TLS.

  3. Remove 200 μl with a pipet and perform the same 200 μl cold PBS/0.5% BSA solution wash three times.

  4. Use the fixative of interest to fix TLSs in the Ibidi plate for the downstream protocol.

  5. Perform the rest of the staining protocol in the Ibidi plate and image the structure with the desired microscope and settings.

    NOTE: We have so far used 4% PFA fixation for whole-mount immunofluorescence (WIFC) as well as whole-mount in situ hybridization (WISH). A detailed description of the protocols used for WIFC and WISH, including method-specific fixation times and downstream processing, is provided in the Supplemental Information of Veenvliet et al. (2020).


D8’’. RNA extraction

  1. Use a P200 pipet set to 50 μl and the cut-off tips to manually pick and transfer each individual TLS that needs to be analyzed to a 1.5-ml tube containing 1 ml cold PBS/0.5% BSA solution.

    NOTE: The number of TLSs that are pooled in one tube depends on the downstream application and experiment.

  2. Centrifuge the TLSs at 200 × g for 1 min at 4°C.

  3. Remove the supernatant with a P1000 pipet while being careful not to disturb the TLS pellet.

  4. Wash the structures with 1 ml cold PBS/0.5% BSA solution.

    NOTE: Ensure loosening of the pellet without aspirating into the pipet tip.

  5. Centrifuge the TLSs at 200 × g for 1 min at 4°C.

  6. Remove the supernatant with a P1000 pipette while being careful not to disturb the TLS pellet.

  7. Add the indicated amount of RNA lysis buffer or TRIzol depending on the desired RNA extraction strategy.


D8’’’. 10× Genomics single-cell RNA sequencing

NOTES:

  1. This section explains how to process TLSs to generate a single cell suspension suitable for efficient Gel Bead-in-Emulsion (GEMs) generation. Follow the manufacturer’s instructions for every step after the single cell suspension has been generated and counted.

  2. Pre-warm TrypLE in the water bath for at least 20 min before starting.


  1. Use a P200 pipette set to 20 μl and the cut-off tips to manually pick each individual TLS that needs to be analyzed in a well of an ultra-low attachment 96-well plate containing 200 μl cold PBS.

  2. Transfer each TLS serially five times to new wells containing 200 μl cold PBS.

    NOTES:

    1. It is CRITICAL to carry over as little volume as possible from the culture to minimize the amount of Matrigel transferred. Carrying over excess amounts of Matrigel can lead to microfluidics clogging during GEM generation.

    2. Since washing of TLSs is performed in PBS without BSA, the structures may become sticky and get stuck to the tip wall. Avoid this by pipetting a very low volume to maintain the structure at the liquid/air interphase in the tip.

  3. After the five washes, transfer all the structures into a single drop of 200 μl pre-warmed TrypLE in the center of a 6-cm plate.

  4. Transfer the plate to the incubator and allow cell dissociation for 25 min with pipetting every 5 min to ensure that a single cell suspension is achieved.

    NOTE: Perform the pipetting steps under a stereoscope to monitor the degree of cell dissociation and ensure no loss of material.

  5. At the end of the 25 min, and after verifying correct achievement of a single cell suspension, transfer the cell suspension to a 1.5-ml tube on ice.

  6. To ensure maximum cell recovery and to quench the trypsinization reaction, wash the part of the plate where the drop was located four times with 200 μl PBS/0.5% BSA solution. Add every wash to the same tube (from step 5) containing the cell suspension.

  7. Filter the cell suspension using a P1000 set to 1 ml through a 40-µm Flowmi Cell Strainer in a new 1.5-ml tube on ice.

  8. Centrifuge the cell suspension at 300 × g for 5 min at 4°C.

  9. Remove 800 μl supernatant with a P1000 while being careful not to disturb the cell pellet.
    NOTE: The cell pellet may be very small and barely visible, so be extremely careful during these steps.

  10. Wash with 1 ml PBS/0.5% BSA solution.

  11. Centrifuge the cell suspension at 300 × g for 5 min at 4°C.

  12. Remove 800 μl supernatant with a P1000 while being careful not to disturb the cell pellet.
    NOTE: The cell pellet may be very small and barely visible, so be extremely careful during these steps.

  13. Resuspend the pellet in the remaining 200 μl left in the tube.

  14. Centrifuge the cell suspension at 300 × g for 5 min at 4°C.

  15. Remove the supernatant with a P200 pipet, leaving ~42 μl in the tube.

    NOTE: Use another tube containing exactly 42 μl PBS/0.5% BSA solution as a guide to evaluate the approximate volume to leave in the tube.

  16. Resuspend the cell pellet in the ~42 μl left and determine the cell suspension concentration using a manual hemocytometer (analyze a 1:5 cell suspension dilution by adding 2 μl cell suspension to 8 μl Trypan Blue).

  17. Proceed with the desired amount of cells for GEM generation following the manufacturer’s instructions.

Data analysis

All data and analysis needed for the development and characterization of this protocol are available in the main text or Supplemental Information of Veenvliet et al. (2020).

Recipes

  1. 0.1% Gelatin solution

    Dilute sterile 2% Gelatin to 0.1% in cell culture grade water. Store at 4°C

  2. Mouse embryonic fibroblast (MEF) medium

    NOTE: Heat inactivate the FCS for 30 min at 56°C before use.

    500 ml Dulbecco’s Modified Eagle's Medium (DMEM)

    55 ml regular FCS (Pan Biotech; catalog number: P30-3306)

    5.5 ml 100× Glutamine

    5.5 ml 100× Penicillin/Streptomycin

    Sterile filter

    Store at 4°C

  3. Mouse embryonic stem cell (mESC) medium

    NOTE: Heat inactivate the FCS for 30 min at 56°C before use.

    400 ml Knockout Dulbecco’s Modified Eagle’s Medium (KO-DMEM)

    75 ml mESC tested FCS (Pan Biotech; catalog number: P30-2602)

    5 ml 100× Glutamine

    5 ml 100× Penicillin/Streptomycin

    5 ml 100× Nucleosides

    1 ml Gibco 2-Mercaptoethanol

    Sterile filter

    Aliquot mESC medium without LIF in 40-ml portions and freeze at -20°C

    Thaw before use, then store at 4°C

    Add 1:10,000 LIF immediately before use

    Store at 4°C

    NOTE: Homemade LIF has also been successfully used; however, the right concentration has to be tested based on the purification protocol and batch concentrations.

  4. PBS/0.5% BSA solution

    PBS with MgCl2/CaCl2

    0.5% BSA powder

    Prepare fresh, sterile filter, keep on ice for the procedure

    Store at 4°C

Acknowledgments

This work is based on and adapted from the method published in Veenvliet et al. (2020). We are grateful for the support and feedback received to develop and characterize our in vitro system from present and past members of the Herrmann & Meissner laboratories, in particular Manuela Scholze-Wittler, Dennis Schifferl, Frederic Koch, Abhishek Sampath Kumar, Milena Pustet, Fabian Tobor, Simon Heimann, Lars Wittler, Stefanie Grosswendt, Zachary Smith and Atsuhiro Taguchi. The work was supported by an Alexander von Humboldt Fellowship (J.V.V.), NIH grant HG006193 (A.M.), and the Max Planck Society.

Competing interests

The authors declare no competing interests.

References

  1. Anlas, K., Baillie-Benson, P., Arato, K., Turner, D. A. and Trivedi, V. (2021). Gastruloids: Embryonic Organoids from Mouse Embryonic Stem Cells to Study Patterning and Development in Early Mammalian Embryos. Methods Mol Biol 2258: 131-147.
  2. Baillie-Benson, P., Moris, N. and Martinez Arias, A. (2020). Pluripotent stem cell models of early mammalian development. Curr Opin Cell Biol 66: 89-96.
  3. Baillie-Johnson, P., van den Brink, S. C., Balayo, T., Turner, D. A. and Martinez Arias, A. (2015). Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro. J Vis Exp(105).
  4. Beccari, L., Moris, N., Girgin, M., Turner, D. A., Baillie-Johnson, P., Cossy, A. C., Lutolf, M. P., Duboule, D. and Arias, A. M. (2018a). Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids. Nature 562(7726): 272-276.
  5. Beccari, L., Girgin, M., Turner, D., Baillie-Johnson, P., Cossy, A.-C., Moris, N., Lutolf, M., Duboule, D. and Martinez Arias, A. (2018b). Generating Gastruloids from Mouse Embryonic Stem Cells. Protocol Exchange. DOI: 10.1038/protex.2018.094.
  6. Cermola, F., D’Aniello, C., Tatè, R., De Cesare, D., Martinez-Arias, A., Minchiotti, G. and Patriarca, E. J. J. B. (2021). Gastruloid development competence discriminates different states of pluripotency between naïve and primed.Stem Cell Reports 16(2): 354-369.
  7. George, S. H., Gertsenstein, M., Vintersten, K., Korets-Smith, E., Murphy, J., Stevens, M. E., Haigh, J. J. and Nagy, A. (2007). Developmental and adult phenotyping directly from mutant embryonic stem cells. Proc Natl Acad Sci U S A 104(11): 4455-4460.
  8. Moris, N., Anlas, K., van den Brink, S. C., Alemany, A., Schroder, J., Ghimire, S., Balayo, T., van Oudenaarden, A. and Martinez Arias, A. (2020). An in vitro model of early anteroposterior organization during human development. Nature 582(7812): 410-415.
  9. Pourquié, O. (2003). The segmentation clock: converting embryonic time into spatial pattern. Science 301(5631): 328-330.
  10. Rossi, G., Broguiere, N., Miyamoto, M., Boni, A., Guiet, R., Girgin, M., Kelly, R. G., Kwon, C. and Lutolf, M. P. (2021). Capturing Cardiogenesis in Gastruloids. Cell Stem Cell 28(2):230-240.e6.
  11. Shahbazi, M. N., Siggia, E. D. and Zernicka-Goetz, M. (2019). Self-organization of stem cells into embryos: A window on early mammalian development. Science 364(6444): 948-951.
  12. Shahbazi, M. N. and Zernicka-Goetz, M. (2018). Deconstructing and reconstructing the mouse and human early embryo. Nat Cell Biol 20(8): 878-887.
  13. Turner, D. A., Girgin, M., Alonso-Crisostomo, L., Trivedi, V., Baillie-Johnson, P., Glodowski, C. R., Hayward, P. C., Collignon, J., Gustavsen, C., Serup, P., Steventon, B., M, P. L. and Arias, A. M. (2017). Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids. Development 144(21): 3894-3906.
  14. van den Brink, S. C., Alemany, A., van Batenburg, V., Moris, N., Blotenburg, M., Vivie, J., Baillie-Johnson, P., Nichols, J., Sonnen, K. F., Martinez Arias, A. and van Oudenaarden, A. (2020). Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. Nature 582(7812): 405-409.
  15. van den Brink, S. C., Baillie-Johnson, P., Balayo, T., Hadjantonakis, A. K., Nowotschin, S., Turner, D. A. and Martinez Arias, A. (2014). Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development 141(22): 4231-4242.
  16. 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).
  17. Veenvliet, J. V. and Herrmann, B. G. (2021). Modeling mammalian trunk development in a dish. Dev Biol. 2021 474:5-15.

简介

[摘要]植入后胚胎的哺乳动物涉及深刻分子,细胞,和形态结构的变化。这些高度动态的过程的研究由于子宫内发育的可及性有限而变得复杂。近年来,几种互补体外系统,其包括小鼠胚胎干细胞的自我组织组件,例如gastruloids ,已经报道了。我们最近证明,通过添加低百分比的Matrigel作为细胞外基质替代物,可以进一步释放类固醇的形态发生潜能。这导致带有神经管的高度组织化的躯干状结构(TLS)形成,该神经管经常两侧为双侧牙节。值得注意的是,在分子和形态发生水平上的发育高度让人联想到天然胚胎。为了方便访问此强大的模型,在此我们提供详细的分步协议,该协议应允许任何实验室都能使用标准细胞培养技术来实施培养系统。这将为用户提供一种以前所未有的时空分辨率研究早期妊娠中期小鼠胚胎发生的方法。

[背景]原肠胚形成和早期器官表示是成功产生功能体计划的关键发育事件。在哺乳动物中,这些过程只在胚胎植入后开始在子宫内,并在几天内,各种各样的形态和功能多样的组织出现。目前,很难在体内研究这些高度动态的变化,并且植入后小鼠胚胎的离体培养费力,昂贵并且需要严格的训练,这对于大多数实验室而言通常是不切实际的。这些障碍导致了大量的努力,以模型植入后和早期妊娠中期人的发展在体外用胚胎干细胞(综述Shahbazi; Shahbazi和Zernicka -戈茨2018 。等,2019;贝利-本森等人。2020年; Veenvliet和Herrmann,2021年)。特别是,植入后的发育可以用自组织的类胃体,小鼠或人类胚胎干细胞(mESC / hESC )聚集体进行建模(van den Brink等人,2014和2020;Moris等人,2020)。原始的小鼠胃二倍体培养方案可产生具有类似于枕后小鼠胚胎的类似胚胎的表达结构域的细长结构,并且三个体轴的位置正确,但形态发生受限(van den Brink等人,2014; Baillie-Johnson等人,2015;贝卡利等人,2018一和201 8B ;特纳。等人,2017)。最近的努力已经设法通过改变细胞环境来引入胚样形态特征,例如形成类似松节状结构或心管(van den Brink et al。,2020; Rossi et al。,202 1 )。进一步的进展已经表明,在加入细胞外基质(ECM)的替代物,以gastruloids Ç一个触发器的更胚样与肠管以及体系结构体节侧翼神经管(Veenvliet等人,2020)。我们戏称这些胚胎组织体干状结构(TLSS),因为它们类似于早期妊娠中期的树干的核心部分人胚胎(〜萌芽阶段(E)8.5-9)。重要的是,在TLS诱导的时间范围内(聚集后96-120小时),基因调控程序与发育中的胚胎高度相似。此外,分割时钟,振荡器驱动的节律沉积体节体内,是活性在胚样步伐在TLS(Pourquié ,2003; Veenvliet 。等人,2020)。

TLS模型易于访问,跟踪,操纵和缩放,这使其成为研究碟中植入后和妊娠中期早期哺乳动物发育的强大工具。在这里,我们提供了一个全面的分步过程,以简化树干状结构的生成。我们还描述了如何处理TLS进行下游分析,包括整个安装的免疫荧光染色和(单细胞)RNA测序。

关键字:像树干的结构, 原肠胚形成, 体节, 自我组织, 形态发生, 类原肠胚, 胚状体, 类器官, 体外模型, 干细胞



材料和试剂


1.移液器吸头,可变容量(Biozym ,SafeSeal SurPhob VT)     

2. 1.5毫升试管(Sarstedt,目录号:72.706)     

3. 15毫升猎鹰管(Sarstedt,目录号:62.554.502)     

4. 50 ml猎鹰管(Sarstedt,目录号:62.547.254)     

5. 6厘米细胞培养板(Sarstedt ,目录号:83.3901.300)     

6.超低附件96孔板(Corning,Costar,目录号:CLS7007)     

7. 6孔细胞培养板(Corning,目录号:3516)     

8. 10厘米细胞培养板(Corning,目录号:430167)     

9. Luna细胞计数玻片(Logos Biosystems,目录号:L12001)     

10. µ-Slide 8孔玻璃底部(Ibidi ,目录号:80827) 

11. 40 µm Flowmi细胞过滤器(Merck,目录号:BAH136800040) 

12.瓶顶真空过滤器单元(Corning,目录号:CLS431096) 

13. KnockOut DMEM(Gibco,目录号:10829018) 

14. 100 ×青霉素(5000U / ml)的-链霉素(5 ,000微克/毫升)(Lonza公司,目录号:DE17-603E) 

15. 100 ×谷氨酰胺,200 mM(Lonza,目录号:BE17-605E) 

16. 100 ×核苷(Sigma,目录号:ES-008D) 

17. Gibco 2-巯基乙醇,在DPBS中的55 mM溶液(Gibco,目录号:21985023) 

18.定期(Pan Biotech,目录号:P30-3306)和经过测试的合格和胚胎干细胞培养物(Pan Biotech,目录号:P30-2602)的胎牛血清(FCS) 

19. TrypLE (Gibco,目录号:12604013)或0.05%胰蛋白酶-EDTA(1 x )(Gibco,目录号:25300-054) 

20. NDiff 227培养基(宝酒,商品目录号:Y40002) 

21. CHIR99021 InSolution (Sigma,目录号:361571)或10mM在二甲SUL ˚F氧化物(DMSO)(Tocris Biosciences公司,目录号:4423) 

22. LDN193189(Reprocell ,目录号:04-0074-10) 

23. DMSO(Sigma,目录号:D2650) 

24.降低的基质胶生长因子(GFR),不含苯酚红(Corning,目录号:356231)–已测试了多个批次/批次,在产生类似树干的结构方面,产生了相似的结果 

25. 2%明胶溶液(Sigma,目录号:G1393) 

26. DPBS,不含MgCl 2 / CaCl 2 (Gibco,目录号:14190144) 

27.含MgCl 2 / CaCl 2的PBS (Sigma,目录号:D8662) 

28.小鼠白血病抑制因子(LIF)ESGRO TM (10 7 U / ml)(Millipore,目录号:ESG1107) 

29.锥虫蓝(Bio-Rad,目录号:1450021) 

30.超纯DNA酶/ RNA酶-免费蒸馏水(Invitrogen,目录号:10977049) 

31.试剂容器60毫升(默克,目录号:BR703411) 

32.牛血清白蛋白粉(BSA)(西格玛,目录号:A2153) 

33.杜比柯克改良的伊格尔培养基(DMEM)4,500 mg / ml葡萄糖,不含丙酮酸钠(Lonza,目录号:BE12-733F) 

34.细胞培养级水(Lonza,目录号:BE17-724Q) 

35. 0.1%明胶溶液(请参阅食谱) 

36.小鼠胚胎成纤维细胞(MEF)培养基(请参阅食谱) 

37.小鼠胚胎干细胞(mESC )培养基(请参阅食谱) 

38. PBS / 0.5%BSA溶液(请参阅食谱) 



设备


1.生物安全柜(Thermo Fisher Scientific,型号:Herasafe KS12)     

2.清洁水平层流罩(Thermo Fisher Scientific,型号:HeraGuard ECO)     

3.细胞培养培养箱(Thermo Fisher Scientific,型号:Heracell Vios 160i)     

4.细胞培养离心机(Eppendorf,型号:Centrifuge 5804R)     

5.可变容积移液管和移液管的多通道(的Eppendorf,型号:研究®加吸管)     

6.水平光源,光环(Nikon,P-DF LED暗场单元)或其他立体显微镜支架     

7.自动化细胞计数器(Logos生物系统,Luna自动化细胞计数器,L10001)     

8.细胞培养水浴(LAUDA Aqualine ,目录号:AL18)     

9.组织培养真空泵(Vacuubrand ,目录号:20727200)     

10.微量离心机(Eppendorf,型号:5424R) 



设备设置:


1.细胞培养箱设置为37°C,5%CO 2 。     

注意:我们还成功以7.5%CO 2生成了TLS ,但通常使用5%。


2.细胞培养水浴(设定为37°C)。     

3.所有离心机通货膨胀步骤在室温下进行,除非另有说明。     



程序


播种小鼠胚胎成纤维细胞(MEF)
笔记:


种子的MEF至少提前一天到播种mESCs 。
开始之前,将MEF介质在水浴中预热至少20分钟。
MEF板应在一周内使用的播种。
1.用3 ml 0.1%明胶溶液涂覆6 cm细胞培养板。     

注:明胶-包被的培养板必须是对接种的MEF的当天新鲜制备,并且可以没有吨被存储。


2.将板在室温下放置15分钟。     

接下来,在水浴中于37°C解冻有丝分裂惰性的MEF小瓶。


笔记:


使用丝裂霉素C处理(在37°C下3小时)可在室内将无活性的MEF进行有丝分裂灭活。
您需要1.0 × 10 6 MEF来覆盖6 cm细胞培养板。解冻适当的数量,取决于所需的6厘米细胞培养板的数量。
3.将MEF添加到装有5 ml预热MEF培养基的15 ml Falcon管中。     

4.将细胞以200 × g离心5分钟。     

5.离心时,从每个6 cm板上吸出明胶,然后加入2 ml MEF培养基。     

6.从装有MEF的15毫升试管中吸出上清液,以每毫升1.0× 10 6个细胞的浓度重悬细胞沉淀。     

注意:冷冻时会计算可行的MEF,解冻后无需再次计算。


7.将1 ml细胞悬液添加到每个准备的6 cm平板中。     

8.播CE板在培养箱,摇动板以使细胞均匀分布。     



播种小鼠胚胎干细胞(mESCs )
笔记:


开始之前,将mESC介质在水浴中预热至少20分钟。
我们通常使用具有F1G4遗传背景的mESC来生成TLS协议(George等,2007)。
1.电镀前,立即在水浴中于37°C解冻一小瓶mESC 。     

注意:6 cm MEF涂层板需要3.5× 10 5 mESC 。解冻适当的数量,取决于所需的6厘米细胞培养板的数量。


2.将mESC加入装有5 ml预热mESC培养基的15 ml Falcon管中。     

3.离心将细胞在200 ×g下于5分钟。     

4.离心时,从每个含MEF的6厘米平板中吸出MEF培养基,然后加入2 ml mESC培养基。     

5.从装有mESC的15毫升试管中吸出上清液,并以每毫升3.5×10 5个细胞的浓度重悬细胞沉淀。     

注意:冷冻时会计算存活的mESC ,解冻后无需再次计数。


6.将1 ml细胞悬液添加到每个涂有MEF的6 cm板上。     

7.将板放入培养箱中并旋转板以确保细胞均匀分布。     

8.替换用3ml新鲜每日介质mESC的平台。     



传代的胚胎干细胞
笔记:


每48小时以1:8-1:10的分流比通过mESC 。菌落密度和形态应该看起来类似于该所示图1A 。不要让您的文化过度发展(图1B )。
此处详细说明的分裂时间和比例已针对Veenvliet等人使用的mESC系进行了优化。(2020)。基于所述的增殖速率mESC的线使用,分裂倍和比M AY需要调整。这可能是,如果转基因品系和/或尤其如此mESCs具有不同遗传背景的小号被使用。
开始之前,将mESC培养基和TrypLE在水浴中预热至少20分钟。
制备MEF -包被的板前一天到传代mESCs 。
代替TrypLE ,可以使用0.05%的胰蛋白酶-EDTA。






图1.成功生成TLS的最佳胚胎干细胞培养密度。A. mESC的培养密度适合能够为TLS代(24 ^ h和接种后48小时)。B. mESC的培养物密度的未套装能够用于TLS代(接种后96小时)。比例尺为所有面板,50微米。


1.从mESC板中吸出培养基,并用3 ml DPBS洗涤。     

2.吸出DPBS并添加1 ml TrypLE 。     

3.确保该板表面被均匀地覆盖着的TrypLE ,并将其放置在培养箱中在37℃下5分钟。     

4.分钟后5,移去具有P1000吸管设置为800菌落μ在板通过上下吸移升20倍。     

5.加入1 ml mESC培养基灭活TrypLE ,并进一步吸移以获得单细胞悬液。     

6.细胞悬液转移到15微米升Falcon管和洗涤板与一个附加3毫升mESC的介质恢复所有细胞。将这些细胞转移到相同的15 ml管中。     

7.将细胞以200 ×g离心5分钟。     

8.离心时,从先前准备的含MEF的6厘米平板中吸出MEF培养基,然后加入2 ml mESC培养基。     

9.从装有mESC的15 ml管中吸出上清液,并将细胞沉淀重悬于2 ml mESCs培养基中。     

10.添加适当的细胞悬浮液的量,以每MEF -涂覆6厘米板(比例为1:8-1:10  200-250 μ 1)。调整最终体积至3ml。 

11.将板放入培养箱中并旋转板以确保细胞均匀分布。 

12.更换培养基每天用3ml新鲜预热的mESC的平台。 



生成类似树干的结构(TLS)
笔记:


此处详述的每个孔的输入细胞数均已针对Veenvliet等人使用的mESC系进行了优化。(2020)。基于所述的增殖率mESC的线路(特别是转基因品系和/或mESCs与不同的遗传背景),细胞量M AY需要进行调整,以达到在所报告的相同的效率Veenvliet等。(2020)。
我们建议第一optimiz荷兰国际集团的标准gastruloid为新的细胞系的协议,使用gastruloid伸长效率作为快速试验读数(Cermola等人,20 21 )。根据我们的经验,良好的gastruloid伸长率(> 95%)是必不可少的实现类似TLS效率到在报道Veenvliet等。(2020)。甲常规优化程序涉及的100个播种- 600 mESCs每孔,用50层的逐步增加的细胞。
开始之前,mESC必须培养至少一代。
开始之前,将mESC培养基和TrypLE在水浴中预热至少20分钟。
在这里,我们使用市售的,质量控制的NDiff 227培养基(N2B27)。我们和其他人成功生成gastruloids自制N2B27(贝卡利等人,201。8B ); ^ h H但是,在我们的手中,更强大的结果gastruloid和TLS协议与获得NDiff 227中。
代替TrypLE ,可以使用0.05%的胰蛋白酶-EDTA。
TLS生成协议的示意图概述指出了关键时间点,如图2所示。






图2. TLS生成协议的示意图。从MEF播种到下游分析,生成树干状结构(TLS)的工作流程。MG,Matrigel;CL,CHIR + LDN;MEF,小鼠胚胎成纤维细胞;mESCs ,小鼠胚胎干细胞。


D1 准备6孔板进行MEF耗尽


1.在6孔板的三个孔上每6 cm的板涂2 ml 0.1%g的Elatin溶液,以用于TLS生成。     

2.在室温下孵育6孔板15分钟。     

3.吸出明胶溶液,并向每个孔中加入1 ml mESC培养基。     

4.将板存放在培养箱中直至使用。     



D2。准备单细胞悬液


1.从mESC板中吸出培养基,并用3 ml DPBS洗涤。     

2.吸出DPBS并添加1 ml TrypLE 。     

3.确保该板表面被均匀地覆盖着的TrypLE ,并将其放置在培养箱中在37℃下进行5分钟。     

4.分钟后5,移去具有P1000吸管设置为800菌落μ在板通过上下吸移升20倍。     

5.加入1 ml mESC培养基和移液混合液,使TrypLE失活。     

6.转移细胞悬液至15ml Falcon管中,并洗涤板与一个附加3毫升mESC的介质恢复所有细胞。将这些细胞转移到相同的15 ml管中。     

7.将细胞以200 ×g离心5分钟。     

8.将细胞沉淀重悬于1 ml mESC培养基中,上下吸移50次。     

注意:在这里,它是实现适当的单细胞悬浮液,以避免丢失关键mESCs (或保留荷兰国际集团的MEF)期间MEF耗尽,并确保最佳的协议性能。我们建议检查荷兰国际集团的一个在显微镜下适当的单细胞悬液。


D3。MEF耗竭


注意:图3中提供了一个示例,其中每个单元的MEF耗尽后,细胞附着在底部的孔的外观如何。






图3. mESC聚集前的MEF消耗。MEF附着在0.1%明胶包被的孔上。比例尺,50μm 。


Ñ OTE :随着连续传输,所述量附着的细胞的减少。第三次孵育后,MEF消耗完成,mESC准备用于聚集。


1.将获得的细胞悬液转移到准备好的6孔板的一个孔中(参见D1)。     

注意:将一块6 cm平板中存在的mESC转移到准备好的6孔平板的一孔中。在P太多细胞resence可能导致降低消耗效率。


2.移液混合10次。     

3.将板放入培养箱中并旋转板以确保细胞均匀分布。静置25分钟。     

4.接下来,使用设置为1 ml的P1000移液器小心地将所有悬浮的细胞转移到另一孔中。     

注:关键是不要撞出MEF中,这是附着在底部井。


5.在新孔中上下吸移10次,以确保单细胞悬浮液。     

注意:孵育过程中细胞可能会结块;因此,一旦将细胞转移到新孔中,就必须用移液器吸移。我们建议,确认在显微镜下是你已经获得适当的单细胞悬液。


6.将板放入培养箱中并旋转板以确保细胞均匀分布。静置20分钟。     

7.接下来,使用设置为1 ml的P1000移液器将所有悬浮的细胞小心地转移到另一孔中。     

注:关键是不要撞出MEF中,这是附着在底部井。


8.吸取上下在新井10次,以确保一个单细胞悬液。     

注意:孵育过程中细胞可能会结块;因此,一旦细胞转移到新孔中,就必须用移液管吸移。我们建议,确认在显微镜下是你已经获得适当的单细胞悬液。


9.将板放入培养箱中并旋转板以确保细胞均匀分布。静置15分钟。     

10.在这个最后的15 -分钟步骤,平衡的requir的ED量NDiff在培养箱10cm皿至少20分钟227。也可以进行更长的孵育(例如,可以将NDiff 227放置在步骤6之后的孵育箱中。请参见表1 ,以获取所需体积作为接种96孔板的数量的函数)。 

注意:NDiff 227对光敏感,应尽可能避免(直射)光。


11.所有MEF转移小心-耗尽mESCs至15ml Falcon管中使用P1000吸管。 

注:关键是不要撞出MEF中,这是附着在底部井。


D4。m ESC聚集(0小时)


注:TLS协议的第一个96小时是相似的gastruloids协议(。贝利-Johnson等人,2015;凌科等人,2014;贝卡利。等人,2011 8B ; Anlas 。等人,2021) 。在其他地方提供了有关形成类固醇的详细协议(包括故障排除)(Baillie-Johnson等人,2015;Beccari等人,201 8b ;Anlas等人,2021)。


1.将细胞以200 ×g离心5分钟。     

2.将细胞沉淀重悬于含MgCl 2 / CaCl 2的5 ml PBS中,上下吸移20次(洗涤1)。     

3.将细胞以200 ×g离心5分钟。     

注意:如果起始单元格数目少,则可以省略步骤2和3。这个米AY然而稍微折衷协议效率。


4.将细胞沉淀重悬于5 ml预先平衡的NDiff 227中,上下吸移20次(洗涤2)。     

5.将细胞以200 ×g离心5分钟。     

6.重悬在500细胞沉淀μ升预平衡NDiff 227和吸管上下30次。注意:在计数和铺板之前,获得单细胞悬液至关重要。     

7.通过将10 2稀释的细胞悬浮液:对于计数,制备1 μ升细胞悬浮液至10 μ升台盼蓝。     

8.使用Luna自动细胞计数器进行以下设置的计数:稀释系数2 ;降噪5 ;实时检测灵敏度5 ;圆度85 %; 最小单元尺寸 10 μ米; 最大小区大小 20 μ米; 整理水平高。     

9.传输量所需的实验到一个新的Falcon管细胞(见表1对于在需要作为96孔板到种子的数量的函数的细胞数)。     

10.添加预培养NDiff 227体积requir编以使细胞悬浮液以5.7的浓度× 10 3个细胞/ ml(参照表1对的体积添加为96孔板到种子的数目的函数)。 

注意:此细胞浓度针对200个细胞/孔的输入量进行了优化,已显示可为所有测试的细胞系提供高TLS生成效率(Veenvliet等,2020)。对于200到250个细胞/孔的输入,获得了相似的结果。


11.剧烈混合新细胞悬液,然后将其转移到储液槽中。 

12.使用多通道移液器以35转μ升至每个孔超低附着96孔板的。每次移液之间,用吸管轻轻向上和向下移液。 

13.轻轻敲击板5次上一个净化台,转移到培养箱中,并允许不受干扰聚合48小时。 

注意:将NDiff 227放在培养箱外的时间较长(超过5分钟)会导致培养基失衡。因此,请尽量避免将NDiff 227或含有NDiff 227中新鲜种子细胞的培养板放置在培养箱外的时间过长。在该情况下的HANDL荷兰国际集团的多个板,我们建议移液之后将每个板放入培养箱直接。


表1细胞数和卷requir编为mESC的聚集。的量被计算为200个细胞/孔在35的输入μ升。对于每块板100个样品(而不是96个),再加上10%的死体积,计算第4列中的体积和细胞数。在柱2的体积NDiff 227平衡来计算基于所需的洗涤量和计数细胞(每个实验5.5毫升,独立的的板的数目,见步骤D4.4和D4.6),加上该第4栏中指出的数量,加上额外的体积以解决培养皿中死角和培养基平衡期间的蒸发。


数的96孔板


NDiff 227达到平衡的体积(毫升)


所需的细胞总数


达到5.7× 10 3个细胞/ ml(ml)所需的NDiff 227体积


1个


12


2.2 × 10 4


3.85减去细胞体积


2个


16


4.4 × 10 4


7.70减去细胞体积


3


20


6.6 × 10 4


11.55减去细胞体积


4


24


8.8 × 10 4


15.40减去细胞体积


5


28岁


11 × 10 4


19.25减去细胞体积




D5。CHIR脉冲(48小时)


笔记:


在48小时结束前至少一小时开始此过程。
在48小时的细胞应该形成有直径测量214一个单轮骨料± 13 μ M(参照图4A )。
CHIR99021对光敏感,应尽可能避免(直射)光。
到达后,应将CHIR99021分成几等分装在棕色(避光)无菌试管中,并且不要反复进行冻融循环。 
 
平衡的所需要的量NDiff在10 227 -在培养箱cm培养皿为至少20分钟。(请参阅表2 ,了解所需体积与实验中使用的96孔板数量的关系)。
转印requir的ED量NDiff 227 50 -毫升Falcon管中(见表2用于将需要作为在实验中使用96孔板的数目的函数体积)。


表2的卷NDiff 227,CHIR99021 ,和Matrigel requir最后三个步骤期间ED(协议步骤D5,D6,D7)的TLS生成协议的。在第2列中,计算出要平衡的NDiff 227的体积,其中包括多余的体积(每个培养皿2 ml用于培养基平衡),以说明培养皿中的死空间和平衡过程中的培养基蒸发。


数的96孔板


NDiff 227的体积以平衡


(D5,D6,D7)


(毫升)


的体积NDiff 227在50转移-毫升管/ s的


(D5,D6)


(毫升)**


的体积NDiff 227在50转移-毫升管/ s的


(D7)


(毫升)**


10mM的CHIR的体积99021添加3 μ M(D5只)(μ升)


最终添加5%的基质胶的体积(仅D7)(ml)***


1个


18岁


16


15.01


4.8


0.99


2个


38 *


32


30.0 3


9.6


1.97


3


52 *


48


45.0 4


14.4


2.96


4


70 *


64


60.05


19.2


3.9 5


5


86 *


80


75.06


24


4.9 4


*对于大于30 ml的体积,请使用一块以上的10 cm平板在培养箱中平衡NDiff 227。


**如果容量超过50毫升,请使用两个50毫升试管。


***注我们计算5%V / V的Matrigel中每个孔(35最终体积的函数μ升+ 150微升= 185微升)(相对于CHIR99021)。这意味着在NDiff 227中,Matrigel的浓度略高于5%(6.17%)。例如,对于1盘TLS,计算的基质胶体积为(0.05 * 16)* (185/150)= 0.987 ml。


添加CHIR99021到所述NDiff 227介质中以获得3的最终浓度μ M.
剧烈混合培养基,然后将其转移到储存器中。
使用多通道移液器以添加150 μ升CHIR 99021 -补充的培养基到每个孔中含有的聚集体的板。
轻轻敲击板10倍一个净化台,转移到培养箱中,并允许进一步不受干扰发展为24小时。
笔记:


敲击对于防止细胞聚集体附着在培养板上至关重要。确保的聚集体被自由地轻敲后立即移动(这可以在显微镜下进行检查)。
轻敲板时,请小心避免介质溅到盖上。






图4.在TLS生成期间的几个时间点,来自m ESC的聚集体的预期形态的示例。甲,B.米ESC衍生的聚集体在48 ħ和聚合72小时后是圆对称无断裂的明显迹象。C.聚集后96小时,这些结构的对称性明显破坏,其水滴形状为d 。白色箭头指示后极,预计在此处出现Brachyury的局部表达。请注意,根据所使用的细胞系,聚集体可在96小时之前建立类似泪滴的形态。在这种情况下,结构应提前(只要嵌入在基质胶的泪滴状形态观察)以实现最佳的效率TLS(详见正文)。D.在添加5%基质胶,所述聚集体将建立一个体系结构让人联想到胚胎躯干,用体节侧翼神经管(绿色(品红色箭头)箭头)。用WNT激动剂(5化学调制μM CHIR99021)和BMP抑制剂(600 nM的LDN193189)导致受损神经管的开发和形成过量的体节状排列“束-的-葡萄”(TLS CL )。比例尺为所有面板,50微米。A,前;P,后部。


D6。介质更换(72小时)


笔记:


在72小时结束前至少一小时开始此过程。
在72小时的聚集体应该像在给定的例子图4B的ð测量244 ± 15 μ直径米。
如果可用,请在干净的工作台上执行此步骤,该工作台包含立体镜或光环,以帮助可视化结构,并避免在移出旧介质时丢失结构。
平衡的requir的ED量NDiff 227在一个10 -在培养箱cm培养皿至少20分钟(参见表2对于在需要作为在实验中使用96孔板的数目的函数体积)。
将所需量的NDiff 227转移到50 ml试管中(所需的体积与实验中使用的96孔板数量的关系,请参见表2 )。
使用多通道移液器以除去150 μ从每个孔升,而不会干扰的结构。
注意:关键步骤。避免丢失结构,同时吸取掉介质。这最好由30-40°的角度下保持板和使移液管尖端抵靠在相对的一侧来实现,以吨帽子,其中聚集体应位于。作为视觉辅助,可以如上所述使用立体镜或光环(有关如何放置板和笔尖的示意图,请参见图5 )。






图5.在TLS生成过程中,用于更换介质的印版位置的示意图。该板是倾斜在一个30-40 °角在净化台上将与媒体小心地用多通道移液器吸出,避免干扰的ANCE聚集小号。


将预先平衡的培养基倒入储液罐中。
使用多通道移液器150添加μ升介质到含有聚集体板的每个孔中。
轻轻敲击板10倍一个净化台,转移到培养箱中,并允许进一步不受干扰发展16- 24小时。
笔记:


敲击对于防止细胞聚集体附着在培养板上至关重要。确保的聚集体被自由地轻敲后立即移动(这可以在显微镜下进行检查)。
轻敲板时,请小心避免介质溅到盖上。
 
在冰上4℃解冻过夜所需基质胶的量翌日(参照表2为对所需的数量的函数的体积在实验中使用96孔板的)。
我们使用了多个Matrigel batc hes,并获得了可比的结果。


D7。TLS生成(88-96小时)


没有测试:


聚集后约88小时开始监视TLS,以观察是否出现“泪滴状”形状(见图4C )。结构应呈现较长的轴(421 ± 33 μ M)和较短的轴线(337 ± 30 μ M) ,用斧头我S比为0.8 ± 0.07。
当在大多数TLS中(或聚集后最近96小时)观察到“泪滴状”形状时,请立即开始此过程,以实现最佳的TLS形成效率。
如果可用,执行此步骤上含有一个立体镜或光环以帮助观察的结构和避免净化台失去结构,同时吸移离原来的培养基。
如果在此步骤执行化学调制,请遵循“变体协议:TLS生成过程中的化学调制” 。”
平衡的requir的ED量NDiff 227在一个10 -在培养箱cm培养皿至少20分钟(参照表2对的需要作为在实验中使用96孔板的数目的函数体积)。
转印requir的ED量NDiff 227 50 -毫升Falcon管中,并将其放置在冰(见表2对于在需要作为在实验中使用96孔板的数目的函数体积)。
培养基冷却后,在冰上补充正确体积的Matrigel,并剧烈混合。
注意:在处理Matrigel时,至关重要的是每一步都要在冰上进行,以免结块。它也建议进行预冷却用于枪头的通过将箱子放进冰箱UNT 100%的基质胶处理IL使用; 所需的体积与实验中使用的96孔板数量的关系,请参见表2 。


将装有基质胶的培养基将猎鹰管移至室温。
使用多通道移液器以除去150 μ从每个孔升,而不会干扰的结构。
笔记:


关键步骤。移出培养基时,避免丢失结构。这最好由30-40°的角度下保持板和使移液管尖端抵靠在相对的一侧实现第在其中聚集体应位于。作为视觉辅助,可以如上所述使用立体镜或光环(有关如何放置板和笔尖的示意图,请参见图5 )。 
 
将基质胶-补充培养基置于室温后,此步骤不应超过5分钟。如果有多个平板,建议在室温下平衡补充了基质胶的培养基之前,先从所有平板中除去培养基,然后将结构保留在培养箱中。
将补充了基质胶的培养基倒入容器中。
使用多通道移液器以添加150 μ在每个升基质胶的培养基以及含有该聚集体的板。
在干净的工作台上轻轻敲打板10次,然后转移到培养箱中。
笔记:


敲击对于防止细胞聚集体附着在培养板上至关重要。敲击后,请确保聚集体立即自由移动(可以在显微镜下检查)。
轻敲板时,请小心避免介质溅到盖上。
如果在嵌入后进行活细胞成像,则在开始成像之前,让TLS在培养箱中静置1小时。


变体协议:TLS嵌入期间的化学调制


遵循“ D7。TLS生成(88-96 h)”,适用于除第3点之外的所有段落。


3'。一旦介质冷却下来,用它补充正确基质胶的数量和适当的化学化合物在冰上的调制和剧烈混合。


笔记:


在处理Matrigel时,至关重要的是每一步都要在冰上进行,以免结块。看表2用于将需要作为在实验中使用96孔板的数目的函数的体积。
执行化学调制时,向对照样品TLS中添加等体积的稀释剂。
在Veenvliet等。(2020) ,我们诱导的过量体节的生产和受损神经管的开发通过补充用5介质μ中号CHIR 99021 ,单独(TLS ç )或与600nM的LDN193189(TLS组合CL )。


预期结果:


TLS小号在120h时应该看起来拉长具有明确的前部和后部结构域(图4D上面板;图6A )。此外,它们应在管状结构(神经管)的一侧或两侧显示清晰的分割(节)。TLS小号进行化学调制(TLS CL )应该显示受损神经管的开发和形成过量的体节状排列“束-的-葡萄”(图4D下面板;图6乙)。






图6. TLS协议的预期结果。A.添加5%Matrigel后24小时(总培养时间120小时)的躯干状结构(TLS)的示例。左上部结构用SOX2抗体免疫染色,并用DAPI复染,分别标记神经管和细胞核。紫红色箭头或S1,S2等表示山梨。(以放大倍数);S1-S8,Somite 1-Somite8。神经管(NT)用绿色箭头指示。A,前;P,后部。B.接受化学调制的TLS的预期结果(TLS CL )损害了神经管的发育,并形成了像“葡萄束”一样排列的多余体节。”体节都标有红色箭头。100μm (整个结构)或20μm (放大倍数)的比例尺。A,前;P,后部。


D8。TLS分析(108-120小时)


笔记:


根据特定的生物学问题,分析米的准确时间AY变化(参见Veenvliet等人,2020年时间分辨表达动力学)。
如果可用,则执行包含立体镜或光环以帮助观察的结构,并避免在净化台上将本步骤中失去结构小号同时处理它们。
根据下游应用程序,执行以下协议变体(D8',D8'',D8''')。
准备与尖端截止P200的提示框在50 -为TLS拾取微升标记(如果使用未标记的提示,切大约为9mm的˚F的尖端)。


D8'。染色和成像


使用P200移液管组50微升和截止提示手动挑每个单独的TLS需要在良好的的被分析Ibidi 8 -室板。
向每个包含TLS的孔中加入200μl冷PBS / 0.5%BSA溶液。
用移液器移出200μl ,并用相同的200μl冷PBS / 0.5%BSA溶液洗涤3次。
使用感兴趣的固定剂来固定TLSS在所述Ibidi板用于下游协议。
在Ibidi板中执行其余的染色方案,并使用所需的显微镜和设置对结构进行成像。
注意:到目前为止,我们已使用4%PFA固定进行全量免疫荧光(WIFC)和全量原位杂交(WISH)。Veenvliet等人的补充信息中提供了用于WIFC和WISH的协议的详细说明,包括特定于方法的固定时间和下游处理。(2020)。


D8''。RNA提取


使用P200移液管组50微升和截止提示手动拾取和传送每个单独的TLS需要被分析,以一个1.5 -含有1ml冷的PBS / 0.5%BSA溶液毫升管。
注意:在一个管中合并的TLS数量取决于下游应用程序和实验。


在4°C下将TLS以200 × g离心1分钟。
用P1000移液器移出上清液,同时注意不要干扰TLS沉淀。
用1 ml冷PBS / 0.5%BSA溶液洗涤结构。
注意:确保松开沉淀,不要吸入移液器吸头。


在4°C下将TLS以200 × g离心1分钟。
用P1000移液器除去上清液,同时注意不要干扰TLS沉淀。
根据所需的RNA提取策略,添加指示量的RNA裂解缓冲液或T RI zol 。


D8''。10 × G ^ enomics单-细胞RNA测序


笔记:


本节说明如何处理TLS,以生成适合高效凝胶珠粒乳液(GEM)生成的单细胞悬液。生成并计数单细胞悬液后,请按照制造商的说明进行每个步骤。
开始之前,将TrypLE在水浴中预热至少20分钟。
使用移液管P200设定为20微升和截止提示手动挑每个单独的TLS需要在良好的超低附着的被分析96孔板含有200微升冷PBS。
将每个TLS连续五次转移至含有200μl冷PBS的新孔中。
笔记:


至关重要的是从培养物中带走尽可能少的体积,以最大程度地减少基质胶的转移量。携带过量的Matrigel可能会导致GEM生成过程中的微流体堵塞。
由于洗涤的TLSS执行在PBS中不含BSA,所述结构中的m AY变粘和卡住到末端壁。通过吸取非常小的体积来避免这种情况,以将结构保持在尖端的液体/空气界面。
五个洗涤后,吨转让(BOT)的所有结构成的200一滴微升预热的TrypLE在一个6的中心-厘米板。
所述板转移到培养箱中,并允许细胞解离25分钟,移取每5分钟,以确保该一个单细胞悬浮液实现。
注:执行移液步骤立体镜,以监察细胞分离的程度,并确保没有了洛小号的材料。


在的25分钟结束时,和验证单细胞悬浮液的正确实现后,将细胞悬浮液转移到1.5 - ml管在冰上。
为了确保最大的细胞回收,并以猝灭胰蛋白酶反应,洗其中滴板的部分位于四次用200微升PBS / 0.5%BSA溶液。将所有洗液添加到装有细胞悬液的同一管中(来自步骤5)。
通过40过滤使用P1000设置为1毫升细胞悬浮液-微米Flowmi细胞过滤器在一个新的1.5 -在冰上毫升管。
将细胞悬液在4°C下以300 ×g离心5分钟。
除去800微升用P1000上清液同时小心不要扰乱细胞沉淀。
注意:细胞沉淀米AY非常小,几乎看不出来,所以要在这些步骤要非常小心。
用1 ml PBS / 0.5%BSA溶液洗涤。
将细胞悬液在4°C下以300 ×g离心5分钟。
除去800微升用P1000上清液同时小心不要扰乱细胞沉淀。
注意:细胞沉淀米AY非常小,几乎看不出来,所以要在这些步骤要非常小心。
将沉淀重悬在试管中剩余的200μl溶液中。
将细胞悬液在4°C下以300 ×g离心5分钟。
删除与P200移液管上清液,留下〜42微升在管中。
注意:使用含有精确的42另一管微升PBS / 0.5%BSA溶液作为引导来评价近似体积在管离开。


重悬细胞沉淀在〜42微升的左和确定的使用手动血球细胞悬浮液的浓度(分析:5细胞悬浮液稀释1通过加入2微升细胞悬浮液至8微升台盼蓝)。
按照制造商的说明,继续进行所需数量的细胞以生成GEM 。


数据分析


Veenvliet等人的正文或补充信息中提供了开发和表征此协议所需的所有数据和分析。(2020)。


菜谱


1. 0.1%明胶溶液     

在细胞培养级水中将无菌2%明胶稀释至0.1%。储存在4°C


2.小鼠胚胎成纤维细胞(MEF)培养基     

注意:使用前,在56°C加热灭活FCS 30分钟。


500毫升Dulbecco's Modified Eagle's Medium(DMEM)


55毫升常规FCS(Pan Biotech;目录号:P30-3306)


5.5毫升100×谷氨酰胺


5.5毫升100×青霉素/链霉素


无菌过滤器


储存在4°C


3.小鼠胚胎干细胞(mESC )培养基     

注意:使用前,在56°C加热灭活FCS 30分钟。


400毫升淘汰赛的Dulbecco改良鹰之培养基(KO-DMEM)


75 ml m ESC测试的FCS(Pan Biotech;目录号:P30-2602)


5毫升100×谷氨酰胺


5毫升100×青霉素/链霉素


5毫升100×核苷


1毫升Gibco 2-巯基乙醇


无菌过滤器


等分试样米ESC 40个无LIF培养基-毫升的部分,并冷冻在-20℃下


使用前解冻,然后在4°C储存


在使用前立即添加1:1 0,0 00 LIF


储存在4°C


注:自制LIF已也已成功地使用; ħ H但是,T他权浓度必须基于该纯化方案和批次的浓度进行测试。


4. PBS / 0.5%BSA溶液     

含MgCl 2 / CaCl 2的PBS


0.5%BSA粉末


准备新鲜的无菌过滤器,保持冰冻状态


储存在4°C


致谢


这个工作是基于并改编自刊登在方法Veenvliet等。(2020)。我们感谢Herrmann&Meissner实验室的现任和过去成员,特别是Manuela Scholze-Wittler ,Dennis Schifferl ,Frederic Koch,Abhishek Sampath Kumar,Milena Pustet ,Fabian所提供的开发和表征我们的体外系统的支持和反馈。Tobor,西蒙·海曼,拉尔斯Wittler ,孙燕姿Grosswendt ,扎卡里·史密斯和笃田口。这项工作得到了亚历山大·冯·洪堡奖学金(JVV),美国国立卫生研究院(NIH)资助HG006193(AM)和马克斯·普朗克学会(Max Planck Society)的支持。


利益争夺


作者宣称没有利益冲突。


参考


Anlas ,K.,贝利-本森,P.,阿拉托,K.,特纳,DA和特里维迪,V.(2021)。胃样体:小鼠胚胎干细胞的胚胎类器官,用于研究早期哺乳动物胚胎的模式和发育。方法分子生物学2258:131-147。
Baillie-Benson,P.,Moris ,N.和Martinez Arias,A.(2020年)。哺乳动物早期发育的多能干细胞模型。Curr Opin Cell Biol 66:89-96。
Baillie-Johnson,P.,van den Brink,SC,Balayo ,T.,Turner,DA和Martinez Arias,A.(2015)。小鼠胚胎干细胞聚集体的产生及其体外对称性破坏,极化和紧急集体行为的产生。视力扩展(105)。
贝卡利,L.,莫里斯,N.,Girgin ,M.,特纳,DA,贝利-约翰逊,P.,Cossy ,AC,Lutolf ,MP,Duboule ,D。和阿里亚斯,AM(2018一)。小鼠胚胎干细胞转变为类胃体的多轴自组织特性。自然562(7726):272-276。              
贝卡利,L.,Girgin ,M.,特纳,D.,贝利-约翰逊,P.,Cossy ,A.-C.,莫里斯,N.,Lutolf ,M.,Duboule ,D。和Martinez的阿里亚斯,A. (201 8b )。从小鼠胚胎干细胞产生类胃异物。协议交换。DOI:10.1038 / protex.2018.094 。             
塞莫拉·F。,达尼洛C.,塔特·R。,德塞萨雷D.,马丁内斯·阿里亚斯A.,Minchiotti G.和帕特里卡,EJJB(2021年)。类胃发育能力区分幼稚和致敏的多能性不同状态。干细胞报告16(2):354-369。
George,SH,Gertsenstein ,M.,Vintersten ,K.,Korets -Smith,E.,Murphy,J.,Stevens,ME,Haigh,JJ和Nagy,A.(2007年)。直接从突变的胚胎干细胞发育和成年表型。PROC国家科科学院科学USA 104(11):4455-4460。              
莫里斯(美国),北卡罗来纳州安拉斯,范登布林克(南卡罗莱纳州),阿勒曼尼(Alemany ),施罗德(S.J.),格西米尔(Ghimire),南卡罗莱纳州(Balayo ),范·奥德纳纳登(A.van Oudenaarden )和马丁内斯·阿里亚斯(A. 。在人类发育过程中早期前后组织的体外模型。自然582(7812):410-415。              
Pourquié ,O。(2003)。分割时钟:将胚胎时间转换为空间模式。科学301(5631):328-330。
罗西(Rossi),布罗吉耶(N. Broguiere ),宫本(Miyamoto),博尼(A.),吉尼(Guet),R。,吉尔金(Girgin ),凯利(Kelly),RG,权汉(Kwon,C.)和路托夫(Lutolf),议员(202 1 )。捕获类胃中的心脏发生。细胞干细胞28(2):230-240.e6。              
Shahbazi ,MN,Siggia ,ED和Zernicka -Goetz,M。(2019)。干细胞自组织成胚胎:早期哺乳动物发育的窗口。科学364(6444):948-951。              
明尼苏达州的Shahbazi和M.的Zernicka-Goetz(2018)。解构和重建小鼠和人类早期胚胎。Nat Cell Biol 20(8):878-887。
Turner,DA,Girgin ,M.,Alonso-Crisostomo,L.,Trivedi,V.,Baillie-Johnson,P.,Glodowski ,CR,Hayward,PC,Collignon,J.,Gustavsen ,C.,Serup ,P. ,史蒂文顿,B.,M,PL和阿里亚斯,AM(2017)。在胚外组织不存在和胚状体空间定位信号缺失的情况下,前后极性和伸长:哺乳动物胚类器官。发展144(21):3894-3906。
van den Brink,SC,Alemany ,A.,van Batenburg ,V.,莫里斯,N.,Blotenburg ,M.,Vivie ,J.,Baillie-Johnson,P.,Nichols,J.,Sonnen ,KF,Martinez Arias ,A。和van Oudenaarden ,A。(2020)。单细胞和空间转录组学揭示了类胚体中的体细胞发生。自然582(7812):405-409。
凌科,SC,贝利-约翰逊,P.,Balayo ,T.,Hadjantonakis ,AK,Nowotschin ,S.,特纳,DA和Martinez的阿里亚斯,A。(2014)。小鼠胚胎干细胞聚集体中的对称性破坏,生殖层规格和轴向组织。发展141(22):4231-4242。
Veenvliet,合资企业,Bolondi,A.,Kretzmer,H.,Haut,L.,Scholze-Wittler,M.,Schifferl,D.,Koch,F.,Guignard,L.,Kumar,AS,Pustet,M. Heimann,S.,Buschow,R.,Wittler,L.,Timmermann,B.,Meissner,A.和Herrmann,BG(2020)。小鼠胚胎干细胞可自组织成具有神经管和体节的躯干状结构。科学370(6522)。             
Veenvliet ,合资公司和Herrmann,BG(202 1 )。在一个盘子里模拟哺乳动物的躯干发育。Dev Biol。2021 474:5-15。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. 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.
  2. 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).
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