This work is funded by an ERC Advanced Investigator Award to AMA (DAT, TB) with a contribution of a Project Grant from the Wellcome Trust to AMA, an EPSRC studentship to PB-J and Erasmus, Stichting dr. Hendrik Muller&#39;s Vaderlandsch Fonds and Fundatie van de Vrijvrouwe van Renswoude te &#8217;s-Gravenhage&#160;to SCvdB. We want to thank J. Briscoe, S. Mu&#241;oz-Descalzo, C. Schr&#246;eter, and T. Rodriguez, for discussions and constructive criticisms, Joaqu&#236;n de Navascu&#233;s for the mounting medium protocol and both A.K. Hadjantonakis and C. Budjan for sharing their laboratory&#8217;s protocols relating to staining of whole-mount embryos. An earlier version of this article was previously made available on the bioRxiv Preprint Server29.

1. Culture Conditions Prior to Aggregation
<ol>
	<li>Maintain mESCs in ESLIF medium (see Table of Specific Materials &#38; Equipment for formulation) on gelatin-coated 25 cm2 tissue-culture-treated flasks in a humidified incubator at 37 &#176;C and 5% CO221-25.</li>
	<li>Grow cells for at least two passages post thawing before use in this protocol; cells at lower passages are generally more successful at generating reproducible characteristics throughout experimental replicates (i.e., no more than 15 passages in culture), however slight variation&#160;between different ES cell-lines is to be expected (see Table 2 for the cell lines tested).</li>
	<li>Grow cells to 40-60% confluency. Do not use over-confluent cells for aggregation.</li>
</ol>
2. Generation of Aggregates
<ol>
	<li>&#160;Pre-warm PBS (+Ca2+, +Mg2+), ESLIF, N2B2726,27 and Trypsin-EDTA in a 37 &#176;C water bath.</li>
	<li>&#160;Aspirate culture medium from the tissue culture flask (step 1.3). Rinse the flask gently with 5 ml PBS, twice. Aspirate the PBS and add 1 to 2 ml of pre-warmed Trypsin-EDTA (0.25%) to dissociate the cells.
	<ol>
		<li>&#160;Place the flask in the incubator for &#60;&#160;5 min or until cells have fully detached from the surface of the flask. Pipette up and down with a 1 ml pipette to generate single-cell suspension for accurate counting and uniform aggregate size formation.</li>
	</ol>
	</li>
	<li>&#160;Neutralize Trypsin with 5-10 ml ESLIF; wash down the growth surface to maximize cell recovery and transfer to a 50 ml centrifuge tube. Remove a 1 ml aliquot from the centrifuge tube and count the cells with a haemocytometer.</li>
	<li>&#160;Determine the volume of suspension required to give 10 cells/&#181;l (a whole 96-well plate requires 5x104 cells in a 5 ml suspension). Count cells accurately, as large deviations from the stated cell number can adversely affect the response of the aggregates to stimuli.</li>
	<li>&#160;Add 5x104 cells to 5 ml pre-warmed PBS in a fresh 50 ml centrifuge tube and spin at ~170 x g for 5 min.</li>
	<li>&#160;Carefully aspirate the PBS and add 5 ml pre-warmed PBS gently; do not disturb the pellet at the bottom of the tube. Centrifuge at ~170 x g for 5 min.</li>
	<li>&#160;Carefully aspirate PBS for a second time. Remove as much PBS as possible without disturbing the pellet as PBS carryover can adversely affect aggregation. Resuspend the pellet firstly in 1 ml warm N2B27 with a P1000 pipette to generate a homogenous cell suspension, followed by further addition of N2B27 to the required volume (e.g., add 4 ml for a 5x104 cells/5 ml suspension).</li>
	<li>&#160;Transfer the cell suspension to a sterile reservoir and pipette a 40 &#181;l droplet into the bottom of each well of a non-tissue-culture treated, &#8216;U&#8217;-bottomed 96-well plate using a multichannel pipette. Cover the 96-well plate with its corresponding lid and confirm the presence of cells with an inverted bench-top microscope (Figure 1B). 
	Note: It is essential that these plates are used to limit the possibility of cells adhering. Do not coat the bottom of the 96-well plate with Gelatin, Fibronectin or any other coating that promotes cell adhesion.</li>
	<li>&#160;Incubate the cells for 48 hr in a humidified incubator at 37 &#176;C and 5% CO2 for aggregation.</li>
</ol>
3. Applying Stimuli and Changing Medium
<ol>
	<li>&#160;Following the 48 hr incubation period, observe the appearance of the mESCs within each well of the 96-well plate to confirm successful aggregation (Figure 1E). Note: Aggregates will appear spherical in nature and approximately 150-200 &#181;m in diameter. Refer to the troubleshooting section if problems arise (Table 1).</li>
	<li>&#160;Add 150 &#181;l fresh secondary medium (3 &#181;M Chi99021 (Chi) in N2B2719; stock prepared at 10 mM in DMSO) to each well using a multichannel pipette. Pipette with enough force to dislodge any aggregates that may have started to adhere to the bottom of the wells. Incubate aggregates in secondary medium for 24 hr in a humidified incubator at 37 &#176;C and 5% CO2 (Figure 1A). 
	Note: Reproducible elongated and polarized aggregates are generated using this secondary medium. Other secondary medium compositions may also be used depending on the experimental conditions required and examples are shown in Figure&#160;3.</li>
	<li>&#160;For subsequent medium changes, use a multichannel pipette held at an angle (approximately 30&#176;) to gently remove 150 &#181;l of the secondary medium from the side of each well. Then, pipette 150 &#181;l fresh N2B27 into each well with enough force to dislodge any aggregates that may have started to adhere to the bottom of the wells.</li>
	<li>&#160;Repeat point 3. 3 every 24 hr until the time-course is complete (the typical length of an aggregate culture experiment is 120 hr). 
	Note: Ensure aggregates are freely moving following medium changes to ensure reproducibility and consistency within and between each 96-well plate. Medium must be changed daily following the aggregation period.</li>
</ol>
4. Preparation of Aggregates for Immunostaining and Analysis by Confocal Microscopy
<ol>
	<li>Fixation and Primary Antibody Incubation 
	Note: The protocol described by A.K. Hadjantonakis28 has been modified to suit immunostaining of mESC aggregates. For the typical antibodies used in our studies and their dilutions, refer to previously published work19,21,24,25,29 and Table 3.
	<ol>
		<li>Use a multichannel pipette held at an angle (approximately 30&#176;) to gently remove 150 &#181;l medium from the side of each well of the 96-well plate. Wash twice by pipetting 150 &#181;l PBS into each well. Leave a couple of minutes between each wash to allow the aggregates to settle.</li>
		<li>Set a P1000 pipette to dispense 200 &#181;l, attach the corresponding pipette tip and cut off approximately 3 mm from the end of the tip with a pair of sterile scissors. Draw a portion of the PBS within each well of the 96-well plate a short way into the tip and expel it to agitate the aggregate.</li>
		<li>Use the same tip to draw up the whole volume within the well including the aggregate and pipette into a small (30 mm diameter) glass Drosophila dissection well. Transfer all the aggregates from the 96-well plate that will undergo identical immunostaining regimes into the same glass dissection well. 
		Note: Use a new cut pipette tip when transferring aggregates from different experimental conditions to prevent carryover of aggregates.</li>
		<li>Place the glass well that contains the aggregates onto a dissection microscope. Swirl the glass well to force the aggregates to the centre and aspirate PBS from one side of the well with a glass Pasteur pipette. Use the microscope to ensure the aggregates are not aspirated. Leave a small volume of PBS within the glass well to cover the aggregates to prevent them from drying out.</li>
		<li>Add 1 ml freshly prepared formaldehyde (4%) dissolved in PBS and incubate for 2 hr at 4 &#176;C on an orbital shaker set to a low speed. Caution: Paraformaldehyde is known to be allergenic, carcinogenic and toxic. Wear appropriate protection whilst handling.</li>
		<li>Following fixation, aspirate the formaldehyde solution in the same manner as described in section 4. 1. 4 and wash the aggregates with 1 ml PBS three times, 10 min for with each wash, on an orbital shaker set to a low speed.</li>
		<li>Aspirate the PBS as described in section 4. 1. 4 and perform a further three, 10 min washes with PBS containing 10% Foetal Bovine Serum and 0.2% Triton X-100 (PBSFT). Perform the 10 min washes on an orbital shaker set to a low speed. 
		Note: Using Foetal Bovine Serum (FBS) results in a clear wash buffer, reducing the number of aggregates that would otherwise be lost during the protocol if milk was used. It is important to note that following fixation, immunohistochemical procedures can be performed in various ways other than those detailed below and should be determined and optimized by the investigator.</li>
		<li>Block the aggregates for 1 hr at 4 &#176;C in PBSFT on an orbital rocker set to a low speed. The protocol may be paused at this point, and aggregates blocked O/N, with constant agitation on a horizontal gyratory rocker&#160;at 4&#176;C.</li>
		<li>Aspirate the PBSFT as previously described (see section 4. 1. 4) and incubate the aggregates with 500 &#181;l of the required primary antibody diluted in PBSFT O/N at 4 &#176;C on an orbital shaker set to low speed. Cover with paraffin film to prevent evaporation.</li>
	</ol>
	</li>
	<li>Secondary Antibody Incubation
	<ol>
		<li>Aspirate the primary antibody solution and wash with 1 ml PBSFT (at 4 &#176;C) in the following manner: twice for 5 min; thrice for 15 min; and four to seven times for 1 hr. Perform each wash step at 4 &#176;C and on an orbital rocker set to low speed. Note: Chill wash medium prior to use. Do not perform the washes on ice as this can cause the aggregates to fragment.</li>
		<li>Aspirate the final wash medium and incubate the aggregates with the required secondary antibody in 500 &#181;l PBSFT&#160;O/N at 4 &#176;C in the dark on a horizontal gyratory rocker. Add nuclear stain such as Hoechst if required.</li>
	</ol>
	</li>
	<li>Mounting and Imaging by Confocal Microcopy
	<ol>
		<li>Wash the aggregates as described in Section 4. 1. 4 with 4 &#176;C PBSFT. After the final wash, rinse the aggregates with 1 ml PBS containing 0.2% FBS and Triton X-100 (PBT) in the following manner: twice for 5 min; thrice for 15 min. Perform washes at RT on an orbital rocker protected from light.</li>
		<li>Aspirate the medium as previously described (section 4. 1. 4) and incubate for 30 min in the dark with a 1:1 solution of glycerol:PBT (1 ml) followed by a second 30 min incubation with a 7:3 solution of glycerol:PBT (1 ml). 
		Note: Mix the glycerol and PBT solutions at 4 &#176;C using a rotator.</li>
		<li>Aspirate the final glycerol:PBT solution and replace with 1 ml mounting medium24. The protocol may be paused at this point prior to mounting (if paused, seal the wells with paraffin film and store the staining wells at 4 &#176;C).</li>
		<li>Mount the aggregates on microscope slides by pipetting them in 17 &#181;l droplets with a cut P20 tip (Figure 2). Fold double-sided tape back on itself four times to generate spacers and attach to each end of the slide. Place the top coverslip (22 mm x 60 mm) upon these spacers (Figure 2). 
		Note: it is essential that a cut tip is used at this stage so as not to damage the samples. The spacers prevent the coverslip crushing the aggregates.</li>
		<li>Once the aggregates are mounted, image the samples using confocal microscopy using protocols previously described19,21,24,25. Once placed on the microscope, leave the slide undisturbed on the stage for a few minutes to allow the aggregates to settle within the mounting medium.</li>
	</ol>
	</li>
</ol>

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<li>Neely, M. D., Litt, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DMH1%2C+a+highly+selective+small+molecule+BMP+inhibitor+promotes+neurogenesis+of+hiPSCs%3A+comparison+of+PAX6+and+SOX1+expression+during+neural+induction%5BTitle%5D)+AND+%22ACS+Chem.+Neurosci%22%5BJournal%5D)">DMH1, a highly selective small molecule BMP inhibitor promotes neurogenesis of hiPSCs: comparison of PAX6 and SOX1 expression during neural induction.</a> ACS Chem. Neurosci. 3 (6), 482-491 (2012).</li>
<li>Schindelin, J., Arganda-Carreras, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li>
<li>Niakan, K. K., Ji, H., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((12+Sox17+promotes+differentiation+in+mouse+embryonic+stem+cells+by+directly+regulating+extraembryonic+gene+expression+and+indirectly+antagonizing+self-renewal%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">12 Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal.</a> Genes Dev. 24 (3), 312-326 (2010).</li>
<li>Chambers, I., Silva, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanog+safeguards+pluripotency+and+mediates+germline+development%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Nanog safeguards pluripotency and mediates germline development.</a> Nature. 450 (7173), 1230-1234 (2007).</li>
<li>Rhee, J. M., Pirity, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((et+al.+In+vivo+imaging+and+differential+localization+of+lipid-modified+GFP-variant+fusions+in+embryonic+stem+cells+and+mice%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">et al. In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice.</a> Genesis. 44 (4), New York, N.Y. 202-218 (2006).</li>
<li>Serup, P., Gustavsen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partial+promoter+substitutions+generating+transcriptional+sentinels+of+diverse+signaling+pathways+in+embryonic+stem+cells+and+mice%5BTitle%5D)+AND+%22Dis.+Models+Mech%22%5BJournal%5D)">Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice.</a> Dis. Models Mech. 5 (6), 956-966 (2012).</li>
<li>Hooper, M., Hardy, K., Handyside, A., Hunter, S., Monk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HPRT-deficient+%28Lesch-Nyhan%29+mouse+embryos+derived+from+germline+colonization+by+cultured+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells.</a> Nature. 326 (6110), 292-295 (1987).</li>
<li>Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D., Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+to+recapitulate+early+embryonic+spatial+patterning+in+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">A method to recapitulate early embryonic spatial patterning in human embryonic stem cells.</a> Nature Chemical Biology. 11 (8), 847-854 (2014).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Human+Fertilisation+and+Embryology+ACT+2008+-+Elizabeth+II%22">Human Fertilisation and Embryology ACT 2008 - Elizabeth II</a>. , The Stationery Office. (2008).</li>
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The authors declare that they have no competing financial interests.

The technique described in this manuscript efficiently and reproducibly generates aggregates of mouse embryonic stem cells (mESCs) that display organized symmetry-breaking and elongation19, combining both the hanging drop culture described by Marikawa et al.14 and EB formation. These aggregates go on to develop axial elongations, complementing existing methods for the generation of anterior organs from ES cells such as optic cups11 and the cerebral cortex13, and contain cells with identities of the three germ layers which display processes similar to those during gastrulation such as rapid cell movement19,21.
It is interesting to speculate on the nature of the symmetry-breaking event, since it occurs in the absence of external patterning tissues and zygotic asymmetry cues19. The spontaneous formation of a rudimentary axis could arise from pre-existing heterogeneities in the starting culture of cells, which form the basis for the development of asymmetry. Indeed, populations of cells cultured in ESLIF conditions display a mixture of self-renewing and differentiating cells, the relative proportions of which may vary from one aggregate to another. Furthermore, preliminary work in our laboratory suggests that aggregates from a wholly pluripotent population of cells show delayed development and defects in patterning, suggesting a role for heterogeneity in organized pattern formation (D.A. Turner &#38; A. Martinez Arias, in preparation). It is also worth considering that a reaction-diffusion mechanism may generate the pattern, with diffusion of an inhibitor away from the surface of the aggregate. Warmflash et al.41 suggest that such a mechanism could be responsible for developing radial asymmetry in adherent culture, making it a possible candidate for patterning in three dimensions.
During the initial 48 hr aggregation period, cells reach a state similar to that within the postimplantation epiblast, in which they are competent to respond to signals from the secondary medium19,21,24. Typically, the protocol described here provides cells with a pulse of secondary medium which contains factors (such as Wnt/&#946;-Catenin agonists) that provide the signals sufficient for elongation. Previous culture methods described by Lancaster et al. (using human ESCs13) and Eiraku et al. (with mESCs11) used a short period of aggregation in low-adhesion 96-well plates before aggregated cells were transferred to Matrigel droplets for on-going culture. Although the time between aggregation and Matrigel insertion was different between the studies, in both cases, large numbers of cells were used for aggregate formation (approximately 3,000-4,500 cells). By contrast, the protocol described here19 uses far fewer cells (approximately 400 cells per aggregate) which has been determined to be absolutely essential to the process of elongation, symmetry-breaking and the polarization that is observed19. In addition, these elongated structures are able to be generated in a much shorter time-period without embedding in artificial matrices: compare the generation of defined brain regions by Lancaster et al. (&#62;20-30 days)13 and the optic cups from Eiraku et al. (~11 days)11 with the 5 days required for the generation of polarized elongated structures.
One of the limitations with the culture method described here however, is that they can only be cultured for approximately 5-6 days post plating until their size renders them competent under gravity to sink to the bottom of the wells and force an adhesion even on non-coated plastic-ware. Further experiments using artificial matrices such as those mentioned previously, may allow us to increase the period of observation and experimentation, and work is on-going to determine the effects of constraining the aggregates in such a manner. A further limitation of this technique is that in order to change the medium without removing the aggregates, a small volume of medium must be left in the bottom of the well. The consequences for chemical genetics approaches are the need to consider this dilution and any residual effects from previous media: on the day of the 3 &#181;M Chi pulse, 2.37 &#181;M solution is actually delivered, and subsequent medium changes result in a carry-over of 0.499 &#181;M (72-96 hr) and 0.105 &#181;M (96-120 hr) between the time-points. Current work has not corrected for dilution and it is assumed that daily medium changes will minimize residual effects.
There are a number of critical steps in the protocol to ensure both intra- and inter-experimental reproducibility. Firstly, the starting culture of mESCs must be well maintained and not over confluent, and medium should always be good quality i.e., fresh and well mixed (Figure 5A,B). Poor culture conditions adversely affect the aggregation (Figure 5Bi). Accurate counting of cells to obtain a ~400 cells/40 &#181;l cell suspension is essential, as larger aggregates fail to self-organize and are more likely to form double aggregates within each well (Figure 5Bii) whereas smaller aggregates are unable to reach the correct density where they are able to respond to the stimulus (Figure 5Biii). A key optimisation step was the inclusion of a second PBS wash that removes contaminant serum from the cell suspension (Step 2.6); this improved the aggregation frequency and accelerated the development of the aggregates by approximately 24 hr. Next, ensuring medium changes are applied with sufficient force to dislodge any aggregates that have the potential to adhere to the surface of the well; failure to do so results in the aggregates &#8216;crashing&#8217; (Figure 5Biv). In addition, and as mentioned previously (and below), it is essential to ensure that the aggregates are maintained in suspension culture and are prevented from adhering to any surface. Once the aggregates have adhered, the pattern of gene expression and their elongation potential are disrupted.
As this technique is relatively straightforward, and well within the remit of individuals with good tissue-culture techniques, most of the problems associated with aggregation can be resolved relatively rapidly if these critical steps are followed; a full table of trouble-shooting is available (Table 1). Once mastered, this technique can be used to study axial development, symmetry breaking and cell-decision events. More specifically, these aggregates have the potential to generate pools of cells that have hitherto been unavailable in culture, such as the spinal cord and motor neurones.
Ethical Note: It should be noted with due caution that the translation of this protocol to human ES or iPS cell culture could bring the experimenter close to the legal basis of the fourteen day limit on human embryo research, namely the generation of a primitive streak, as detailed in the Human Fertilisation and Embryology Act 2008 (UK)42. Since the Act covers all live human embryos regardless of their manner of creation, culture of human gastruloids beyond the primitive-streak like stage would be beyond the scope of licensable research.

The study and understanding of cell-fate decisions in early mammalian development can make use of cultures of Embryonic Stem Cells (ESCs), clonal populations derived from blastocysts which have the ability to self renew and differentiate into all cell types of an organism i.e., they are pluripotent1,2. While these cultures have been and are continuing to be useful for the understanding of the molecular basis of cell-fate decisions, they are unable to reproduce some of the spatial arrangements and global behaviours that are generated in embryos during gastrulation. In the embryo, the process of gastrulation transforms a single epithelial layer into the three distinct germ layers and endows the embryo with an overt anteroposterior organization3-5. Attempts to recapitulate these events ex vivo have been based on the generation of three-dimensional aggregates of ESCs, referred to as embryoid bodies (EBs), and subjecting them to differentiation conditions6,7. These aggregates can be coaxed to differentiate into many different cell types, some of which are either unable to be obtained or induced with low efficiency in adherent culture or cannot be produced at all; e.g., blood8 and primordial germ cells9. A limitation in the use of EBs however, is that they are unable to display the morphogenetic behaviour, germ layer distribution or axial organization that are major characteristics of the developing embryo, resulting in spatial disorganization6,10. In one report, treatment of EBs with Wnt leads to a weak polarization in gene expression in some aggregates but no clear morphogenesis is observed7. In more recent reports, EBs that have been cultured for extended periods of time develop anterior structures such as retinas, cortex and inner ear sensory cells, which mimic their embryonic counterparts but develop without the context of an axial organization11-13.
A report by Marikawa et al.14, whilst working with aggregates of mouse P19 Embryo Carcinoma (EC) cells formed by the hanging-drop method, reported the emergence of elongated structures of mesodermal origin reminiscent of the elongations that are observed with exogastrulae in amphibian and sea urchin embryos and Keller explants15-18. As this had not been observed with mouse embryonic stem cells (mESCs), we attempted to reproduce the behaviour observed with P19 EC cells using aggregates of mESCs19 and we report culture conditions that lead to their symmetry breaking and axial elongation. An important difference from the work with P19 cells is that the aggregation protocol described here is performed in a 96-well plate, similar to that described by Eiraku et al.20, instead of hanging drops. This change resulted in an increased efficiency in terms of aggregate recovery and in both intra- and inter-experimental reproducibility. Importantly, maintaining aggregates in individual wells ensures that fusion&#160;between aggregates (common when pooling aggregates from hanging drops) does not occur. In addition, a key feature of the protocol is 24 hr exposure to the GSK3 inhibitor CHI99021 (Chi), a potent activator of Wnt/&#946;-Catenin signalling, following aggregation.
The method described here provides a basis for understanding the processes of self-organization, axial organization and germ layer specification in culture, allowing inferences to be made concerning axial development in vivo19,21. For these reasons the method has the potential to allow detailed mechanistic analysis of processes that may be difficult to study in the embryo. Furthermore, a potential application is in the generation of tissues and organs that are not easily obtainable in adherent culture due to the lack of a structured cellular niche such as spinal cord precursors21 and motor neurones. Three-dimensional aggregate culture offers a physical structure and signalling environment that may not be achievable by conventional means, leading to a new approach for the derivation of embryonic lineages in a spatially organized manner.

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Gibco/Invitrogen</td><td>31350-010</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Cell Culture Flask</td><td>Grenier Bio-one</td><td>690 175</td><td></td></tr><tr><td>Benchtop Centrifuge</td><td>MSE</td><td>MSB020.CX1.5</td><td></td></tr><tr><td>BES Buffered Saline (BBS)</td><td>Sigma-Aldrich</td><td>B2891</td><td>BBS+CaCl&lt;sub&gt;2&lt;/sub&gt; made by disolving 50 mM BES Sodium Salt, 280 mM NaCl, 1.5 mM Na2HPO4 in distilled water with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt; adjusted to pH 6.96 with 1 M HCl&amp;nbsp;</td></tr><tr><td>Centrifuge tubes (15 or 50 ml)</td><td>Greiner Bio-One&amp;nbsp;</td><td>118271 or 227261</td><td></td></tr><tr><td>CHIR 99021 (Chi)</td><td>CSCR</td><td></td><td>Bought through the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. Stock concentration: 10 mM in DMSO</td></tr><tr><td>Dissection Well (30 mm Internal Diameter)</td><td>Fisher Scientific</td><td>12678586</td><td></td></tr><tr><td>ESLIF</td><td></td><td></td><td>500 ml GMEM, 5 ml Sodium Pyruvate, 5 ml Non-Essential Amino Acids, 5 ml Glutamax, 1 ml beta-mercaptoethanol, 50 ml FBS and 550 &amp;micro;l LIF (1,000 units).</td></tr><tr><td>Foetal Bovine Serum (FBS)</td><td>Biosera</td><td>FB-1090/500</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td>Dissolved in distilled water to a 1 % (w/v) solution, autoclaved and diluted to 0.1% in PBS (see below) for individual use.</td></tr><tr><td>Glasgow&#039;s Minimal Essential Medium (GMEM)</td><td>Gibco</td><td>11710-035</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Improved Neubauer Haemocytometer</td><td>Hawksley</td><td>AS1000</td><td></td></tr><tr><td>Leukaemia Inhibitory Factor (LIF), Recombinant</td><td>CSCR</td><td></td><td>Produced in house by the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute.</td></tr><tr><td>n-Propyl-gallate</td><td>Sigma-Aldrich</td><td>02370</td><td></td></tr><tr><td>N2B27/NDiff 227</td><td>Stemcells, Inc.</td><td>SCS-SF-NB-02</td><td>&lt;a href=&quot;http://www.stemcellsinc.com/_literature_40366/Technical_Specifications_Ndiff&quot;&gt;http://www.stemcellsinc.com/_literature_40366/Technical_Specifications_Ndiff&lt;/a&gt;</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco/Invitrogen</td><td>11140-035</td><td></td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma-Aldrich</td><td>P6148</td><td>Dissolved in water to form a 8% Formaldehyde stock solution. Diluted with BBS&lt;sup&gt;+&lt;/sup&gt;CaCl&lt;sub&gt;2&lt;/sub&gt; to give a 4% working solution</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8662</td><td>With Calcium and Magnesium</td></tr><tr><td>Sodium Pyruvate</td><td>Invitrogen</td><td>11360-039</td><td></td></tr><tr><td>Sterile Reservoir</td><td>STARLAB</td><td>E2310-1010</td><td></td></tr><tr><td>Sterile, Non-Tissue Culture Treated, U-Bottomed 96-well Plate</td><td>Grenier Bio-one</td><td>650185</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr></tbody></table>

generation of aggregates of mouse embryonic stem cells that show symmetry breaking, polarization and emergent collective behaviour in vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Appearance of Cells Immediately After Plating and After Aggregation
Immediately after plating, one can observe the presence of single cells within each well of the 96-well plate with a standard inverted tissue-culture microscope (Figure 1B). Within 8 hr of plating, these cells will have begun to descend to the bottom of the well due to gravity and will have started to coalesce into discrete clusters (Figure 1C). After 24 hr, the coalescing cells will have completed the primary aggregation, and will have compacted into well defined, yet ragged aggregates where individual cells are indistinct from one another (Figure 1D). By the end of the second day (48 hr), the aggregates should look &#8216;clean&#8217;, having taken up all the cells within the well (Figure 1E); the bottom of the well may have some cells that have been shed from the aggregate at this stage. Providing that the aggregates have been aggregated in N2B27, at this time-point, they should be spherical, approximately 150-200 &#181;m in diameter and freely moving within the well (i.e., not adhered to the bottom of the wells). Aggregation in other media (i.e., ESLIF or N2B27 with specific factors) has the potential to alter both the initial aggregate morphology and the response to subsequent stimuli (Turner et al., in preparation).
Representative Morphological Changes
Addition of a 24 hr pulse of secondary medium containing Chi between 48 and 72 hr (Figure 1A, 5A) generates well defined elongated aggregates that show polarized expression in specific markers of the germ layers19,21. Immediately after the Chi pulse (72 hr), the aggregates will begin to shed cells and start to show their response to these signals towards the end of this day (Figure 3A). These responses are manifested by changes in gene expression and morphology, both of which are dependent on the treatment that the aggregates have received following the initial 48 hr aggregation period in N2B27 (Figure 3B). If only an endpoint analysis is required, the optimum time to image the aggregates is at approximately 96-120 hr. At this point the aggregates will have developed clear morphologies and gene expression patterns (Figure 3A), and their size and mass are still low enough so that ejection of medium from P200 pipette is sufficient to dislodge any that may have attached. Failure to prevent attachment of the aggregate to the well will adversely affect aggregate formation (Figure 5Biv). The morphologies and gene expression pattern of the aggregates can be altered depending on the treatment. Representative results for sustained or pulsed regimes with either single treatment or combinations of factors are shown for BMP4, Dorsomorphin H1 (BMP receptor inhibitor), ActivinA, SB43 (Activin/Nodal inhibitor), bFGF or PD03 (MEK inhibitor) (Figure 3B).
Aggregate Imaging and Quantitative Image Analysis
Aggregates are amenable to imaging by either widefield microscopy (principally for time-lapse imaging19), or fixed and immunostained for confocal imaging19,21 (Figure 2). The current protocol and imaging description above allows quantitative information to be gained from these aggregates. Following confocal or wide-field image capture, the length and the corresponding gene expression along the diameter or spine of the aggregate (spherical or elongated respectively; Figure 4A, B) can be measured. These analyses are also directly applicable to time-lapse images, where one can obtain information of the rate of growth, size and elongation of the aggregates under different conditions (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/53252/53252fig1.jpg" /> 
Figure 1. Typical time-course and early morphologies. (A) The typical time-course for aggregation experiments. Cells are aggregated for 48 hr in N2B27 containing a suspension of mouse ES cells (10 cells/&#181;l) in a 96-well plate (p1, p2). (B) Immediately after plating (t = ~5min), individual cells can be seen in the suspension and have started to form clumps by 8h (C).&#160;(D) After 24 hr the cells have formed a single aggregate in each well which is approximately 100 &#181;m in diameter. (E) After 48 hr aggregation, the aggregate diameter ranges from 150-200 &#181;m. At this point, the aggregation medium (N2B27) is removed and a secondary medium is added either for the rest of the experiment (p1 in part A) or for 24 hr before being changed back to N2B27 (p2 in part A). Scale bar represents 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53252/53252fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53252/53252fig2.jpg" /> 
Figure 2. Mounting aggregates onto microscope slides. Once the aggregates have been fixed, immunostained and placed in mounting medium, each aggregate is pipetted onto a microscope slide as a 17 &#181;l droplet (A,B,B&#8217;). Enough space is left between the aggregate droplets to prevent their merging. Spacers are made with double sided tape and placed at each corner of the microscope slide (A, A&#8217;, B). It is upon these that a glass coverslip is placed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53252/53252fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53252/53252fig3.jpg" /> 
Figure 3. The effect of different treatments on the morphology of aggregated ES cells. Following 2 days in N2B27, ES cells have formed aggregates. Addition of specific factors either as a 1 day pulse (A) or continuously (B) can alter the phenotype of the aggregates with respect to the polarity of gene expression, elongation potential, or their overall shape. The examples in this figure are aggregates formed from either Bra::GFP30 (A) or Sox1::GFP27 (B) mouse ES cells imaged after 120 hr and treated as indicated. Chi: CHIR 9902131; SB43: SB 43154232;DM: DorsomorphinH133,34; PD03: PD0325901. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53252/53252fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53252/53252fig4.jpg" /> 
Figure 4. Quantitative analysis of Aggregates. A typical aggregate formed from Bra::GFP mESCs25,30 following a 24 hr pulse of Chi and imaged at 120 hr. Merged image of the bright-field and GFP channel is shown with segmented line marking the &#8216;spine&#8217; of the aggregate from point A to B (A), along which the fluorescence and length of the aggregate can be measured (B). The length (C) and &#8216;roundness&#8217; (D) from 24 aggregates within the same experiment are shown. Shape descriptors such as the roundness (D), circularity, perimeter and area can be measured using the Image Analysis Cookbook plugin from the image analysis software FIJI35. Mean indicated by horizontal line at each time-point; error bars indicate standard deviation; scale bar in (A) indicates 200 &#181;m. As there are subtle differences between reporter cell lines, it is expected that after a pulse of Chi, the average maximum length and average minimum roundness of the aggregates will vary. Between different cell lines, we find that the average maximum length can be within the range of ~400-800 &#181;m and the average minimum roundness between 0.4 and 0.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53252/53252fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53252/53252fig5.jpg" /> 
Figure 5. Examples of failures in aggregate formation. The ability to generate reproducible aggregates (A) depends on critical factors such as (Bi) fresh and well mixed secondary medium, (Bii, Biii) the accuracy in counting the initial number of cells and (Biv) ensuring the aggregates do not form adherent colonies i.e., &#8216;crash&#8217; into the surface of the well. Typical examples of the errors in aggregate formation for each of the mentioned conditions (B) are shown. Scale-bar as indicated; see trouble-shooting table for details (Table 1). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53252/53252fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/53252/53252table1.jpg" /> 
Table 1. Guide to troubleshooting. Typical errors associated with the aggregation protocol are given with suggestions as to their resolution.
<img alt="Table 2" src="/files/ftp_upload/53252/53252table2.jpg" /> 
Table 2. Table of cell lines tested for the formation of aggregates. A number of cell lines19,22,27,30,36-40 have been characterized for the formation of aggregates and, although subtle differences between cell lines are expected and have been observed, all the above lines show similar dynamics in terms of elongation and morphology. In terms of gene expression, the expression pattern is specific to the gene expressed, but within each cell line, the expression pattern is generally consistent over a number of passages. For consistency, we generate aggregates from cells that have not exceeded 15 passages in culture.
<img alt="Table 3" src="/files/ftp_upload/53252/53252table3.jpg" /> 
Table 3. List of representative antibodies used in these studies. A selection of the antibodies and the dilution factors used for aggregate immunostaining.

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Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

17.8K Views.  University of Cambridge. We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

Video: Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Bioengineering

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Biology

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Source: Hubbard, K. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Forebrain-Type Cerebral Organoids from Human-Induced Pluripotent Stem Cells

To generate pluripotent stem cell aggregates, when the stem cell cultures reach 70% to 90% confluency, add 500 microliters of cell dissociation reagent to one well of the six-well culture plate to detach the cells.
Induced pluripotent stem cells, which are adapted to monolayer culture conditions, are a good cell type to generate aggregates as they are less prone to stress-induced cell death during the dissociation and aggregation procedure.
After 5 to 10 minutes at 37 degrees Celsius, gently tap the plate, and use 2 milliliters of DMEM/F-12 to wash the cells from the bottom of the well. Transfer the resulting cell suspension into a 15-milliliter conical tube, and bring the volume of the cell solution up to 10 milliliters with more DMEM/F-12 medium.
After counting, transfer 4.5 times 10 to the third cells per pluripotent stem cell aggregate into a new 15-milliliter tube, and collect the cells by centrifugation. Resuspend the pellet in the appropriate volume of pluripotent stem cell medium, supplemented with 50 micromolar ROCK inhibitor to achieve a 4.5 x 103 cells per 150 microliters of medium concentration.
Next, add 150 microliters of cells to individual wells of a 96-well low-attachment U-bottom plate, and place the plate in a 37 degrees Celsius and 5% carbon dioxide incubator. For monitoring anterior neuroectoderm induction, use a tissue culture microscope to closely observe the morphologic changes of the pluripotent stem cell aggregates every day under low magnification.
On day 1, cell aggregates with clear borders should be observed. On day 2, carefully aspirate approximately 2/3 of the medium without disturbing the cell aggregates at the bottom of each well, and replace the discarded medium with 100 microliters of fresh pluripotent stem cell medium.
Between four and six days later, when the cell aggregates reach 350 to 450 micrometers in diameter and exhibit smooth edges, use a modified 100-microliter pipette tip to transfer up to 20 aggregates into a single 6-centimeter low-attachment culture plate containing 5 milliliters of cortical induction medium.
Replace the cortical induction medium with fresh medium once every three days, monitoring the morphology of the aggregates daily under the 4x objective. After four to five days in the cortical induction medium, the edges of the cell aggregates should begin to brighten at the surface, indicating neuroectodermal differentiation, and the radial organization of a pseudostratified epithelium should emerge.
To embed the neuroectodermal aggregates in a matrix scaffold, fill a basement membrane extract on ice for two to three hours. While the extract is thawing, use sterile scissors to cut plastic paraffin film into one 4-by-4 centimeter square per 16 organoids, and place each piece of film over an empty 100-microliter micropipette tip tray.
Press the paraffin film with a gloved fingertip so that small dimples appear, and clean the film with 70% ethanol. After UV irradiation in a closed, sterile biosafety cabinet for 30 minutes, use a modified 100-microliter pipette tip with a 1 and 1/2 to 2-millimeter opening to transfer each cell aggregate into one dimple in the film.
When all of the aggregates have been transferred, use an uncut 100-microliter pipette tip to carefully aspirate the medium from each dimple, and add 40 microliters of undiluted basement membrane extract to each cell aggregate.
Be careful not to damage the organoids by rough manipulation or aspiration into the pipette tip, as this will damage the developing neuroepithelium.
Use the pipette tip to position the aggregates into the middle of each drop, and use sterile forceps to carefully transfer the plastic paraffin film sheet into a 10-centimeter Petri dish. Place the dish in the incubator for 15 to 20 minutes.
While the extract is solidifying, add 5 milliliters of fresh cortical induction medium to one six-centimeter low attachment culture dish. At the end of the incubation, flip the paraffin film sheet over, and use sterile forceps to gently squeeze up to 16 polymerized droplets into each 6-centimeter dish. Then, return the aggregates to the cell culture incubator.
The next day, transfer the organoid culture dishes onto a rocking cell culture shaker tilted at a 5-degree angle at 14 RPM within a cell culture incubator with daily light microscopy monitoring until the aggregates reach the differentiation stage of interest.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

Summary

Explore More Videos

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Generation of Mice Derived from Induced Pluripotent Stem Cells

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Generating Forebrain-Type Cerebral Organoids from Human-Induced Pluripotent Stem Cells