Name: generation of aggregates of mouse embryonic stem cells that show symmetry breaking, polarization and emergent collective behaviour in vitro
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Description: Watch this Scientific Journal Video about Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro at JoVE.com

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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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SB-431542+is+a+potent+and+specific+inhibitor+of+transforming+growth+factor-beta+superfamily+type+I+activin+receptor-like+kinase+(ALK)+receptors+ALK4,+ALK5,+and+ALK7.">SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.</a> Mol Pharmacol. 62 (1), 65-74 (2002).</li><li>Hao, J., Daleo, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin,+a+selective+small+molecule+inhibitor+of+BMP+signaling,+promotes+cardiomyogenesis+in+embryonic+stem+cells.">Dorsomorphin, a selective small molecule inhibitor of BMP signaling, promotes cardiomyogenesis in embryonic stem cells.</a> PLoS ONE. 3 (8), e2904 (2008).</li><li>Neely, M. D., Litt, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMH1,+a+highly+selective+small+molecule+BMP+inhibitor+promotes+neurogenesis+of+hiPSCs:+comparison+of+PAX6+and+SOX1+expression+during+neural+induction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=12+Sox17+promotes+differentiation+in+mouse+embryonic+stem+cells+by+directly+regulating+extraembryonic+gene+expression+and+indirectly+antagonizing+self-renewal.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanog+safeguards+pluripotency+and+mediates+germline+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+In+vivo+imaging+and+differential+localization+of+lipid-modified+GFP-variant+fusions+in+embryonic+stem+cells+and+mice.">et al. In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice.</a> Genesis. 44 (4), 202-218 (2006).</li><li>Serup, P., Gustavsen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+promoter+substitutions+generating+transcriptional+sentinels+of+diverse+signaling+pathways+in+embryonic+stem+cells+and+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPRT-deficient+(Lesch-Nyhan)+mouse+embryos+derived+from+germline+colonization+by+cultured+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+recapitulate+early+embryonic+spatial+patterning+in+human+embryonic+stem+cells.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Fertilisation and Embryology ACT 2008 - Elizabeth II. , (2008).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">2-Mercaptoethanol</td>
			<td style="width:116px;">Gibco/Invitrogen</td>
			<td style="width:87px;">31350-010</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">25cm2 Cell Culture Flask</td>
			<td style="width:116px;">Grenier Bio-one</td>
			<td style="width:87px;">690 175</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Benchtop Centrifuge</td>
			<td style="width:116px;">MSE</td>
			<td style="width:87px;">MSB020.CX1.5</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:235px;">BES Buffered Saline (BBS)</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:87px;">B2891</td>
			<td style="width:188px;">BBS+CaCl2 made by disolving 50 mM BES Sodium Salt, 280 mM NaCl, 1.5 mM Na2HPO4 in distilled water with 1 mM CaCl2 adjusted to pH 6.96 with 1 M HCl&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:235px;">Centrifuge tubes (15 or 50ml)</td>
			<td style="width:116px;">Greiner Bio-One&#160;</td>
			<td style="width:87px;">118271 or 227261</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:235px;">CHIR 99021 (Chi)</td>
			<td style="width:116px;">CSCR</td>
			<td style="width:87px;">n/a</td>
			<td style="width:188px;">Bought through the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. Stock concentration: 10mM in DMSO</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:235px;">Dissection Well (30mm Internal Diameter)</td>
			<td style="width:116px;">Fisher Scientific</td>
			<td style="width:87px;">12678586</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:235px;">ESLIF</td>
			<td style="width:116px;"></td>
			<td style="width:87px;"></td>
			<td style="width:188px;">500 mL GMEM, 5mL Sodium Pyruvate, 5 mL Non-Essential Amino Acids, 5 mL Glutamax, 1 mL beta-mercaptoethanol, 50 mL FBS and 550 &#181;L LIF (1000 units).</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:116px;">Biosera</td>
			<td style="width:87px;">FB-1090/500</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:235px;">Gelatin</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:87px;">G1890-100G</td>
			<td style="width:188px;">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 height="34">
			<td height="34" style="height:34px;width:235px;">Glasgow&#39;s Minimal Essential Medium (GMEM)</td>
			<td style="width:116px;">Gibco</td>
			<td style="width:87px;">11710-035</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">GlutaMAX</td>
			<td style="width:116px;">Gibco</td>
			<td style="width:87px;">35050-038</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Glycerol</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:87px;">G5516</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Improved Neubauer Haemocytometer</td>
			<td style="width:116px;">Hawksley</td>
			<td style="width:87px;">AS1000</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:235px;">Leukaemia Inhibitory Factor (LIF), Recombinant</td>
			<td style="width:116px;">CSCR</td>
			<td style="width:87px;">n/a</td>
			<td style="width:188px;">Produced in house by the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">n-Propyl-gallate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:87px;">02370</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:235px;">N2B27/NDiff 227</td>
			<td style="width:116px;">Stemcells, Inc.</td>
			<td style="width:87px;">SCS-SF-NB-02</td>
			<td style="width:188px;"><a href="http://www.stemcellsinc.com/_literature_40366/Technical_Specifications_Ndiff">http://www.stemcellsinc.com/_literature_40366/Technical_Specifications_Ndiff</a></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Non-Essential Amino Acids</td>
			<td style="width:116px;">Gibco/Invitrogen</td>
			<td style="width:87px;">11140-035</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:235px;">Paraformaldehyde powder</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:87px;">P6148</td>
			<td style="width:188px;">Dissolved in water to form a 8% Formaldehyde stock solution. Diluted with BBS+CaCl2 to give a 4% working solution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:87px;">D8662</td>
			<td style="width:188px;">With Calcium and Magnesium</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Sodium Pyruvate</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:87px;">11360-039</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Sterile Reservoir</td>
			<td style="width:116px;">STARLAB</td>
			<td style="width:87px;">E2310-1010</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:235px;">Sterile, Non-Tissue Culture Treated, U-Bottomed 96-well Plate</td>
			<td style="width:116px;">Grenier Bio-one</td>
			<td style="width:87px;">650185</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Triton X-100</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:87px;">X100</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:235px;">Trypsin-EDTA</td>
			<td style="width:116px;">Gibco</td>
			<td style="width:87px;">25300-054</td>
			<td style="width:188px;"></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/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/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/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/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/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.

Watch this Scientific Journal Video about Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro at JoVE.com

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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Research

JoVE Journal

Developmental Biology

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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 ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.