Updates

We have recently published an essay/review (Turner, D.A., Baillie-Johnson, P. & Martinez Arias, A. Organoids and the genetically encoded self-assembly of embryonic stem cells. Bioessays 38, 181-191 (2016)) in which we discuss how embryonic organoids can be used to understand the principles of embryonic patterning. In this essay we put a focus on some interesting observations of M. Lutolf’s and E. Tanaka’s laboratories in the engineered patterning of neural cysts from Embryonic Stem cells (Meinhardt et al. 3D reconstruction of patterned neural tubes from ES cells. Stem cells reports 3, 967-999 (2014)). These observations, we believe, challenge well established notions, often attributed to V. Hamburger, that only the embryo ‘knows’ how to make an organ or a tissue. Much as it is the case in engineering, there might be more than one way to a goal. This might come handy.

We have been putting efforts in making the system reproducible i.e. that if one starts the experiment, it will work with more than 80% efficiency. We are making progress on this and are preparing a manuscript in which we shall discuss this as well as show how the events in the Gastruloids parallel the events in embryos. In the meantime, here you have an example of how far we are getting with reproducibility:

for website-01 Figure 1: An example of Gastruloid reproducibility. Gastruloids were formed from Bra::GFP mESCs and incubated for 120h in N2B27 with a pulse of the Wnt/β-Catenin agonist CHIR between 48 and 72h and imaged daily by widefield fluorescence microscopy. Following image acquisition, the fluorescence along the spine of every Gastruloid within the experiment was quantified. The fluorescence is normalised to the maximum and the length of each Gastruloid re-scaled to length 1unit.

8/04/2016

Initial conditions for the establishment of Gastruloids (embryonic organoids)

A very curious and probably important feature of our embryonic organoids is their sensitivity to initial conditions. In our work we have used a number of different ES cell lines and have observed that while our initial observation of a critical number of starting ES cells, this number needs to be titrated for every cell line. Perhaps not surprisingly, the number does not vary wildly and is around 475 ± 50 but every cell lines has its number and, if you are interested in working with these organoids, you need to get it right. A telling milestone of the process is the diameter of the aggregate after 48 hrs in N2B27 (this 48 hrs in N2B27 is also a crucial step in the protocol). At this stage, the aggregate has to be around 150µm ± 30µm.  Find below a table with the cell lines we have used so far and the parameters that we have found are needed for them to undergo symmetry breaking (monitored by T/Brachyury expression), polarization and axial extension.

It is important to point out that like any experimental biological process, this one is sensitive to the conditions and that what we provide here is a guideline. We shall be interested to hear, if you try them, what you find. We sometimes have issues with the N2B27 which can complicate the process. Still, we are aware of other laboratories trying the protocol and finding a similar range than we do for the process to go forward.

ref_table

 

2 thoughts on “Updates

  1. One additional question regarding the starting conditions that I’m wondering about: all protocols (J Vis Exp, Development, Biorxiv) describe the use of mESCs kept under ES+LIF (w/o feeders). However, cells kept for at least two passages tend to be a very heterogeneous population, with lots of cells already primed towards certain lineages. Also, culture of mESCs under ES+LIF w/o feeders is not the ‘typical’ EB starting condition. Have you tried starting with mESCs cultured under ES+LIF /w feeders? Or 2i+LIF as a starting condition?

    • Hi Jesse, thank you for your comments. We have never generated Gastruloids from mESCs that were cultured on feeders, however we have a number of experiments using cells cultured in 2i conditions. The responses of the Gastruloids following 2i culture are similar in a number of ways to what is obtained from ESL medium, however we have noticed some striking differences which we think is due to the differences in the heterogeneity of the system between cells cultured in 2i and ESL. We’re currently working on a manuscript which will present and discuss these results in much more detail, however, feel free to drop us an email (ama11@cam.ac.uk or dat40@cam.ac.uk) if you would like more details or whether you’d like to discuss any experiments you’ve done with the Gastruloids in the conditions you’ve mentioned.

Leave a Reply

Your email address will not be published. Required fields are marked *