During embryonic development, cells can integrate information in space and time to build the complex structure of a full organism in a remarkably reproducible way. The possibility to emulate some of the ‘logics’ of embryonic development to rationally control tissue formation in vitro is one of the main goals of the Laboratory of Stem Cell Bioengineering at EPFL. 

A tricky problem in constructing tissues outside of an organism is how to present key signalling molecules, also termed morphogens, to the cells in culture at the right time and dose. Simply exposing a cell ensemble to a uniform concentration of a morphogen, as is commonly done in organoid cultures, results in uncontrolled morphogenesis, because the cells lack important spatial and temporal instructions. In a developing organism, cells are exposed to highly dynamic and spatially variable signalling environments that are generated by so-called ‘signalling centres’. These centres act as sources of morphogens (or morphogen inhibitors) that play an essential role in the formation of cell fate patterns. We hypothesized that engineering of an artificial signalling centre ‘ex vivo’ could allow us to steer the self-organization of a stem cell population towards a desired outcome, with obvious advantages for tissue and organ engineering.  

The initial idea for our paper came from a really cool Nature Methods article reporting the surprising self-organization capacity of Pluripotent Stem Cell (hPSC) colonies grown under geometric confinement¹. In this paper, Ali Brivanlou and Eric Siggia with their colleagues at Rockefeller University showed that hPSC colonies, stimulated with the morphogen BMP4 and forced to grow on circular adhesive patches, self-organized into radial patterns of different cell types. This system recapitulated some features of embryonic gastrulation such as the ordered presence of cells from multiple germ layers. Because of their robust and predictable outcome, we felt that micropatterned hPSC colonies could be an ideal testbed to study whether artificial signalling centres could be built and used to influence the patterning of a self-organizing multicellular system.  

But how to expose hPSC colonies to localized signalling sources? Based on the lab’s previous experience (e.g.^2,3), we decided to turn to microfluidic technology. Together with my colleague Yoji Tabata, I designed and fabricated a microfluidic device that could contain geometrically confined hPSC colonies and allow for the exposure of the cells to gradients of morphogens diffusing from localized signalling sources. Although everything looked straightforward on paper, we had to go through a painfully long period of trial and error in which we designed, fabricated and tested several prototypes. One of the major problems was the incompatibility of the devices with stable hPSC culture.  

After months of optimization work, we ended up with a functioning prototype device that we fabricated from poly(dimethylsiloxane) (PDMS). An important feature of this system was a set of hydrogel walls that separated the circular hPSC culture chamber from the external channels used for medium perfusion (Fig. 1a, b). The hydrogel walls thus shielded the cells from convective flow present in the external channels, yet allowed for the diffusion of molecules across the culturing chamber. In this way, we managed to generate dynamic concentration gradients of morphogens inside the chamber in a predictable and reproducible manner (Fig. 1c, d).

Figure 1. (a) Scheme of one unit of the microfluidic device. (b) Picture of the microfluidic device. (c) Experimentally visualized concentration gradient of fluorescently-labelled 40kDa Dextran at 48 hours from the beginning of the perfusion. (d) Quantification of the concentration gradient of fluorescently-labelled 40kDa Dextran at the indicated time points from the beginning of the perfusion (source on the left).

Excitingly, this microfluidic device enabled us to control the patterning of hPSCs and led to a number of interesting findings. For example:

• we showed how localized signalling sources of BMP4 could break the radial symmetry of hPSC colonies (Fig. 2a, b), usually with the formation of a more posterior domain close to the source.
• we observed interesting “bi-phasic” dynamics of BMP pathway activity during the exposure to a localized signalling source.
• we observed the sensitivity of the patterning to the morphogen source concentration (Fig. 2c). We also obtained data suggesting that patterning under a localized signalling source depends on the balance between the number of cells receiving the morphogen and the quantity of morphogen provided (in a way similar to that shown for the uniform exposure to BMP4 by other groups⁴).
• we demonstrated how it is possible to increase the spatial control on the patterning by implementing counteracting gradients of a morphogen (BMP4) and a related inhibitor (NOGGIN) (Fig. 2d).

Figure 2. (a) hPSC colony exposed to a uniform 50ng/ml BMP4 concentration. (b) hPSC colony exposed to a localized 50ng/ml BMP4 source.  (c) hPSC colony exposed to a localized 250ng/ml BMP4 source. (d) hPSC colony exposed to counteracting sources of 50ng/ml BMP4 and 200ng/ml NOGGIN. 

We think that this system is broadly applicable to study how localized signalling sources and morphogen gradients operate in determining cell fate patterning. The approach may be useful for building more complex physiological signalling environments to quantitatively study symmetry breaking or other complex cell behaviours that occur during early embryonic development and organogenesis. Ultimately, this system could elucidate important principles that can be applied to increase the control on in vitro morphogenesis for applications in other fields such as tissue engineering.  

This post was written by Andrea Manfrin and Matthias P. Lutolf

References:

1. Warmflash A. et al. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat Methods 11, 847-854 (2014).
2. Cosson S. & Lutolf M.P. Hydrogel microfluidics for the patterning of pluripotent stem cells. Sci Rep., 4 4462 (2015).
3. Tabata Y. and Lutolf M.P. Multiscale microenvironmental perturbation of pluripotent stem cell fate and self-organization. Sci Rep. 7, 44711 (2017).
4. Etoc F. et al.  A balance between secreted inhibitors and edge sensing controls gastruloid self-organization. Dev. Cell 39, 302-315 (2016).