A synthetic mammalian network to compute population borders based on engineered reciprocal cell-cell communication
© Kolar et al. 2015
Received: 7 March 2015
Accepted: 15 December 2015
Published: 30 December 2015
Multicellular organisms depend on the exchange of information between specialized cells. This communication is often difficult to decipher in its native context, but synthetic biology provides tools to engineer well-defined systems that allow the convenient study and manipulation of intercellular communication networks.
Here, we present the first mammalian synthetic network for reciprocal cell-cell communication to compute the border between a sender/receiver and a processing cell population. The two populations communicate via L-tryptophan and interleukin-4 to highlight the population border by the production of a fluorescent protein. The sharpness of that visualized edge can be adjusted by modulating key parameters of the network.
We anticipate that this network will on the one hand be a useful tool to gain deeper insights into the mechanisms of tissue formation in nature and will on the other hand contribute to our ability to engineer artificial tissues.
Multicellular organisms strongly depend on the communication between specialized cells starting from their development throughout their entire lifespan . This communication between cell populations is often hard to decipher in its native context due to the presence of perturbing factors that are not central to the communication but attenuate it in various ways and because of a crosstalk with other signaling components. To gain deeper insight into such complex processes, synthetic biology provides us with tools to engineer artificial intercellular communication systems with a clearly defined, finite number of well-characterized components [2–5]. Such synthetic systems do not only open up the possibility to study and manipulate the communication between cell populations without any external perturbing influences, but are also important steps towards the engineering of artificial tissues. To date, several intercellular communication systems have been constructed in bacteria and in unicellular eukaryotes [2, 6], but the implementation of such systems in mammalian backgrounds is lagging behind, likely due to their unequally greater complexity. The few existing synthetic sender-receiver systems for mammalian cells use acetaldehyde , nitric oxide , hepatocyte growth factor , L-tryptophan  or Delta-Notch-mediated direct cell-cell contact  as signals for communication. Notably, one system for two-way communication between mammalian cells has been implemented that is based on L-tryptophan and acetaldehyde as signaling molecules. This system has been applied to coordinate the expression of highly regulated factors required for the maturation of blood vessels, namely vascular endothelial growth factor and angiopoietin-1 .
One particular example of intercellular communication is the exchange of information between adjacent cell populations in order to define and detect population borders. The importance of this mechanism is probably best illustrated in cancer cells that lose the ability to detect and react to such borders during unregulated tumor growth and metastasis [11, 12]. In this study, we aimed to engineer a synthetic system that emulates the detection of the border between two distinct populations of cells.
Results and discussion
Beyond this proof-of-principle, our synthetic network opens up the possibility to emulate intercellular communication systems that require the computation of population borders as sharper or wider edges, simply by tuning key parameters of the system. The high flexibility of our system allows the visualized edge to be decreased in broadness by 1) decreasing the production of L-tryptophan in the sender/receiver population, 2) decreasing the sensitivity of the processing cell population towards L-tryptophan, 3) decreasing the production of interleukin-4 by the processing cell population, 4) decreasing the sensitivity of the sender/receiver population towards interleukin-4 and by 5) limiting the diffusion of interleukin-4. Analogously, the spread of the detected edge may be increased by adjusting the parameters in the opposite direction.
We have established the first synthetic mammalian system for two-way cell-cell communication to compute and visualize the border between cell populations. Communication between the populations takes place via L-tryptophan and interleukin-4 and results in the production of a fluorescent protein at the population border. Formation and maintenance of borders between functionally different mammalian cell populations are highly interesting and complex phenomena, which are especially vital in the context of embryonic development and homeostasis [15, 16]. It can be expected that this network will be useful as a tool and a blueprint to help emulate these processes in order to increase our understanding of the molecular mechanisms that underlie tissue formation, as well as contribute to the engineering of artificial tissues.
The construction of expression vectors is given in detail in Additional file 2: Table S1.
Cell culture and transfection
All experiments were conducted in human embryonic kidney fibroblasts (HEK-293T ). Unless indicated, the cells were maintained in Dulbecco’s modified Eagle’s medium (PAN, cat. no. P03-0710) supplemented with 10 % (v/v) FBS (PAN, cat. no. P30-3602, batch no. P101003TC), 100 U ml−1 of penicillin and 0.1 mg ml−1 of streptomycin (PAN, cat no. P06-07001). In L-tryptophan-sensitive experiments the cells were cultured in L-tryptophan free InVitrus medium (Cell Culture Technologies, custom-made) supplemented with 10 % (v/v) FBS, 100 U ml−1 of penicillin, 0.1 mg ml−1 of streptomycin and 0.5 μM L-tryptophan. Where indicated, L-tryptophan or indole was added to the culture medium from a 39.17 mM stock in H2O or from a 500 mM stock in ethanol, respectively.
Cells were transfected using an optimized polyethylene-imine-based method (PEI, linear, MW: 25 kDa) (Polyscience) . In brief, 1 mg ml−1 PEI solution in H2O was adjusted to pH 7.0 with HCl, sterile filtered and stored at −80 °C in aliquots. Next, 70,000 cells were seeded per well of a 24-well plate and cultivated overnight. Aliquots of 0.75 μg of DNA were diluted in 50 μl of OptiMEM (Invitrogen) and mixed with 2.5 μl of PEI solution in 50 μl of OptiMEM by vortexing (amounts scaled to one well). After 20 min incubation at room temperature, the precipitate was added to the cells. The culture medium was replaced 5 h after the transfection. Unless indicated, plasmids were transfected in equal amounts (w:w).
Production of fibronectin
His-tagged fibronectin (domains 7–10) was expressed from pET15bFN-III7-10RGE  in E.coli BL21(DE3)pLysS (Promega, cat. no. L1195) and purified using Ni-NTA chromatography. Aliquots of the purified protein were frozen in liquid nitrogen and stored in PBS (pH 7.4) at a concentration of 1 mg ml−1 at −80 °C.
Computation of the interface between adjacent cell populations
To set up a culture system with two compartments, the inner wall of a culture insert (Ibidi, cat. no. 80209) was removed and the modified insert was placed in the middle of a 60 mm cell culture dish. Next, the surface of the culture dish was coated with fibronectin by applying 37.5 μl of fibronectin solution (80 μg ml−1) to the central area and 1.5 ml to the outer area. After incubation for 1 h at room temperature the processing cell population (HEK-293T transfected with pWB024 and pHW073, 24 h post transfection) was seeded in the inner compartment (70,000 cells in 125 μl InVitrus medium), while the sender/receiver population (HEK-293T transfected with pHW074, pHW040, pSTAT6 and pMK47 – as a transfection control plasmid for constitutive expression of mCherry; 1.5 μg per 10 cm culture dish transfection – 24 h post transfection) was seeded in the outer compartment (2.75 million cells in 5 ml InVitrus medium). Twenty-four hours later, the inserts were removed and the cells were overlaid with 5.25 ml agarose-solidified InVitrus medium (1 % w/v) supplemented with 500 μM indole. After 35 min at room temperature for solidification of the overlay, the cells were cultivated at 37 °C for 48 h before microscopic detection of the fluorescent reporter proteins.
The reporter SEAP was quantified in the cell culture medium, using a colorimetric assay as described elsewhere . Interleukin-4 (IL-4) and L-tryptophan were quantified in the culture medium using an IL-4 ELISA kit (Pepro Tech, cat. no. 900-K14) or the Bridge-IT L-Tryptophan Fluorescence Assay (Mediomics, cat. no. 1-1-1002A) according to the manufacturer’s instructions, respectively.
The fluorescence intensity of mammalian cells was quantified in cell lysates. First, the cells were lysed by the addition of 250 μl lysis buffer (25 mM Tris–HCl pH 7.8, 1 % (v/v) Triton X-100, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT) per well of a 24-well plate. Then, 100 μl of each lysate was transferred to a 96-well flat-bottom black plate and YFP fluorescence intensity was quantified using a Synergy 4 multimode microplate reader (BioTek Instruments) or a Tecan infinite 200Pro microplate reader (Tecan Group), with excitation at 490 nm and emission at 527 nm.
A fluorescence microscope (Zeiss Cell Observer, Carl Zeiss) was used to acquire YFP and mCherry mosaic images of 60 mm dishes with a Plan-Neofluar pol. 2.5× objective lens (NA 0.075). The same exposure time was used for all samples. Mosaic images were stitched with XuvStitch (v1.8.1-beta5) and processed with Fiji (ImageJ v2.0.0-rc-41/1.50b), where heat maps were produced using the 3D Surface Plot function.
Availability of supporting data
The data sets supporting the results of this article are included within the article (and its additional files).
We thank Prof. Dr. Peter L. Graumann for providing the E. coli K-12 MG1655 strain, Prof. Dr. Reinhard Fässler for providing the plasmid pET15bFN-III7-10RGE, and Prof. Dr. Martin Fussenegger for providing the plasmid pWB024.
This work was supported by the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement no 259043-CompBioMat and the Excellence Initiative of the German Federal and State Governments (EXC 294).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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