A library of mammalian effector modules for synthetic morphology

Abstract

Background: In mammalian development, the formation of most tissues is achieved by a relatively small repertoire of basic morphogenetic events (e.g. cell adhesion, locomotion, apoptosis, etc.), permutated in various sequences to form different tissues. Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms. The control of tissue morphogenesis by populations of engineered cells is a potentially very powerful but neglected aspect of synthetic biology.

Results: We have assembled a modular library of synthetic morphogenetic driver genes to control (separately) mammalian cell adhesion, locomotion, fusion, proliferation and elective cell death. Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command.

Conclusions: Building on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

Keywords: Development; Human embryonic kidney cells; Morphogenesis; Morphogenetic effectors; Synthetic biology; Synthetic morphology.

Figure 1

The morphogenetic effectors used in this study. p27 ^kip1 (control of proliferation), Casp2 (elective cell death), Cdh1 (cell-cell adhesion), p14 (fusion) and Crk-II (locomotion) were inserted downstream of a tetracycline-responsive promoter and coupled with different fluorescent reporters in pTREx destination vectors.

Figure 2

Control of cell proliferation through p27 ^kip1 inducible expression. (a) Control T-REx-293 cells were seeded in 6-well plates and cultured for 72 h with or without 0.05 μg/mL tetracycline. (b) Cell counts in triplicate wells showed that tetracycline itself had no effect on wild-type cell growth. (c) Cells from clone THGA-17 (a representative clone of T-REx-293 cells carrying the growth arrest module) showed clear growth rate differences after 72 h of culture with tetracycline. (d) After 48 h of growth inhibition with tetracycline, culture in tetracycline-free medium released THGA-17 cells from proliferation inhibition (green line). Scale bars: 100 μm. Standard deviation bars: n = 3.

Figure 3

Tetracycline-induced cell death in Casp2 -engineered T-REx-293 cells. (a) Uninduced cells from clone THAP2-2 (a representative clone of T-REx-293 cells carrying the elective cell death module) showed normal growth, but extensive cell death when induced with tetracycline (1 μg/mL) for 48 h. (b) Numbers of adherent, uninduced and induced THAP2-2 cells were monitored over 48 h after induction in 24-well plates. Standard deviation bars: n = 3. (c) When medium was supplemented with caspase inhibitor Q-VD-OPh (50 μM), cell death was largely blocked. Graph: percentages of area covered by adherent cells in various image fields: under normal growth conditions, after tetracycline induction, after tetracycline induction in the presence of Q-VD-OPh or in the presence of Q-V-D-OPh alone. Scale bars: 100 μm.

Figure 4

Effect of the adhesion effector on engineered T-REx-293 cells. (a) Levels of murine Cdh1 mRNA expression in cells of clone THAD1-34 (a representative clone of T-REx-293 cells carrying the adhesion module), uninduced or induced with 1 μg/mL tetracycline for 48 h; and in wild-type T-REx-293 cells treated with tetracycline, as a control, for 48 h. (b) Morphology of wild-type T-REx-293 cells and THAD1-34 cells growing with or without tetracycline for 48 h: wild-type cells with or without tetracycline and uninduced THAD1-34 cells spread across the culture substrate with only loose mutual association, whereas induced THAD1-34 cells adhere to one another strongly to produce dense islands separated by clear space. Scale bars: 100 μm.

Figure 5

Tetracycline-induced cell fusion in p14 -engineered T-REx-293 cells. (a) Bright-field microscopy, DAPI staining (white or magenta) and phalloidin-FITC (green) of THFU-4 cells (a representative clone of T-REx-293 cells carrying the fusion module), treated or not with tetracycline for 24 h. Untreated cells (left column) grow normally, without obvious signs of fusion, nuclei remaining separate and surrounded individually by cell membranes, shown by phalloidin stain of cortical actin filaments in the bottom row. Cells in which the fusion construct is induced show formation of large, multinucleate cells in bright field illumination (top row), formation of ‘rosettes’ of tightly apposed nuclei (middle row), with cortical actin (green) surrounding the whole groups of nuclei rather than each individual one (bottom row). (b) Cells grown in calcium-free medium showed fewer fusion events, as expected (33). (c) Evidence of fusion between tetracycline-induced cells from clone THFU-10 (another representative clone) and MDCK cells transiently expressing mCherry: unfused MDCK cells appear as small, deep red individual cells (white arrow). The red is also seen (black arrows) in large syncytia, implying that the THFU-10 cells can fuse with cells not themselves containing the fusion module. Scale bars: 100 μm.

Figure 6

Enhanced locomotion induced by tetracycline in T-REx-293 cells engineered with Crk-II. (a) DAPI staining (blue) and phalloidin-FITC (green) during scratch closing assays showing enhanced lamellipodia formation in THLOII-15 cells (a representative clone of T-REx-293 cells carrying the locomotion module) when induced with tetracycline: the induced cells show more frequent formation of lamellipodia, shown in green. Scale bars: 100 μm. (b) Distance travelled by cells in scratch closing assays with or without induction of the locomotion module. Images were acquired every 10 min and coordinates of 4 groups of cells on each side of wounds were recorded (6 to 18 h post-induction). Tetracycline increased the migration of THLOII-15 cells (trail plot and graph, *p <0.001) but had no effect on wild-type cells (graph). Statistical analysis was performed using a two-sample Student’s t-Test with a two-tailed distribution. Standard deviation bars: n = 8.