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A library of mammalian effector modules for synthetic morphology.

Cachat E, Liu W, Hohenstein P, Davies JA - J Biol Eng (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Centre for Integrative Physiology, Hugh Robson Building, George Square, Edinburgh, EH8 9XD UK.

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.

No MeSH data available.


Related in: MedlinePlus

Tetracycline-induced cell fusion inp14-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.
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Fig5: Tetracycline-induced cell fusion inp14-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.

Mentions: Control T-REx-293 cells (see Figures 2a and 4b), and T-REx-293 cells carrying the fusion module but without induction (Figure 5a), showed no evidence of cell-cell fusion. Cells overexpressing p14FAST after tetracycline induction formed multinucleated syncytia as shown in Figure 5a: these could be detected as large, multinucleate cells in bright field that showed formation of tight ‘rosettes’ of nuclei visible with DAPI staining. In uninduced (unfused) cells, cortical actin surrounds each individual nucleus, indicating that the nucleus is in cytoplasm surrounded by its own plasma membrane: in the induced (fused cells), cortical actin instead surrounds complete rosettes of nuclei rather than individual ones, implying that the nuclei share one common cytoplasm. Syncytia formed through p14-induced fusion started dying around 20 h after tetracycline induction. Fusion events were rare when cells were grown in calcium-free medium (Figure 5b), confirming that the fusion process is calcium-dependent as expected [37]. To test whether the construct needed to be expressed in both neighbouring cells for fusion to take place, we mixed T-REx-293 cells carrying the fusion construct with MDCK canine kidney cells that had been transiently transfected with pTREx-mCherry as a marker (but not with the fusion construct). MDCK cells were chosen as they are well-characterized cells routinely used in our laboratory, and are from a different cell type and a different species than HEK cells (dog). They do not naturally form syncytia. In the mixed culture (Figure 5c), while some normal (unfused) MDCK cells could be seen as intensely red individual cells, some of the large syncytia contained red fluorescent patches suggesting that syncytia had formed in the mixed culture, and contained cytoplasms originating from both cell types. Potential users of this module should note that this result implies a risk that cells carrying the fusion module might fuse with cells of living humans, and potentially carry dangerous genetic material such as oncogenes and viruses into them (even basic cells lines carry mutations making them proliferative and immortal). Appropriate precautions should therefore be taken when handling the cultures.Figure 5


A library of mammalian effector modules for synthetic morphology.

Cachat E, Liu W, Hohenstein P, Davies JA - J Biol Eng (2014)

Tetracycline-induced cell fusion inp14-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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Tetracycline-induced cell fusion inp14-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.
Mentions: Control T-REx-293 cells (see Figures 2a and 4b), and T-REx-293 cells carrying the fusion module but without induction (Figure 5a), showed no evidence of cell-cell fusion. Cells overexpressing p14FAST after tetracycline induction formed multinucleated syncytia as shown in Figure 5a: these could be detected as large, multinucleate cells in bright field that showed formation of tight ‘rosettes’ of nuclei visible with DAPI staining. In uninduced (unfused) cells, cortical actin surrounds each individual nucleus, indicating that the nucleus is in cytoplasm surrounded by its own plasma membrane: in the induced (fused cells), cortical actin instead surrounds complete rosettes of nuclei rather than individual ones, implying that the nuclei share one common cytoplasm. Syncytia formed through p14-induced fusion started dying around 20 h after tetracycline induction. Fusion events were rare when cells were grown in calcium-free medium (Figure 5b), confirming that the fusion process is calcium-dependent as expected [37]. To test whether the construct needed to be expressed in both neighbouring cells for fusion to take place, we mixed T-REx-293 cells carrying the fusion construct with MDCK canine kidney cells that had been transiently transfected with pTREx-mCherry as a marker (but not with the fusion construct). MDCK cells were chosen as they are well-characterized cells routinely used in our laboratory, and are from a different cell type and a different species than HEK cells (dog). They do not naturally form syncytia. In the mixed culture (Figure 5c), while some normal (unfused) MDCK cells could be seen as intensely red individual cells, some of the large syncytia contained red fluorescent patches suggesting that syncytia had formed in the mixed culture, and contained cytoplasms originating from both cell types. Potential users of this module should note that this result implies a risk that cells carrying the fusion module might fuse with cells of living humans, and potentially carry dangerous genetic material such as oncogenes and viruses into them (even basic cells lines carry mutations making them proliferative and immortal). Appropriate precautions should therefore be taken when handling the cultures.Figure 5

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Centre for Integrative Physiology, Hugh Robson Building, George Square, Edinburgh, EH8 9XD UK.

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.

No MeSH data available.


Related in: MedlinePlus