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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 death inCasp2-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.
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Fig3: Tetracycline-induced cell death inCasp2-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.

Mentions: Uninduced T-REx-293 cells carrying the elective cell death module (Figure 3a, Additional file 1: Movie 1), as well as control wild-type T-REx-293 cells treated with tetracycline (see Figure 2a), showed little cell death and their number increased after 48 h in culture due to proliferation. After induction, the cells carrying pTREx-Casp2 showed extensive apoptotic death, detectable by microscopy (Figure 3a, Additional file 1: Movie 1) and by counting the declining number of cells still adhering to the wells (Figure 3b). To verify that the mechanism of cell death was that intended, we used Q-VD-OPh, a pan-caspase inhibitor. When added to the growth medium, it largely blocked cell death after tetracycline induction of the module (Figure 3c), confirming that the cell death observed previously (in absence of the inhibitor) was indeed caspase-driven.Figure 3


A library of mammalian effector modules for synthetic morphology.

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

Tetracycline-induced cell death inCasp2-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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4255936&req=5

Fig3: Tetracycline-induced cell death inCasp2-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.
Mentions: Uninduced T-REx-293 cells carrying the elective cell death module (Figure 3a, Additional file 1: Movie 1), as well as control wild-type T-REx-293 cells treated with tetracycline (see Figure 2a), showed little cell death and their number increased after 48 h in culture due to proliferation. After induction, the cells carrying pTREx-Casp2 showed extensive apoptotic death, detectable by microscopy (Figure 3a, Additional file 1: Movie 1) and by counting the declining number of cells still adhering to the wells (Figure 3b). To verify that the mechanism of cell death was that intended, we used Q-VD-OPh, a pan-caspase inhibitor. When added to the growth medium, it largely blocked cell death after tetracycline induction of the module (Figure 3c), confirming that the cell death observed previously (in absence of the inhibitor) was indeed caspase-driven.Figure 3

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