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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

Control of cell proliferation through p27kip1inducible 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.
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Fig2: Control of cell proliferation through p27kip1inducible 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.

Mentions: Control T-REx-293 cells treated with 0.05 μg/mL tetracycline showed population growth rates similar to untreated wild-type cells (Figure 2a,b). On the other hand, clones stably transfected with p27Kip1 and induced with tetracycline for 72 h showed less proliferation than their uninduced (but still p27Kip1-carrying) counterparts (Figure 2c). Arrested cells appeared rounded and attached to the culture plate surface. It was important to test that the great reduction in proliferation that we observed was due to a reversible brake on cell cycling, and not as a result of cell damage or cell death. To verify this, we exploited the flexibility of the Tet induction system and observed the behaviour of cells following release from p27Kip1-mediated inhibition of proliferation. Cells were induced with tetracycline for 48 h, when the medium was replaced by the tetracycline-free version: released cells resumed proliferation after a delay of about one day (Figure 2d), although somewhat slower than their never-inhibited counterparts. Arrested cells could withstand tetracycline exposure for as long as 72 h before resuming growth. Longer exposures resulted in cell death.Figure 2


A library of mammalian effector modules for synthetic morphology.

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

Control of cell proliferation through p27kip1inducible 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Control of cell proliferation through p27kip1inducible 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.
Mentions: Control T-REx-293 cells treated with 0.05 μg/mL tetracycline showed population growth rates similar to untreated wild-type cells (Figure 2a,b). On the other hand, clones stably transfected with p27Kip1 and induced with tetracycline for 72 h showed less proliferation than their uninduced (but still p27Kip1-carrying) counterparts (Figure 2c). Arrested cells appeared rounded and attached to the culture plate surface. It was important to test that the great reduction in proliferation that we observed was due to a reversible brake on cell cycling, and not as a result of cell damage or cell death. To verify this, we exploited the flexibility of the Tet induction system and observed the behaviour of cells following release from p27Kip1-mediated inhibition of proliferation. Cells were induced with tetracycline for 48 h, when the medium was replaced by the tetracycline-free version: released cells resumed proliferation after a delay of about one day (Figure 2d), although somewhat slower than their never-inhibited counterparts. Arrested cells could withstand tetracycline exposure for as long as 72 h before resuming growth. Longer exposures resulted in cell death.Figure 2

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