A synthetic mammalian electro-genetic transcription circuit.

Electric signal processing has evolved to manage rapid information transfer in neuronal networks and muscular contraction in multicellular organisms and controls the most sophisticated man-built devices. Using a synthetic biology approach to assemble electronic parts with genetic control units engineered into mammalian cells, we designed an electric power-adjustable transcription control circuit able to integrate the intensity of a direct current over time, to translate the amplitude or frequency of an alternating current into an adjustable genetic readout or to modulate the beating frequency of primary heart cells. Successful miniaturization of the electro-genetic devices may pave the way for the design of novel hybrid electro-genetic implants assembled from electronic and genetic parts.

Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats.

Cell-based therapies and tissue engineering initiatives are gathering clinical momentum for next-generation treatment of tissue deficiencies. By using gravity-enforced self-assembly of monodispersed primary cells, we have produced adult and neonatal rat cardiomyocyte-based myocardial microtissues that could optionally be vascularized following coating with human umbilical vein endothelial cells (HUVECs). Within myocardial microtissues, individual cardiomyocytes showed native-like cell shape and structure, and established electrochemical coupling via intercalated disks. This resulted in the coordinated beating of microtissues, which was recorded by means of a multi-electrode complementary metal-oxide-semiconductor microchip. Myocardial microtissues (microm3 scale), coated with HUVECs and cast in a custom-shaped agarose mold, assembled to coherent macrotissues (mm3 scale), characterized by an extensive capillary network with typical vessel ultrastructures. Following implantation into chicken embryos, myocardial microtissues recruited the embryo's capillaries to functionally vascularize the rat-derived tissue implant. Similarly, transplantation of rat myocardial microtissues into the pericardium of adult rats resulted in time-dependent integration of myocardial microtissues and co-alignment of implanted and host cardiomyocytes within 7 days. Myocardial microtissues and custom-shaped macrotissues produced by cellular self-assembly exemplify the potential of artificial tissue implants for regenerative medicine.

Modulation of cardiomyocyte electrical properties using regulated bone morphogenetic protein-2 expression.

Because cardiomyocytes lose their ability to divide after birth, any subsequent cell loss or dysfunction results in pathologic cardiac rhythm initiation or impulse conduction. Strategies to restore and control the electrophysiological activity of the heart may, therefore, greatly affect the regeneration of cardiac tissue functionality. Using lentivirus-derived particles to regulate the bone morphogenetic protein-2 (BMP-2) gene expression in a pristinamycin- or gaseous acetaldehyde-inducible manner, we demonstrated the adjustment of cardiomyocyte electrophysiological characteristics. Complementary metal oxide semiconductor-based high-density microelectrode arrays (HD-MEAs) were used to monitor the electrophysiological activity of neonatal rat cardiomyocytes (NRCs) cultured as monolayers (NRCml) or as microtissues (NRCmt). NRCmt more closely resembled heart tissue physiology than did NRCml and could be conveniently monitored using HD-MEAs because of their ability to detect low-signal events and to sub-select the region of interest, namely, areas where the microtissues were placed. Cardiomyocyte-forming microtissues, transduced using lentiviral vectors encoding BMP-2, were capable of restoring myocardial microtissue electrical activity. We also engineered NRCmt to functionally couple within a cardiomyocyte monolayer, thus showing pacemaker-like activity upon local regulation of transgenic BMP-2 expression. The controlled expression of therapeutic transgenes represents a crucial advance for clinical interventions and gene-function analysis.

Adenoviral vector platform for transduction of constitutive and regulated tricistronic or triple-transcript transgene expression in mammalian cells and microtissues.

BACKGROUND: Adenoviral particles can efficiently transduce a broad spectrum of cell types, so they are widely used in basic research and clinical trials. METHODS: We have developed a novel adenoviral vector platform for delivery of constitutive or streptogramin-inducible expression of up to three therapeutic transgenes into a variety of murine and human cell lines, primary cells and microtissues. RESULTS: Coordinated expression of three independent transgenes in a compact genetic format was achieved by two different expression configurations: (i) The multicistronic expression format consisting of a single constitutive (simian virus 40 promoter, P(SV40); murine or human cytomegalovirus immediate-early promoter, P(mCMV), P(hCMV)) or regulated (streptogramin-inducible) promoters (P(PIR)ON2) driving the expression of a single multicistronic transcript of which the first cistron is translated in a cap-dependent manner and the two subsequent ones by internal ribosome entry site (IRES)-mediated translation initiation. (ii) The triple-transcript expression configuration, in which a combination of well-established (P(SV40), P(hCMV), P(mCMV)) and novel synthetic constitutive promoters (P(GTX)) control transcription of three expression units. The constitutive multigene expression design enabled coordinated high-level expression of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY), the human vascular endothelial growth factor 121 (VEGF(121)) and the human placental secreted alkaline phosphatase (SEAP) in monolayer populations and microtissues of Chinese hamster ovary cells (CHO-K1), human fibrosarcoma cells (HT-1080), primary neonatal rat cardiomyocytes (NRCs) and primary human aortic fibroblasts (HAFs). Streptogramin-inducible tricistronic SAMY-VEGF(121)-SEAP expression provided excellent regulation performance-high-level induction in the presence of the streptogramin antibiotic pristinamycin I (PI), near-undetectable basal expression in the absence of PI, optimal adjustability and perfect reversibility-in all cell types, in particular in NRCs and NRC-derived myocardial microtissues. CONCLUSIONS: Triple-transcript and tricistronic expression configurations conserve the DNA packaging capacity of the size-constrained viral transduction systems and enable coordinated and regulated expression of up to three therapeutic transgenes for concerted clinical interventions in future gene therapy scenarios.

Heterologous protein production capacity of mammalian cells cultivated as monolayers and microtissues.

A precise understanding of processes managing heterologous protein production in vitro and in vivo is essential for the manufacture of sophisticated biopharmaceuticals as well as for future gene therapy and tissue engineering initiatives. Capitalizing on the gravity-enforced self-assembly of monodispersed cells into coherent (multicellular) microtissues we studied heterologous protein production of microtissues and monolayers derived from cell lines and primary cells engineered/transduced for (i) constitutive, (ii) proliferation-controlled, (iii) macrolide-, or (iv) gas-inducible expression of the human placental secreted alkaline phosphatase (SEAP) and of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Specific productivity of cells assembled in microtissues was up to 20-fold higher than isogenic monolayer cultures. Diffusion across microtissues could be further increased by HUVEC-mediated vascularization. As well as higher specific protein productivities, microtissues were also more efficient than monolayer cultures in assembling transgenic lentiviral particles. Our results showed that mammalian cells embedded in a tissue-like three-dimensional (3D) microenvironment exhibit increased production capacity. This observation should be considered for gene therapy and tissue engineering scenarios as well as for biopharmaceutical manufacturing.