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 part

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

Ectopic expression of delta FBJ murine osteosarcoma viral oncogene homolog B mediates transdifferentiation of adipose-like spheroids into osteo-like microtissues

Differentiation and transdifferentiation strategies have a large role in the manipulation of cells in replacing dysfunctional cells and tissues. We developed adipose-like microtissues using gravity-enforced self-assembly of monodispersed human primary preadipocytes to determine their transdifferentiation capacity to form bone-like tissues. Using lentivirus-derived particles to induce ectopic bone morphogenetic protein (BMP)-2 and delta FBJ murine osteosarcoma viral oncogene homolog B (DeltaFosB) gene expression, we demonstrated a time-dependent induction of osteoblast-specific genes and properties such as calcium deposits, bone-like extracellular matrix (ECM), and matrix mineralization. DeltaFosB was able to trigger partial Pref-1-mediated de-differentiation of adipocytes, which also retained their adipocytic cell phenotype. Osteoblast-specific structures could be co-localized in the ECM of lipid-containing cells analyzed using immunofluorescence and transmission electron microscopy when BMP-2 and DeltaFosB were co-expressed, suggesting that differentiated adipocytes are able to transdifferentiate into osteoblasts via a transient hybrid adipocyte-preadipocyte-osteoblast cell phenotype. Microtissues transgenic for BMP-2 and DeltaFosB expression were able to reproduce bone matrix, which occurs to a lesser extent in conventional two-dimensional (2D) cultures but is known to play a decisive role in the development and function of bone in vivo. This demonstrates that ECM-inclusive studies are essential for future characterization assays. Therefore, 3D cultures provide a superior ex vivo system for the improved characterization of phenotypical and functional alterations resulting from interventions directed toward differentiation processes. Precise control of transdifferentiation of adipocytes into osteoblasts in a 3D culture mimicking in vivo tissue conditions as closely as possible will foster important advances in regenerative medicine and tissue engineering

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

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