Barcoded microbial system for high-resolution object provenance.

Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.

Single-Nucleotide-Resolution Computing and Memory in Living Cells.

The ability to process and store information in living cells is essential for developing next-generation therapeutics and studying biology in situ. However, existing strategies have limited recording capacity and are challenging to scale. To overcome these limitations, we developed DOMINO, a robust and scalable platform for encoding logic and memory in bacterial and eukaryotic cells. Using an efficient single-nucleotide-resolution Read-Write head for DNA manipulation, DOMINO converts the living cells' DNA into an addressable, readable, and writable medium for computation and storage. DOMINO operators enable analog and digital molecular recording for long-term monitoring of signaling dynamics and cellular events. Furthermore, multiple operators can be layered and interconnected to encode order-independent, sequential, and temporal logic, allowing recording and control over the combination, order, and timing of molecular events in cells. We envision that DOMINO will lay the foundation for building robust and sophisticated computation-and-memory gene circuits for numerous biotechnological and biomedical applications.

Small-molecule control of antibody N-glycosylation in engineered mammalian cells.

N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG. We knocked out two key glycosyltransferase genes, α-1,6-fucosyltransferase (FUT8) and ß-1,4-galactosyltransferase (ß4GALT1), genomically integrated circuits expressing synthetic glycosyltransferase genes under constitutive or inducible promoters and generated antibodies with concurrently desired fucosylation (0-97%) and galactosylation (0-87%) levels. Simultaneous and independent control of FUT8 and ß4GALT1 expression was achieved using orthogonal small molecule inducers. Effector function studies confirmed that glycosylation profile changes affected antibody binding to a cell surface receptor. Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.

Optical High Content Nanoscopy of Epigenetic Marks Decodes Phenotypic Divergence in Stem Cells.

While distinct stem cell phenotypes follow global changes in chromatin marks, single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics, we developed a novel approach, termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States), to discern chromatin organizational changes, demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness, thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall, EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks.

Bioreducible-Cationic Poly(amido amine)s for Enhanced Gene Delivery and Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine.

Osteogenic priming of mesenchymal stem cells by chondrocyte-conditioned factors and mineralized matrix.

Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8 weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson's trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs.

Induced myogenic commitment of human chondrocytes via non-viral delivery of minicircle DNA.

Lineage conversion from one somatic cell type to another is an attractive approach for deriving specific therapeutic cell generation. In order to bypass inducing pluripotent stage, transdifferentiation/direct conversion technologies have been recently developed. We report the development of a direct conversion methodology in which cells are transdifferentiated through a plastic intermediate state induced by exposure to non-integrative minicircle DNA (MCDNA)-based reprogramming factors, followed by differentiation into myoblasts. In order to increase the MCDNA delivery efficiency, reprogramming factors were delivered into the chondrocytes via electroporation followed by poly (ß-amino esters) (PBAE) transfection. We used this approach to convert human chondrocytes to myoblast, and with treatment of SB-431542, an inhibitor of the activin receptor-like kinase receptors, to enhance myogenic commitment. Differentiated cells exhibited expression of myogenic markers such as MyoD and Myog. This methodology for direct lineage conversion from chondrocytes to myoblast represents a novel non-viral Method to convert hard-to-transfect cells to other lineage.

Extracellular matrix-immobilized nanotopographical substrates for enhanced myogenic differentiation.

Extracellular matrix (ECM) components such as fibronectin (FN) and laminin (LMN) play prominent roles in controlling cellular behaviors. Many attempts have been made to explore cellular behaviors on combinatorial ECM arrays in high-throughput systems. However these studies were limited to physical adsorption of ECM, which does not guarantee a lasting effect of ECM in vitro. Here, we demonstrate ECM immobilization on polyurethane acrylate (PUA) substrate fabricated in 24 well-plate platforms to effectively differentiate C2C12. Our study demonstrate that co-immobilization of FN and LMN was found to enhance myogenic differentiation of C2C12 cells compared to single immobilization of either FN or LMN alone. Furthermore, utilizing nano-imprint lithography technique, 300 nm and 5 µm line-patterned substrates were fabricated on 24-well plates. FN and LMN co-immobilized substrates with line-patterns additionally provided the directionality for mimicking musculoskeletal structure and enhanced the myogenic differentiation.

Application of magnetic nanoparticle for controlled tissue assembly and tissue engineering.

Magnetic nanoparticles have been subjected to extensive studies in the past few decades owing to their promising potentials in biomedical applications. The versatile intrinsic properties of magnetic nanoparticles enable their use in many biomedical applications. Recently, magnetic nanoparticles were utilized to control the cell's function. In addition, intracellular delivery of magnetic nanoparticles allowed cell's positioning by appropriate use of magnetic field and created cellular cluster. Furthermore, magnetic nanoparticles have been utilized to assemble more complex tissue structures than those that are achieved by conventional scaffold-based tissue engineering strategies. This review addresses recent work in the use magnetic nanoparticle for controlled tissue assembly and complex tissue formation.

Gold nanoparticle (AuNP)-based drug delivery and molecular imaging for biomedical applications.

Gold nanoparticles (AuNPs) can be readily synthesized and modified with chemical and biological molecules, making them attractive inorganic biomaterials for drug delivery and molecular diagnostics. The surface of AuNPs supports the efficient attachment of various biomacromolecules via chemisorption, chemical conjugation and electrostatic interaction. Based on advantages of facile surface modification and unique optical properties, AuNPs have been extensively used as drug carriers for the intracellular delivery of therapeutics as well as molecular nanoprobes for detection and monitoring of the target molecules of interest. In this review, we highlight advanced approaches in the biomedical applications of AuNPs such as gene and drug therapy, molecular diagnostics and imaging.