Advancing in vivo reprogramming with synthetic biology.

Reprogramming cells will play a fundamental role in shaping the future of cell therapies by developing new strategies to engineer cells for improved performance and higher-order physiological functions. Approaches in synthetic biology harness cells' natural ability to sense diverse signals, integrate environmental inputs to make decisions, and execute complex behaviors based on the health of the organism or tissue. In this review, we highlight strategies in synthetic biology to reprogram cells, and discuss how recent approaches in the delivery of modified mRNA have created new opportunities to alter cell function in vivo. Finally, we discuss how combining concepts from synthetic biology and the delivery of mRNA in vivo could provide a platform for innovation to advance in vivo cellular reprogramming.

In Vitro Generation of Megakaryocytes from Engineered Mouse Embryonic Stem Cells.

The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase-mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.

Reprogramming megakaryocytes to engineer platelets as delivery vehicles.

We developed a method for the efficient generation of engineered platelets that can be filled with any recombinant therapeutic protein during the differentiation process by reprogramming megakaryocytes, the progenitor cells of platelets. To demonstrate the versatility of this approach, we loaded cytoplasmic and secreted proteins that can be delivered as active enzymes to recipient cells, be released upon platelet activation, or be continuously secreted by platelets over time.

In vitro generation of megakaryocytes from engineered mouse embryonic stem cells.

The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

Design and development of engineered receptors for cell and tissue engineering.

Advances in synthetic biology have provided genetic tools to reprogram cells to obtain desired cellular functions that include tools to enable the customization of cells to sense an extracellular signal and respond with a desired output. These include a variety of engineered receptors capable of transmembrane signaling that transmit information from outside of the cell to inside when specific ligands bind to them. Recent advances in synthetic receptor engineering have enabled the reprogramming of cell and tissue behavior, controlling cell fate decisions, and providing new vehicles for therapeutic delivery.

Enhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator.

Expanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. Here, we reverse the current trend of moving genetic parts from prokaryotes to eukaryotes and demonstrate that the activating eukaryotic transcription factor QF and its corresponding DNA-binding sequence can be moved to E. coli to introduce transcriptional activation, in addition to tight off states. We further demonstrate that the QF transcription factor can be used in genetic devices that respond to low input levels with robust and sustained output signals. Collectively, we show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. These genetic tools hold the potential to improve biotechnology applications for medical science and research.

High-throughput enrichment and isolation of megakaryocyte progenitor cells from the mouse bone marrow.

Megakaryocytes are a rare population of cells that develop in the bone marrow and function to produce platelets that circulate throughout the body and form clots to stop or prevent bleeding. A major challenge in studying megakaryocyte development, and the diseases that arise from their dysfunction, is the identification, classification, and enrichment of megakaryocyte progenitor cells that are produced during hematopoiesis. Here, we present a high throughput strategy for identifying and isolating megakaryocytes and their progenitor cells from a heterogeneous population of bone marrow samples. Specifically, we couple thrombopoietin (TPO) induction, image flow cytometry, and principal component analysis (PCA) to identify and enrich for megakaryocyte progenitor cells that are capable of self-renewal and directly differentiating into mature megakaryocytes. This enrichment strategy distinguishes megakaryocyte progenitors from other lineage-committed cells in a high throughput manner. Furthermore, by using image flow cytometry with PCA, we have identified a combination of markers and characteristics that can be used to isolate megakaryocyte progenitor cells using standard flow cytometry methods. Altogether, these techniques enable the high throughput enrichment and isolation of cells in the megakaryocyte lineage and have the potential to enable rapid disease identification and diagnoses ahead of severe disease progression.

High-Throughput Production of Platelet-Like Particles.

The in vitro production of platelets could provide a life-saving intervention for patients that would otherwise require donor-derived platelets. Producing large numbers of platelets in vitro from their progenitor cells, megakaryocytes, remains remarkably difficult and inefficient. Here, a human megakaryoblast leukemia cell line (MEG-01) was used to assess the maturation of megakaryocytes and to develop a new methodology for producing high numbers of platelet-like particles from mature MEG-01 cells in vitro.

Biological Cells as Therapeutic Delivery Vehicles.

One of the significant challenges remaining in the field of drug delivery is insufficient targeting of diseased tissues or cells. While efforts to perform targeted drug delivery by engineered nanoparticles have shown some success, there are underlying targeting, toxicity, and immunogenicity challenges. By contrast, live cells usually have innate targeting mechanisms, and can be used as drug-delivery vehicles to increase the efficiency with which a drug accumulates to act on the intended tissue. In some cases, when no native cell types exhibit the desired therapeutic phenotype, preferred outcomes can be achieved by genetically modifying and reprogramming cells with gene circuits. This review highlights recent advances in the use of cells to deliver therapeutics. Specifically, we discuss how red blood cells (RBCs), platelets, neutrophils, mesenchymal stem cells (MSCs), and bacteria have been utilized to advance drug delivery.

Rosa26 docking sites for investigating genetic circuit silencing in stem cells.

Approaches in mammalian synthetic biology have transformed how cells can be programmed to have reliable and predictable behavior, however, the majority of mammalian synthetic biology has been accomplished using immortalized cell lines that are easy to grow and easy to transfect. Genetic circuits that integrate into the genome of these immortalized cell lines remain functional for many generations, often for the lifetime of the cells, yet when genetic circuits are integrated into the genome of stem cells gene silencing is observed within a few generations. To investigate the reactivation of silenced genetic circuits in stem cells, the Rosa26 locus of mouse pluripotent stem cells was modified to contain docking sites for site-specific integration of genetic circuits. We show that the silencing of genetic circuits can be reversed with the addition of sodium butyrate, a histone deacetylase inhibitor. These findings demonstrate an approach to reactivate the function of genetic circuits in pluripotent stem cells to ensure robust function over many generations. Altogether, this work introduces an approach to overcome the silencing of genetic circuits in pluripotent stem cells that may enable the use of genetic circuits in pluripotent stem cells for long-term function.

Synthetic biology for improving cell fate decisions and tissue engineering outcomes.

Synthetic biology is a relatively new field of science that combines aspects of biology and engineering to create novel tools for the construction of biological systems. Using tools within synthetic biology, stem cells can then be reprogrammed and differentiated into a specified cell type. Stem cells have already proven to be largely beneficial in many different therapies and have paved the way for tissue engineering and regenerative medicine. Although scientists have made great strides in tissue engineering, there still remain many questions to be answered in regard to regeneration. Presented here is an overview of synthetic biology, common tools built within synthetic biology, and the way these tools are being used in stem cells. Specifically, this review focuses on how synthetic biologists engineer genetic circuits to dynamically control gene expression while also introducing emerging topics such as genome engineering and synthetic transcription factors. The findings mentioned in this review show the diverse use of stem cells within synthetic biology and provide a foundation for future research in tissue engineering with the use of synthetic biology tools. Overall, the work done using synthetic biology in stem cells is in its early stages, however, this early work is leading to new approaches for repairing diseased and damaged tissues and organs, and further expanding the field of tissue engineering.

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications.

Synthetic biologists use engineering principles to design and construct genetic circuits for programming cells with novel functions. A bottom-up approach is commonly used to design and construct genetic circuits by piecing together functional modules that are capable of reprogramming cells with novel behavior. While genetic circuits control cell operations through the tight regulation of gene expression, a diverse array of environmental factors within the extracellular space also has a significant impact on cell behavior. This extracellular space offers an addition route for synthetic biologists to apply their engineering principles to program cell-responsive modules within the extracellular space using biomaterials. In this review, we discuss how taking a bottom-up approach to build genetic circuits using DNA modules can be applied to biomaterials for controlling cell behavior from the extracellular milieu. We suggest that, by collectively controlling intrinsic and extrinsic signals in synthetic biology and biomaterials, tissue engineering outcomes can be improved.

Adoption of the Q Transcriptional System for Regulating Gene Expression in Stem Cells.

The field of mammalian synthetic biology seeks to engineer enabling technologies to create novel approaches for programming cells to probe, perturb, and regulate gene expression with unprecedented precision. To accomplish this, new genetic parts continue to be identified that can be used to build novel genetic circuits to re-engineer cells to perform specific functions. Here, we establish a new transcription-based genetic circuit that combines genes from the quinic acid sensing metabolism of Neorospora crassa and the bacterial Lac repressor system to create a new orthogonal genetic tool to be used in mammalian cells. This work establishes a novel genetic tool, called LacQ, that functions to regulate gene expression in Chinese hamster ovarian (CHO) cells, human embryonic kidney 293 (HEK293) cells, and in mouse embryonic stem (ES) cells.

Tools and applications in synthetic biology.

Advances in synthetic biology have enabled the engineering of cells with genetic circuits in order to program cells with new biological behavior, dynamic gene expression, and logic control. This cellular engineering progression offers an array of living sensors that can discriminate between cell states, produce a regulated dose of therapeutic biomolecules, and function in various delivery platforms. In this review, we highlight and summarize the tools and applications in bacterial and mammalian synthetic biology. The examples detailed in this review provide insight to further understand genetic circuits, how they are used to program cells with novel functions, and current methods to reliably interface this technology in vivo; thus paving the way for the design of promising novel therapeutic applications.

A noisy tug of war: the battle between transcript production and degradation in the liver.

Genetically identical cells in culture often exhibit significant variations, or noise, in gene expression, largely due to transcriptional bursting. Halpern et al. (2015) have developed methods to study gene bursting in tissues to find that this transcriptional bursting also occurs in the mammalian liver and may contribute to functional plasticity in hepatocytes.

Photomodulation of Cellular Gene Expression in Hydrogels.

Biomaterials are designed to mimic aspects of various extracellular matrix environments, through chemical modifications to input biological or chemical signals. However, the dynamic nature and timing of gene expression during cellular events is much more difficult to mimic and control in these synthetic environments. Here, we utilized concepts of photochemistry combined with click chemistry for synthetic biology applications to modulate cellular gene expression in poly(ethylene glycol) (PEG) hydrogels. Specifically, a genetic inducer, isopropyl ß-d-1-thiogalactopyranoside (IPTG), is covalently linked to PEG via a biocompatible and easy to synthesize 2-(2-azido-6-nitrophenyl)ethoxycarbonyl (ANPEOC) photocleavable moiety that, on a short exposure to UV light, effectively releases IPTG and activates gene expression of enhanced green fluorescence protein (EGFP). We anticipate that combining concepts of material chemistry with synthetic biology will further enable the construction of highly defined engineered niches that are capable of controlling both intrinsic and extrinsic cellular events.

Regulating synthetic gene networks in 3D materials.

Combining synthetic biology and materials science will enable more advanced studies of cellular regulatory processes, in addition to facilitating therapeutic applications of engineered gene networks. One approach is to couple genetic inducers into biomaterials, thereby generating 3D microenvironments that are capable of controlling intrinsic and extrinsic cellular events. Here, we have engineered biomaterials to present the genetic inducer, IPTG, with different modes of activating genetic circuits in vitro and in vivo. Gene circuits were activated in materials with IPTG embedded within the scaffold walls or chemically linked to the matrix. In addition, systemic applications of IPTG were used to induce genetic circuits in cells encapsulated into materials and implanted in vivo. The flexibility of modifying biomaterials with genetic inducers allows for patterned placement of these inducers that can be used to generate distinct patterns of gene expression. Together, these genetically interactive materials can be used to characterize genetic circuits in environments that more closely mimic cells' natural 3D settings, to better explore complex cell-matrix and cell-cell interactions, and to facilitate therapeutic applications of synthetic biology.