Electrogenetics: Bridging synthetic biology and electronics to remotely control the behavior of mammalian designer cells.

Electrogenetics, the combination of electronics and genetics, is an emerging field of mammalian synthetic biology in which electrostimulation is used to remotely program user-designed genetic elements within designer cells to generate desired outputs. Here, we describe recent advances in electro-induced therapeutic gene expression and therapeutic protein secretion in engineered mammalian cells. We also review available tools and strategies to engineer electro-sensitive therapeutic designer cells that are able to sense electrical pulses and produce appropriate clinically relevant outputs in response. We highlight current limitations facing mammalian electrogenetics and suggest potential future directions for research.

Engineering mammalian living materials towards clinically relevant therapeutics.

Engineered living materials represent a new generation of human-made biotherapeutics that are highly attractive for a myriad of medical applications. In essence, such cell-rich platforms provide encodable bioactivities with extended lifetimes and environmental multi-adaptability currently unattainable in conventional biomaterial platforms. Emerging cell bioengineering tools are herein discussed from the perspective of materializing living cells as cooperative building blocks that drive the assembly of multiscale living materials. Owing to their living character, pristine cellular units can also be imparted with additional therapeutically-relevant biofunctionalities. On this focus, the most recent advances on the engineering of mammalian living materials and their biomedical applications are herein outlined, alongside with a critical perspective on major roadblocks hindering their realistic clinical translation. All in all, transposing the concept of leveraging living materials as autologous tissue-building entities and/or self-regulated biotherapeutics opens new realms for improving precision and personalized medicine strategies in the foreseeable future.

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

The Effect of Hyperbaric Oxygen Therapy on Human Adipose-Derived Stem Cells.

BACKGROUND: Adipose-derived stem cells are considered as candidate cells for regenerative plastic surgery. Measures to influence cellular properties and thereby direct their regenerative potential remain elusive. Hyperbaric oxygen therapy-the exposure to 100% oxygen at an increased atmospheric pressure-has been propagated as a noninvasive treatment for a multitude of indications and presents a potential option to condition cells for tissue-engineering purposes. The present study evaluates the effect of hyperbaric oxygen therapy on human adipose-derived stem cells. METHODS: Human adipose-derived stem cells from healthy donors were treated with hyperbaric oxygen therapy at 2 and 3 atm. Viability before and after each hyperbaric oxygen therapy, proliferation, expression of surface markers and protein contents of transforming growth factor (TGF)-ß, tumor necrosis factor-α, hepatocyte growth factor, and epithelial growth factor in the supernatants of treated adipose-derived stem cells were measured. Lastly, adipogenic, osteogenic, and chondrogenic differentiation with and without use of differentiation-inducing media (i.e., autodifferentiation) was examined. RESULTS: Hyperbaric oxygen therapy with 3 atm increased viability, proliferation, and CD34 expression and reduced the CD31/CD34/CD45 adipose-derived stem cell subset and endothelial progenitor cell population. TGF-ß levels were significantly decreased after two hyperbaric oxygen therapy sessions in the 2-atm group and decreased after three hyperbaric oxygen therapy sessions in the 3-atm group. Hepatocyte growth factor secretion remained unaltered in all groups. Although the osteogenic and chondrogenic differentiation were not influenced, adipogenic differentiation and autodifferentiation were significantly enhanced, with osteogenic autodifferentiation significantly alleviated by hyperbaric oxygen therapy with 3 atm. CONCLUSION: Hyperbaric oxygen therapy with 3 atm increases viability and proliferation of adipose-derived stem cells, alters marker expression and subpopulations, decreases TGF-ß secretion, and skews adipose-derived stem cells toward adipogenic differentiation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Liver microsystems in vitro for drug response.

Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

Cellular engineering of Artemisia annua and Artemisia dubia with the rol ABC genes for enhanced production of potent anti-malarial drug artemisinin.

BACKGROUND: Malaria is causing more than half of a million deaths and 214 million clinical cases annually. Despite tremendous efforts for the control of malaria, the global morbidity and mortality have not been significantly changed in the last 50 years. Artemisinin, extracted from the medicinal plant Artemisia sp. is an effective anti-malarial drug. In 2015, elucidation of the effectiveness of artemisinin as a potent anti-malarial drug was acknowledged with a Nobel prize. Owing to the tight market and low yield of artemisinin, an economical way to increase its production is to increase its content in Artemisia sp. through different biotechnological approaches including genetic transformation. METHODS: Artemisia annua and Artemisia dubia were transformed with rol ABC genes through Agrobacterium tumefacienes and Agrobacterium rhizogenes methods. The artemisinin content was analysed and compared between transformed and untransformed plants with the help of LC-MS/MS. Expression of key genes [Cytochrome P450 (CYP71AV1), aldehyde dehydrogenase 1 (ALDH1), amorpha-4, 11 diene synthase (ADS)] in the biosynthetic pathway of artemisinin and gene for trichome development and sesquiterpenoid biosynthetic (TFAR1) were measured using Quantitative real time PCR (qRT-PCR). Trichome density was analysed using confocal microscope. RESULTS: Artemisinin content was significantly increased in transformed material of both Artemisia species when compared to un-transformed plants. The artemisinin content within leaves of transformed lines was increased by a factor of nine, indicating that the plant is capable of synthesizing much higher amounts than has been achieved so far through traditional breeding. Expression of all artemisinin biosynthesis genes was significantly increased, although variation between the genes was observed. CYP71AV1 and ALDH1 expression levels were higher than that of ADS. Levels of the TFAR1 expression were also increased in all transgenic lines. Trichome density was also significantly increased in the leaves of transformed plants, but no trichomes were found in control roots or transformed roots. The detection of significantly raised levels of expression of the genes involved in artemisinin biosynthesis in transformed roots correlated with the production of significant amounts of artemisinin in these tissues. This suggests that synthesis is occurring in tissues other than the trichomes, which contradicts previous theories. CONCLUSION: Transformation of Artemisia sp. with rol ABC genes can lead to the increased production of artemisinin, which will help to meet the increasing demand of artemisinin because of its diverse pharmacological and anti-malarial importance.

Synthetic Biology--Toward Therapeutic Solutions.

Higher multicellular organisms have evolved sophisticated intracellular and intercellular biological networks that enable cell growth and survival to fulfill an organism's needs. Although such networks allow the assembly of complex tissues and even provide healing and protective capabilities, malfunctioning cells can have severe consequences for an organism's survival. In humans, such events can result in severe disorders and diseases, including metabolic and immunological disorders, as well as cancer. Dominating the therapeutic frontier for these potentially lethal disorders, cell and gene therapies aim to relieve or eliminate patient suffering by restoring the function of damaged, diseased, and aging cells and tissues via the introduction of healthy cells or alternative genes. However, despite recent success, these efforts have yet to achieve sufficient therapeutic effects, and further work is needed to ensure the safe and precise control of transgene expression and cellular processes. In this review, we describe the biological tools and devices that are at the forefront of synthetic biology and discuss their potential to advance the specificity, efficiency, and safety of the current generation of cell and gene therapies, including how they can be used to confer curative effects that far surpass those of conventional therapeutics. We also highlight the current therapeutic delivery tools and the current limitations that hamper their use in human applications.

Engineering cell-compatible paper chips for cell culturing, drug screening, and mass spectrometric sensing.

Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis.

Plant nanobionic materials with a giant temperature response mediated by pectin-Ca2+.

Conventional approaches to create biomaterials rely on reverse engineering of biological structures, on biomimicking, and on bioinspiration. Plant nanobionics is a recent approach to engineer new materials combining plant organelles with synthetic nanoparticles to enhance, for example, photosynthesis. Biological structures often outperform man-made materials. For example, higher plants sense temperature changes with high responsivity. However, these properties do not persist after cell death. Here, we permanently stabilize the temperature response of isolated plant cells adding carbon nanotubes (CNTs). Interconnecting cells, we create materials with an effective temperature coefficient of electrical resistance (TCR) of -1,730% K(-1), ∼2 orders of magnitude higher than the best available sensors. This extreme temperature response is due to metal ions contained in the egg-box structure of the pectin backbone, lodged between cellulose microfibrils. The presence of a network of CNTs stabilizes the response of cells at high temperatures without decreasing the activation energy of the material. CNTs also increase the background conductivity, making these materials suitable elements for thermal and distance sensors.

De novo generation of HSCs from somatic and pluripotent stem cell sources.

Generating human hematopoietic stem cells (HSCs) from autologous tissues, when coupled with genome editing technologies, is a promising approach for cellular transplantation therapy and for in vitro disease modeling, drug discovery, and toxicology studies. Human pluripotent stem cells (hPSCs) represent a potentially inexhaustible supply of autologous tissue; however, to date, directed differentiation from hPSCs has yielded hematopoietic cells that lack robust and sustained multilineage potential. Cellular reprogramming technologies represent an alternative platform for the de novo generation of HSCs via direct conversion from heterologous cell types. In this review, we discuss the latest advancements in HSC generation by directed differentiation from hPSCs or direct conversion from somatic cells, and highlight their applications in research and prospects for therapy.

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.

N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.

Generación in vitro de un sustituto de pared vesical humana obtenido mediante ingeniería tisular / In vitro generation of a human bladder wall substitute by tissue engineering

Objetivos. Numerosas patologías que afectan a la vejiga, de origen congénito (extrofia) o adquirido (traumatismos, tumores), requieren la reconstrucción de la pared vesical utilizando intestino delgado, sigma o estómago, los cuales no están exentos de complicaciones. Por ese motivo, en el presente trabajo pretendemos desarrollar un nuevo modelo de pared vesical humana mediante ingeniería tisular que pudiese tener una utilidad clínica. Material y métodos. En primer lugar, se procedió a generar cultivos primarios de células epiteliales y estromales de la mucosa vesical a partir de pequeñas biopsias de la pared vesical humana, utilizando para ello técnicas de digestión enzimática mediante tripsina-EDTA y colagenasa. Posteriormente, se generó un sustituto tridimensional de la mucosa vesical utilizando como soporte biomateriales de fibrina-agarosa. El análisis de las muestras se realizó a los 14 días mediante examen histológico de muestras teñidas con hematoxilina-eosina. Resultados. La aplicación de los métodos de digestión enzimática permitió generar eficientemente cultivos primarios de células epiteliales y estromales de la mucosa vesical humana, comprobándose que la tasa de proliferación de las células estromales era superior a la de las células epiteliales. Una vez generados los sustitutos de la pared vesical, se comprobó el adecuado nivel de biocompatibilidad del biomaterial y las células estromales y epiteliales. La estructura histológica de los sustitutos de pared vesical presentaba una gran analogía con la mucosa vesical humana nativa. Conclusiones. El tejido vesical generado por ingeniería tisular muestra importantes similitudes estructurales e histológicas con el tejido vesical nativo. Estos resultados sugieren que los tejidos generados mediante ingeniería tisular podrían tener utilidad terapéutica en el futuro (AU)

Introduction. Certain urological congenital conditions, such as bladder exstrophy and acquired conditions such as trauma and tumors may require the use of different tissues like small bowel, sigmoid colon or stomach for bladder reconstruction. However, these tissues are often associated to important complications. The aim of this study is to develop a novel substitute of the human bladder wall by tissue engineering. Material and methods. We first generated primary cell cultures of epithelial and stromal bladder mucosa cells from small tissue biopsies of human bladder by using enzymatic methods based on trypsin-EDTA and collagenase I. Then, a three-dimensional substitute of the bladder mucosa was generated using fibrin-agarose biomaterials. The analysis of the tissue substitutes was carried out at day 14th of development by histological examination of samples stained with hematoxylin-eosin. Results. The use of enzymatic digestion methods allowed us to efficiently generate primary cell cultures of the human bladder epithelial and stromal cells. The proliferation rate was higher in stromal cells as compared to epithelial cells. Once the bladder mucosa substitutes were generated, a good biocompatibility of the stromal and epithelial cells into the biomaterial was found. The histological structure of the bladder wall substitutes was analogue to that of the native human bladder mucosa. Conclusions. The bladder mucosa substitute generated by tissue engineering showed structural and histological similarities with the native human bladder tissues and open the door to the future therapeutic use of these bioengineered tissues (AU)

Chemical biology in stem cell research.

Stem cells are offering a considerable range of prospects to the biomedical research including novel platforms for disease models and drug discovery tools to cell transplantation and regenerative therapies. However, there are several obstacles to overcome to bring these potentials into reality. First, robust methods to maintain stem cells in the pluripotent state should be established and factors that are required to direct stem cell fate into a particular lineage should be elucidated. Second, both allogeneic rejection following transplantation and limited cell availability issues must be circumvented. These challenges are being addressed, at least in part, through the identification of a group of chemicals (small molecules) that possess novel activities on stem cell biology. For example, small molecules can be used both in vitro and/or in vivo as tools to promote proliferation of stem cells (self-renewal), to direct stem cells to a lineage specific patterns (differentiation), or to reprogram somatic cells to a more undifferentiated state (de-differentiation or reprogramming). These molecules, in turn, have provided new insights into the signaling mechanisms that regulate stem cell biology, and may eventually lead to effective therapies in regenerative medicine. In this review, we will introduce recent findings with regards to small molecules and their impact on stem cell self-renewal and differentiation.

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2-EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine-sensitive K(+) current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.