RESUMEN
Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary, we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.
Asunto(s)
Células Endoteliales , Técnicas de Sustitución del Gen , Células Madre Pluripotentes Inducidas , Receptor TIE-2 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Células Endoteliales/metabolismo , Mutación/genética , Sistemas CRISPR-Cas/genética , Malformaciones Vasculares/genética , Malformaciones Vasculares/patología , Malformaciones Vasculares/metabolismoRESUMEN
CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.
Asunto(s)
Sistemas CRISPR-Cas , Células Madre Pluripotentes Inducidas , Expresión Génica , Humanos , Línea PrimitivaRESUMEN
Advances in synthetic biology have enabled robust control of cell behavior by using tunable genetic circuits to regulate gene expression in a ligand-dependent manner. Such circuits can be used to direct the differentiation of pluripotent stem cells (PSCs) towards desired cell types, but rational design of synthetic gene circuits in PSCs is challenging due to the variable intracellular environment. Here, we provide a framework for implementing synthetic gene switches in PSCs based on combinations of tunable transcriptional, structural, and posttranslational elements that can be engineered as required, using the vanillic acid-controlled transcriptional activator (VanA) as a model system. We further show that the VanA system can be multiplexed with the well-established reverse tetracycline-controlled transcriptional activator (rtTA) system to enable independent control of the expression of different transcription factors in human induced PSCs in order to enhance lineage specification towards early pancreatic progenitors. This work represents a first step towards standardizing the design and construction of synthetic gene switches for building robust gene-regulatory networks to guide stem cell differentiation towards a desired cell fate.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Diferenciación Celular/genética , Redes Reguladoras de Genes/genética , Genes Sintéticos , HumanosRESUMEN
Orthogonal tools for controlling protein function by post-translational modifications open up new possibilities for protein circuit engineering in synthetic biology. Phosphoregulation is a key mechanism of signal processing in all kingdoms of life, but tools to control the involved processes are very limited. Here, we repurpose components of bacterial two-component systems (TCSs) for chemically induced phosphotransfer in mammalian cells. TCSs are the most abundant multi-component signal-processing units in bacteria, but are not found in the animal kingdom. The presented phosphoregulated orthogonal signal transduction (POST) system uses induced nanobody dimerization to regulate the trans-autophosphorylation activity of engineered histidine kinases. Engineered response regulators use the phosphohistidine residue as a substrate to autophosphorylate an aspartate residue, inducing their own homodimerization. We verify this approach by demonstrating control of gene expression with engineered, dimerization-dependent transcription factors and propose a phosphoregulated relay system of protein dimerization as a basic building block for next-generation protein circuits.
Asunto(s)
Histidina Quinasa/metabolismo , Transducción de Señal , Tejido Adiposo/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Femenino , Regulación de la Expresión Génica , Células HEK293 , Histidina/química , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Nanotecnología , Fosforilación , Dominios Proteicos , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Biología Sintética , Factores de Transcripción/metabolismoRESUMEN
We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.
Asunto(s)
Proteínas Fúngicas/metabolismo , Genes Reporteros , Oxigenasas/metabolismo , Ácidos Picolínicos/metabolismo , Análisis de la Célula Individual/métodos , Absorción de Radiación , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas Fúngicas/genética , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Oxigenasas/genética , Ácidos Picolínicos/efectos de la radiación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia/métodos , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Rayos UltravioletaRESUMEN
Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.
Asunto(s)
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Estimulación Eléctrica/instrumentación , Secreción de Insulina/genética , Células Secretoras de Insulina/metabolismo , Tecnología Inalámbrica/instrumentación , Animales , Biónica , Canales de Calcio Tipo L/genética , Señalización del Calcio , Ingeniería Celular , Células HEK293 , Humanos , Masculino , Ratones , Canales de Potasio de Rectificación Interna/genética , Prótesis e Implantes , Transcripción Genética , TransgenesRESUMEN
The ability to safely control transgene expression with simple synthetic gene switches is critical for effective gene- and cell-based therapies. In the present study, the signaling pathway controlled by human transient receptor potential (TRP) melastatin 8 (hTRPM8), a TRP channel family member1, is harnessed to control transgene expression. Human TRPM8 signaling is stimulated by menthol, an innocuous, natural, cooling compound, or by exposure to a cool environment (15-18 °C). By functionally linking hTRPM8-induced signaling to a synthetic promoter containing elements that bind nuclear factor of activated T cells, a synthetic gene circuit was designed that can be adjusted by exposure to either a cool environment or menthol. It was shown that this gene switch is functional in various cell types and human primary cells, as well as in mice implanted with engineered cells. In response to transdermal delivery of menthol, microencapsulated cell implants harboring this gene circuit, coupled to expression of either of two therapeutic proteins, insulin or a modified, activin type IIB, receptor ligand trap protein (mActRIIBECD-hFc), could alleviate hyperglycemia in alloxan-treated mice (a model of type 1 diabetes) or reverse muscle atrophy in dexamethasone-treated mice (a model of muscle wasting), respectively. This fully human-derived orthogonal transgene switch should be amenable to a wide range of clinical applications.
Asunto(s)
Receptores de Activinas Tipo II/sangre , Insulina/biosíntesis , Canales Catiónicos TRPM/fisiología , Transgenes , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/uso terapéutico , Fosfatasa Alcalina/genética , Animales , Frío , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Insulina/genética , Insulina/uso terapéutico , Mentol/farmacología , Ratones , Ratones Endogámicos C57BL , Distrofias Musculares/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
Exosomes are cell-derived nanovesicles (50-150 nm), which mediate intercellular communication, and are candidate therapeutic agents. However, inefficiency of exosomal message transfer, such as mRNA, and lack of methods to create designer exosomes have hampered their development into therapeutic interventions. Here, we report a set of EXOsomal transfer into cells (EXOtic) devices that enable efficient, customizable production of designer exosomes in engineered mammalian cells. These genetically encoded devices in exosome producer cells enhance exosome production, specific mRNA packaging, and delivery of the mRNA into the cytosol of target cells, enabling efficient cell-to-cell communication without the need to concentrate exosomes. Further, engineered producer cells implanted in living mice could consistently deliver cargo mRNA to the brain. Therapeutic catalase mRNA delivery by designer exosomes attenuated neurotoxicity and neuroinflammation in in vitro and in vivo models of Parkinson's disease, indicating the potential usefulness of the EXOtic devices for RNA delivery-based therapeutic applications.
Asunto(s)
Cerebro/patología , Sistemas de Liberación de Medicamentos , Exosomas/metabolismo , Enfermedad de Parkinson/terapia , Regiones no Traducidas 3' , Animales , Encéfalo/metabolismo , Catalasa/metabolismo , Comunicación Celular , Línea Celular Tumoral , Cerebro/metabolismo , Citosol/metabolismo , Electroporación , Femenino , Terapia Genética , Células HEK293 , Células HeLa , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs , Nanopartículas , ARN Mensajero/metabolismo , Biología SintéticaRESUMEN
Synthetic biology, the synthesis of engineering and biology, has rapidly matured and has dramatically increased the complexity of artificial gene circuits in recent years. The deployment of intricate synthetic gene circuits in mammalian cells requires the establishment of very precise and orthogonal control of transgene expression. In this chapter, we describe methods of modulating the expression of transgenes at the transcriptional level. Using cAMP-response element-binding protein (CREB)-dependent promoters as examples, a tool for the precise tuning of gene expression by using different core promoters and by varying the binding affinity of transcription factor operator sites is described.
Asunto(s)
Redes Reguladoras de Genes , Regiones Promotoras Genéticas , Activación Transcripcional , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Transducción de Señal , Biología Sintética/métodos , Transfección/métodosRESUMEN
We previously reported novel technology to differentiate induced pluripotent stem cells (IPSCs) into glucose-sensitive insulin-secreting beta-like cells by engineering a synthetic lineage-control network regulated by the licensed food additive vanillic acid. This genetic network was able to program intricate expression dynamics of the key transcription factors Ngn3 (neurogenin 3, OFF-ON-OFF), Pdx1 (pancreatic and duodenal homeobox 1, ON-OFF-ON) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A, OFF-ON) to guide the differentiation of IPSC-derived pancreatic progenitor cells to beta-like cells. In the present study, we show for the first time that this network can also program the expression dynamics of Ngn3, Pdx1 and MafA in human embryonic stem cell (hESC)-derived pancreatic progenitor cells and drive differentiation of these cells into glucose-sensitive insulin-secreting beta-like cells. Therefore, synthetic lineage-control networks appear to be a robust methodology for differentiating pluripotent stem cells into somatic cell types for basic research and regenerative medicine.
Asunto(s)
Redes Reguladoras de Genes/genética , Células Madre Embrionarias Humanas , Células Secretoras de Insulina , Biología Sintética/métodos , Línea Celular , Glucosa/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Chronically deregulated blood-glucose concentrations in diabetes mellitus result from a loss of pancreatic insulin-producing ß cells (type 1 diabetes, T1D) or from impaired insulin sensitivity of body cells and glucose-stimulated insulin release (type 2 diabetes, T2D). Here, we show that therapeutically applicable ß-cell-mimetic designer cells can be established by minimal engineering of human cells. We achieved glucose responsiveness by a synthetic circuit that couples glycolysis-mediated calcium entry to an excitation-transcription system controlling therapeutic transgene expression. Implanted circuit-carrying cells corrected insulin deficiency and self-sufficiently abolished persistent hyperglycemia in T1D mice. Similarly, glucose-inducible glucagon-like peptide 1 transcription improved endogenous glucose-stimulated insulin release and glucose tolerance in T2D mice. These systems may enable a combination of diagnosis and treatment for diabetes mellitus therapy.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/terapia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animales , Biomimética , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Ingeniería Celular , Diabetes Mellitus Experimental/terapia , Péptido 1 Similar al Glucagón/genética , Células HEK293 , Humanos , Hiperglucemia/terapia , Secreción de Insulina , Células Secretoras de Insulina/trasplante , Transcripción Genética , TransgenesRESUMEN
BACKGROUND & AIMS: The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. METHODS: By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. RESULTS: After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. CONCLUSIONS: Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. LAY SUMMARY: Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.
Asunto(s)
Hígado/lesiones , Animales , Ácidos y Sales Biliares , Enfermedad Hepática Inducida por Sustancias y Drogas , Humanos , Hepatopatías , Ratones , Biología SintéticaRESUMEN
Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Glucosa/farmacología , Células Madre Pluripotentes Inducidas/citología , Células Secretoras de Insulina/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Persona de Mediana Edad , Reproducibilidad de los Resultados , Factores de Transcripción/metabolismo , Ácido Vanílico/farmacologíaRESUMEN
Graves' disease is an autoimmune disorder that causes hyperthyroidism because of autoantibodies that bind to the thyroid-stimulating hormone receptor (TSHR) on the thyroid gland, triggering thyroid hormone release. The physiological control of thyroid hormone homeostasis by the feedback loops involving the hypothalamus-pituitary-thyroid axis is disrupted by these stimulating autoantibodies. To reset the endogenous thyrotrophic feedback control, we designed a synthetic mammalian gene circuit that maintains thyroid hormone homeostasis by monitoring thyroid hormone levels and coordinating the expression of a thyroid-stimulating hormone receptor antagonist (TSHAntag), which competitively inhibits the binding of thyroid-stimulating hormone or the human autoantibody to TSHR. This synthetic control device consists of a synthetic thyroid-sensing receptor (TSR), a yeast Gal4 protein/human thyroid receptor-α fusion, which reversibly triggers expression of the TSHAntag gene from TSR-dependent promoters. In hyperthyroid mice, this synthetic circuit sensed pathological thyroid hormone levels and restored the thyrotrophic feedback control of the hypothalamus-pituitary-thyroid axis to euthyroid hormone levels. Therapeutic plug and play gene circuits that restore physiological feedback control in metabolic disorders foster advanced gene- and cell-based therapies.
Asunto(s)
Modelos Animales de Enfermedad , Redes Reguladoras de Genes , Genes Sintéticos , Enfermedad de Graves/genética , Hipófisis/fisiopatología , Glándula Tiroides/fisiopatología , Animales , Células Cultivadas , Retroalimentación , Enfermedad de Graves/fisiopatología , Humanos , Ratones , Hormonas Tiroideas/sangreRESUMEN
Synthetic biology, an engineering, circuit-driven approach to biology, has developed whole new classes of therapeutics. Unfortunately, these advances have thus far been undercapitalized upon by basic researchers. As discussed herein, using synthetic circuits, one can undertake exhaustive investigations of the endogenous circuitry found in nature, develop novel detectors and better temporally and spatially controlled inducers. One could detect changes in DNA, RNA, protein or even transient signaling events, in cell-based systems, in live mice, and in humans. Synthetic biology has also developed inducible systems that can be induced chemically, optically or using radio waves. This induction has been re-wired to lead to changes in gene expression, RNA stability and splicing, protein stability and splicing, and signaling via endogenous pathways. Beyond simple detectors and inducible systems, one can combine these modalities and develop novel signal integration circuits that can react to a very precise pre-programmed set of conditions or even to multiple sets of precise conditions. In this review, we highlight some tools that were developed in which these circuits were combined such that the detection of a particular event automatically triggered a specific output. Furthermore, using novel circuit-design strategies, circuits have been developed that can integrate multiple inputs together in Boolean logic gates composed of up to 6 inputs. We highlight the tools available and what has been developed thus far, and highlight how some clinical tools can be very useful in basic science. Most of the systems that are presented can be integrated together; and the possibilities far exceed the number of currently developed strategies.
Asunto(s)
Redes Reguladoras de Genes , Ingeniería Genética/métodos , Biología Sintética/métodos , Animales , ADN/química , Regulación de la Expresión Génica , Genoma , Humanos , Cinética , Ligandos , Luz , Ratones , Oscilometría , ARN/química , Empalme del ARN , Transducción de SeñalRESUMEN
Previous studies reported much heterogeneity in baseline neural marker expression by undifferentiated mesenchymal stem cells (MSCs) of animal and human origin, which could confound reproducibility of neural differentiation experiments with MSCs. Nevertheless, basic donor characteristics such as age and gender were unspecified in these previous studies; and relative levels of baseline neural marker expression amongst primary MSCs of different tissue and donor origin have not been compared by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, which is the focus of this study. The results showed that amongst a mixed group of primary adipose and bone marrow-derived MSCs (12-50 years), the observed variability in baseline neural marker expression may be correlated to donor age. Adipose-derived MSCs from the youngest donor (male, 12 years old) displayed the highest expression of all four early neural markers (Pax6, Nestin, Musashi 1 and ßIII-tubulin), and three out of four mature neural markers (NCAM, NSE and NFM) analyzed by qRT-PCR. Conversely, adipose MSCs of the oldest donor (female, 50 years old) displayed the lowest expression of three out of four early neural markers (Pax6, Musashi 1 and ßIII-tubulin), and three out of four mature neural markers (MAP2, NCAM and NSE) analyzed by qRT-PCR.