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1.
Mol Syst Biol ; 18(11): e10886, 2022 11.
Article in English | MEDLINE | ID: mdl-36366891

ABSTRACT

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an "hPSC ON" circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an "hPSC-Off" circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and fine-tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.


Subject(s)
MicroRNAs , Pluripotent Stem Cells , Humans , Genes, Synthetic , Cell Differentiation/genetics , Pluripotent Stem Cells/metabolism , Gene Regulatory Networks , MicroRNAs/genetics , MicroRNAs/metabolism , Proteins/metabolism
2.
Sci Rep ; 11(1): 6137, 2021 03 17.
Article in English | MEDLINE | ID: mdl-33731744

ABSTRACT

Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.


Subject(s)
Endoderm , Gene Expression Regulation, Developmental , Human Embryonic Stem Cells , Wnt Signaling Pathway , Cell Differentiation , Cell Line , Endoderm/cytology , Endoderm/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans
3.
PLoS Biol ; 17(10): e3000081, 2019 10.
Article in English | MEDLINE | ID: mdl-31634368

ABSTRACT

In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous Nodal expression, with up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.


Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Cell Lineage/drug effects , Gene Expression Regulation, Developmental , Nodal Protein/genetics , Pluripotent Stem Cells/drug effects , Biomechanical Phenomena , Body Patterning/genetics , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolism , Cell Culture Techniques , Cell Differentiation/drug effects , Cell Line , Cell Lineage/genetics , Gastrulation/drug effects , Gastrulation/genetics , Gene Expression Profiling , Genetic Heterogeneity , High-Throughput Screening Assays , Humans , Models, Biological , Neurogenesis/drug effects , Neurogenesis/genetics , Nodal Protein/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Signal Transduction , Surface Properties
4.
ACS Synth Biol ; 7(2): 474-489, 2018 02 16.
Article in English | MEDLINE | ID: mdl-29257672

ABSTRACT

Cell classifier gene circuits that integrate multiple molecular inputs to restrict the expression of therapeutic outputs to cancer cells have the potential to result in efficacious and safe cancer therapies. Preclinical translation of the hitherto developments requires creating the conditions where the animal model, the delivery platform, in vivo expression levels of the inputs, and the efficacy of the output, all come together to enable detailed evaluation of the fully assembled circuits. Here we show an integrated workflow that addresses these issues and builds the framework for preclinical classifier studies using the design framework of microRNA (miRNA, miR)-based classifier gene circuits. Specifically, we employ HCT-116 colorectal cancer cell xenograft in an experimental mouse metastatic liver tumor model together with Adeno-associated virus (AAV) vector delivery platform. Novel engineered AAV-based constructs are used to validate in vivo the candidate inputs miR-122 and miR-7 and, separately, the cytotoxic output HSV-TK/ganciclovir. We show that while the data are largely consistent with expectations, crucial insights are gained that could not have been obtained in vitro. The results highlight the importance of detailed stepwise interrogation of the experimental parameters as a necessary step toward clinical translation of synthetic gene circuits.


Subject(s)
Colorectal Neoplasms , Gene Regulatory Networks , Genes, Neoplasm , Liver Neoplasms, Experimental , MicroRNAs , RNA, Neoplasm , Animals , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Dependovirus , Genetic Vectors , Humans , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/metabolism , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism
5.
Development ; 144(23): 4298-4312, 2017 12 01.
Article in English | MEDLINE | ID: mdl-28870989

ABSTRACT

How position-dependent cell fate acquisition occurs during embryogenesis is a central question in developmental biology. To study this process, we developed a defined, high-throughput assay to induce peri-gastrulation-associated patterning in geometrically confined human pluripotent stem cell (hPSC) colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient. We developed a reaction-diffusion (RD)-based computational model and observed that the self-organization of pSMAD1 signaling was consistent with the RD principle. Consequent fate acquisition occurred as a function of both pSMAD1 signaling strength and duration of induction, consistent with the positional-information (PI) paradigm. We propose that the self-organized peri-gastrulation-like fate patterning in BMP4-treated geometrically confined hPSC colonies arises via a stepwise model of RD followed by PI. This two-step model predicted experimental responses to perturbations of key parameters such as colony size and BMP4 dose. Furthermore, it also predicted experimental conditions that resulted in RD-like periodic patterning in large hPSC colonies, and rescued peri-gastrulation-like patterning in colony sizes previously thought to be reticent to this behavior.


Subject(s)
Body Patterning/physiology , Gastrulation/physiology , Models, Biological , Body Patterning/genetics , Bone Morphogenetic Protein 4/physiology , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/physiology , Cell Differentiation/physiology , Cells, Cultured , Colony-Forming Units Assay/methods , Gastrulation/genetics , High-Throughput Screening Assays/methods , Humans , Nodal Protein/physiology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/physiology , RNA, Small Interfering/genetics , Signal Transduction , Smad1 Protein/physiology
6.
Nat Commun ; 7: 10709, 2016 Feb 16.
Article in English | MEDLINE | ID: mdl-26880188

ABSTRACT

Development of drug discovery assays that combine high content with throughput is challenging. Information-processing gene networks can address this challenge by integrating multiple potential targets of drug candidates' activities into a small number of informative readouts, reporting simultaneously on specific and non-specific effects. Here we show a family of networks implementing this concept in a cell-based drug discovery assay for miRNA drug targets. The networks comprise multiple modules reporting on specific effects towards an intended miRNA target, together with non-specific effects on gene expression, off-target miRNAs and RNA interference pathway. We validate the assays using known perturbations of on- and off-target miRNAs, and evaluate an ∼700 compound library in an automated screen with a follow-up on specific and non-specific hits. We further customize and validate assays for additional drug targets and non-specific inputs. Our study offers a novel framework for precision drug discovery assays applicable to diverse target families.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery/methods , High-Throughput Screening Assays/methods , MicroRNAs/drug effects , Cell Line, Tumor , Computer Simulation , Drug Screening Assays, Antitumor/methods , Escherichia coli , Flow Cytometry , Gene Library , Humans , Microscopy, Fluorescence , Molecular Targeted Therapy , Small Molecule Libraries
7.
Nat Commun ; 5: 4729, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25311543

ABSTRACT

Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare. A possible solution lies in the 'bow-tie' architecture, which stipulates a focal component-a 'knot'-uncoupling circuits' inputs and outputs, simplifying component swapping, and introducing additional layer of control. Here we construct, in cultured human cells, synthetic bow-tie circuits that transduce microRNA inputs into protein outputs with independently programmable logical and dynamic behaviour. The latter is adjusted via two different knot configurations: a transcriptional activator causing the outputs to track input changes reversibly, and a recombinase-based cascade, converting transient inputs into permanent actuation. We characterize the circuits in HEK293 cells, confirming their modularity and scalability, and validate them using endogenous microRNA inputs in additional cell lines. This platform can be used for biotechnological and biomedical applications in vitro, in vivo and potentially in human therapy.


Subject(s)
Gene Regulatory Networks , Proteins/genetics , HEK293 Cells , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Proteins/metabolism
8.
Science ; 333(6047): 1307-11, 2011 Sep 02.
Article in English | MEDLINE | ID: mdl-21885784

ABSTRACT

Engineered biological systems that integrate multi-input sensing, sophisticated information processing, and precisely regulated actuation in living cells could be useful in a variety of applications. For example, anticancer therapies could be engineered to detect and respond to complex cellular conditions in individual cells with high specificity. Here, we show a scalable transcriptional/posttranscriptional synthetic regulatory circuit--a cell-type "classifier"--that senses expression levels of a customizable set of endogenous microRNAs and triggers a cellular response only if the expression levels match a predetermined profile of interest. We demonstrate that a HeLa cancer cell classifier selectively identifies HeLa cells and triggers apoptosis without affecting non-HeLa cell types. This approach also provides a general platform for programmed responses to other complex cell states.


Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , MicroRNAs/genetics , RNA Interference , Biomarkers, Tumor , Cell Line , HeLa Cells , Humans , Synthetic Biology/methods , Transfection , bcl-2-Associated X Protein/genetics
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