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      In vitro generation of megakaryocytes from engineered mouse embryonic stem cells

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      bioRxiv
      Cold Spring Harbor Laboratory

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          Abstract

          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.

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          Synthetic biology: applications come of age

          Key Points Early synthetic biology designs, namely the genetic toggle switch and repressilator, showed that regulatory components can be characterized and assembled to bring about complex, electronics-inspired behaviours in living systems (for example, memory storage and timekeeping). Through the characterization and assembly of genetic parts and biological building blocks, many more devices have been constructed, including switches, memory elements, oscillators, pulse generators, digital logic gates, filters and communication modules. Advances in the field are now allowing expansion beyond small gene networks to the realm of larger biological programs, which hold promise for a wide range of applications, including biosensing, therapeutics and the production of biofuels, pharmaceuticals and biomaterials. Synthetic biosensing circuits consist of sensitive elements that bind analytes and transducer modules that mobilize cellular responses. Balancing these two modules involves engineering modularity and specificity into the various circuits. Biosensor sensitive elements include environment-responsive promoters (transcriptional), RNA aptamers (translational) and protein receptors (post-translational). Biosensor transducer modules include engineered gene networks (transcriptional), non-coding regulatory RNAs (translational) and protein signal-transduction circuits (post-translational). The contributions of synthetic biology to therapeutics include: engineered networks and organisms for disease-mechanism elucidation, drug-target identification, drug-discovery platforms, therapeutic treatment, therapeutic delivery, and drug production and access. In the microbial production of biofuels and pharmaceuticals, synthetic biology has supplemented traditional genetic and metabolic engineering efforts by aiding the construction of optimized biosynthetic pathways. Optimizing metabolic flux through biosynthetic pathways is traditionally accomplished by driving the expression of pathway enzymes with strong, inducible promoters. New synthetic approaches include the rapid diversification of various pathway components, the rational and model-guided assembly of pathway components, and hybrid solutions.
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            From haematopoietic stem cells to complex differentiation landscapes.

            The development of mature blood cells from haematopoietic stem cells has long served as a model for stem-cell research, with the haematopoietic differentiation tree being widely used as a model for the maintenance of hierarchically organized tissues. Recent results and new technologies have challenged the demarcations between stem and progenitor cell populations, the timing of cell-fate choices and the contribution of stem and multipotent progenitor cells to the maintenance of steady-state blood production. These evolving views of haematopoiesis have broad implications for our understanding of the functions of adult stem cells, as well as the development of new therapies for malignant and non-malignant haematopoietic diseases.
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              The incredible journey: From megakaryocyte development to platelet formation

              Circulating blood platelets are specialized cells that prevent bleeding and minimize blood vessel injury. Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets. Given the need for platelets in many pathological situations, understanding how this process occurs is an active area of research with important clinical applications.
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                Author and article information

                Journal
                bioRxiv
                BIORXIV
                bioRxiv
                Cold Spring Harbor Laboratory
                01 March 2023
                : 2023.03.01.530476
                Affiliations
                Department of Biomedical Engineering, University of Utah, Salt Lake City, UT 84112, USA
                Author notes
                [* ]Correspondence: tara.deans@ 123456utah.edu
                Article
                10.1101/2023.03.01.530476
                10002726
                36909620
                6efe3ec2-41b5-414f-98a6-67e8ac95afea

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.

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