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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., Competing Interests: Declaration of interests J.T. and L.B. acknowledge outside interest in Stylus Medicine., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

There is intense worldwide effort in generating kidney organoids from pluripotent stem cells, for research, for disease modelling and, perhaps, for making transplantable organs. Organoids generated from pluripotent stem cells (PSC) possess accurate micro-anatomy, but they lack higher-organization. This is a problem, especially for transplantation, as such organoids will not be able to perform their physiological functions. In this study, we develop a method for generating murine kidney organoids with improved higher-order structure, through stages using chimaeras of ex-fetu and PSC-derived cells to a system that works entirely from embryonic stem cells. These organoids have nephrons organised around a single ureteric bud tree and also make vessels, with the endothelial network approaching podocytes., (© 2022. The Author(s).)

Mast cells are tissue-resident immune cells. Their overgrowth/overactivation results in a range of common distressing, sometimes life-threatening disorders, including asthma, psoriasis, anaphylaxis, and mastocytosis. Currently, drug discovery is hampered by use of cancer-derived mast cell lines or primary cells. Cell lines provide low numbers of mature mast cells and are not representative of in vivo mast cells. Mast cell generation from blood/bone marrow gives poor reproducibility, requiring 8-12 weeks of culture. Here we report a method for the rapid/robust production of mast cells from pluripotent stem cells (PSCs). An advantageous Gata2Venus reporter enriches mast cells and progenitors as they differentiate from PSCs. Highly proliferative mouse mast cells and progenitors emerge after 2 weeks. This method is applicable for rapid human mast cell generation, and could enable the production of sufficient numbers of physiologically relevant human mast cells from patient induced PSCs for the study of mast cell-associated disorders and drug discovery., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Branching morphogenesis of epithelia involves division of cells into leader (tip) and follower (stalk) cells. Published work on cell lines in culture has suggested that symmetry-breaking takes place via a secreted autocrine inhibitor of motility, the inhibitor accumulating more in concave regions of the culture boundary, slowing advance of cells there, and less in convex areas, allowing advance and a further exaggeration of the concave/convex difference. Here we test this hypothesis using a two-dimensional culture system that includes strong flow conditions to remove accumulating diffusible secretions. We find that, while motility does indeed follow boundary curvature in this system, flow makes no difference: this challenges the hypothesis of control by a diffusible secreted autocrine inhibitor., (© 2017 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance., (© 2015 Vukovic et al.)