7 research outputs found
Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling
Muscle satellite stem cells (MuSCs) are responsible for skeletal muscle growth and regeneration. Despite their differentiation potential, human MuSCs have limited in vitro expansion and in vivo migration capacity, limiting their use in cell therapies for diseases affecting multiple skeletal muscles. Several protocols have been developed to derive MuSC-like progenitors from human induced pluripotent stem (iPS) cells (hiPSCs) to establish a source of myogenic cells with controllable proliferation and differentiation. However, current hiPSC myogenic derivatives also suffer from limitations of cell migration, ultimately delaying their clinical translation. Here we use a multi-disciplinary approach including bioinformatics and tissue engineering to show that DLL4 and PDGF-BB improve migration of hiPSC-derived myogenic progenitors. Transcriptomic analyses demonstrate that this property is conserved across species and multiple hiPSC lines, consistent with results from single cell motility profiling. Treated cells showed enhanced trans-endothelial migration in transwell assays. Finally, increased motility was detected in a novel humanised assay to study cell migration using 3D artificial muscles, harnessing advanced tissue modelling to move hiPSCs closer to future muscle gene and cell therapies
An in vitro stem cell model of human epiblast and yolk sac interaction.
Human embryogenesis entails complex signalling interactions between embryonic and extra-embryonic cells. However, how extra-embryonic cells direct morphogenesis within the human embryo remains largely unknown due to a lack of relevant stem cell models. Here, we have established conditions to differentiate human pluripotent stem cells (hPSCs) into yolk sac-like cells (YSLCs) that resemble the post-implantation human hypoblast molecularly and functionally. YSLCs induce the expression of pluripotency and anterior ectoderm markers in human embryonic stem cells (hESCs) at the expense of mesoderm and endoderm markers. This activity is mediated by the release of BMP and WNT signalling pathway inhibitors, and, therefore, resembles the functioning of the anterior visceral endoderm signalling centre of the mouse embryo, which establishes the anterior-posterior axis. Our results implicate the yolk sac in epiblast cell fate specification in the human embryo and propose YSLCs as a tool for studying post-implantation human embryo development in vitro.</i
PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications
Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs—mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)—that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation
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An in vitro model of human yolk sac and epiblast crosstalk
Embryogenesis entails complex signalling interactions between embryonic and extraembryonic lineages. Improved embryo culture techniques have recently allowed human embryos to be cultured in vitro past the point at which they would implant in vivo, until the internationally accepted culture limit of 14 days. Nonetheless, the reliance on surplus in vitro fertilised (IVF) embryos, along with the ethical and legal considerations associated with genetic manipulation of human embryos, present barriers to the study of postimplantation human development. However, human stem cell-based embryo models offer a system that is amenable to genetic and molecular manipulation.
Within mouse development, the anterior visceral endoderm (AVE), formed from the extraembryonic endoderm, plays a crucial role in restricting primitive streak formation to the posterior epiblast, thereby establishing the anterior-posterior axis of the mouse embryo. Recent studies of human embryos cultured to postimplantation stages have alluded to the presence of a human equivalent of the mouse AVE in the anterior hypoblast, the hypoblast being the portion of the human yolk sac that lies adjacent to the epiblast. However, the functional role of such a structure within the context of human development is yet to be understood. In vitro stem cell modelling of the human yolk sac- epiblast interaction offers a system through which the role of the yolk sac in postimplantation epiblast morphogenesis can be interrogated. Consequently, the aim of this study was to derive yolk sac-like cells (YSLCs) that could be used to model human epiblast-yolk sac crosstalk in vitro.
Following a screen of developmentally relevant signalling pathway activators, this project identified that the coactivation of NODAL, WNT and JAK/STAT signalling via ACTIVIN-A, CHIRON, and leukaemia inhibitory factor (LIF) (ACL) induced an endodermal fate in human pluripotent stem cells (hPSCs). Functional and transcriptional analysis of ACL-treated cell populations revealed that the pluripotent state harboured by hPSCs determines the type of endoderm that ACL treatment induces. Human embryonic stem cells (hESCs), which harbour a primed pluripotent state, were found to respond to ACL by adopting a definitive endoderm-like fate, whereas Rset hPSCs, which harbour a pluripotent state that is an intermediary between naïve and primed, gave rise to a yolk sac-like population. Accordingly, Rset hPSCs treated with ACL were coined YSLCs.
YSLCs were found to express inhibitors of BMP, WNT and NODAL signalling, while YSLC conditioned medium was shown to inhibit downstream activity of BMP and WNT signalling in hESCs in both 2D and 3D culture conditions. Similarly, YSLC conditioned medium supported the coexpression of pluripotency and anterior ectoderm markers in hESCs, at the expense of posterior epiblast markers. These results indicated that YSLCs share functional characteristics with the mouse AVE, and also potentially the human anterior hypoblast. Collectively, therefore, this project proposes YSLCs as a tool for studying human epiblast-yolk sac crosstalk
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Proposal for a Cambridgeshire Carbon Advisory Service and Strategic Business Case for a Cambridgeshire Decarbonisation Fund: Executive Summary
The imperative and urgency to reach net-zero has never been clearer. Decarbonising our local environment and practices is a momentous task; however, Cambridgeshire County Council and its various public sector partners and stakehold- ers together are uniquely placed to collaborate positively and holistically towards tackling the climate crisis at a local level. Thus, the Cam- bridgeshire local system has an exciting and crit- ical opportunity to drive the achievement of a net-zero Cambridgeshire by 2045 and serve as a model for other local areas across the country and elsewhere. This report recommends the es- tablishment of a Carbon Advisory Service, which will support local businesses to decarbonise. In conjunction, this report sets out the strategic business case for a Cambridgeshire Decarboni- sation Fund, which will offset residual ‘hard to
reduce’ emissions and support investment in lo- cal community infrastructure and nature-based projects which will avoid, reduce, or sequester carbon
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PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications.
Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation