Meat and Medicine: Regenerative programming of animal and human stem cells for engineered skeletal muscle

背景：多能干细胞（PSC）和多能干细胞（如间充质干细胞；MSCs）在治疗人类骨骼肌创伤和疾病方面有着巨大的希望，同时也为产生用于食物消费的组织提供了新的途径。然而，将PSCs或MSCs转化为骨骼肌细胞仍然具有挑战性，并且主调控转录因子促进肌肉干（卫星）细胞身份的机制尚未完全了解。然而，很明显，病毒或转基因转录因子（TFs）如Pax7或MyoD的表达可以有效地诱导这些细胞类型的肌肉程序。方法：我们已经开发了一种非病毒的方法来传递基因到组织、细胞或干细胞。这将是博士学位的重点，将TF基因传递到干细胞中，以介导其编程为肌生成前体，并扩大和使用这些细胞来产生再生移植物组织，并作为肉类替代品。您将使用PSC和MSC线作为概念证明，使用来自猪，羊和牛的iPSCs来生成用于食品应用的物种特定肌肉组织。对于临床应用，您将使用人类iPSCs， MSCs和骨髓和吸脂器将这些组织干细胞编程成肌肉，用于细胞疗法或组织工程和植入应用。基因，表达时间，如果需要连续剂量，培养条件将需要优化和评估。将生成的祖细胞植入支架或直接在3D中编程将需要生成组织，这也将被研究。有趣的是，我们有电刺激装置，可用于工程肌肉的生理收缩测试，根据RCSI（爱尔兰都柏林）和JHU（美国巴尔的摩）的合作者的项目，有可能转移到体内测试。地点：该项目将在大学公园（James Dixon）的新生物发现研究所和萨顿博宁顿校区（Ramiro Alberio）的生物科学学院进行。该应用程序对人类和动物细胞的使用将具有变革性，可能具有重大的商业价值，并与利益相关者进行互动。目前，国防部（通过DSTL）正在资助Dixon小组使用基因传递的骨骼和脂肪重建技术，该博士可以为这些项目增加影响和协同作用。技术和机会：该项目将使用细胞/干细胞培养、QPCR、免疫标记、流式细胞术、单细胞RNAseq、载体设计和构建、基因传递、支架、组织工程、电刺激、体内翻译。了解更多信息的参考资料：Lilja等人。Pax7重塑骨骼肌干细胞的染色质景观。科学通报，12(4):e0176190.2。Darabi R和Perlingueiro R（2016）。利用PAX7的条件表达从人多能干细胞衍生骨骼肌前体。方法：Mol Biol 1357:423-439

Background: Pluripotent (PSC) and multipotent (such as mesenchymal; MSCs) stem cells hold great promise for the treatment of human skeletal muscle trauma and diseases, but also for the generation of new approaches to generate tissue for food consumption. However, it remains challenging to convert PSCs or MSCs to skeletal muscle cells, and the mechanisms by which the master regulatory transcription factors, promotes muscle stem (satellite) cell identity are not yet fully understood. However it is clear that viral or transgenic expression of transcription factors (TFs) such as Pax7 or MyoD can effectively induce the muscle program in these cell types.Approach: We have developed a non-viral method to deliver genes to tissues, cells or stem cells. This will be the focus of the PhD, to deliver TF genes to stem cells in order to mediate there programming into myogenic precursors and to expand and use those cells to generate tissue of regenerative grafts and as meat-substitutes. You will use PSC and MSC lines as proof of concept, employing iPSCs from pig, sheep and cow to generate species specific muscle tissue for food applications. For clinical use you will employ human iPSCs, MSCs and bone marrow and liposuction aspirates to program these tissue stem cells into muscle for with cell-therapies or for tissue engineering and implant applications. The genes, duration of expression, if serial dosages are required and the culture conditions will require optimisation and assessment. The seeding of scaffolds with generated progenitors or directly programming in 3D will be needed to generate tissue which will also be studied. Interestingly we have electrical stimulation set ups that can be used to physiologically testcontraction of engineered muscle and there is potential to move to in vivo testing depending on the project with collaborators at the RCSI (Dublin, Ireland) and JHU (Baltimore, USA).Location: The project will be carried out in the new state-of-the-art Biodiscovery institute at University Park (James Dixon) and the School of Biosciences at Sutton Bonington Campus (Ramiro Alberio). The use of human and animal cells of this application will be transformative and is likely to have significant commercial value and interaction with stakeholders. Presently the MoD (through the DSTL) are funding bone and fat reconstructive technologies using gene delivery in the Dixon group and this PhD could add impact and synergy to these projects reciprocally.Techniques and opportunities: The project will use cell/stem cell culture, QPCR, immunolabelling, flow cytometry, single cell RNAseq, vector design and construction, gene delivery, scaffolds, tissue engineering, electrical stimulation, in vivo translation.References to learn more:1. Lilja et al. Pax7 remodels the chromatin landscape in skeletal muscle stem cells. PLoS ONE 12(4):e0176190.2. Darabi R and Perlingueiro R (2016). Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7. Methods in Mol Biol 1357:423-439