Biocompatible Sterilisable Worm Gels: An Enabling Technology for the Development of Pluripotent Human Stem Cell-based Therapies

人类胚胎干细胞（hES）是多能细胞，既可以自我更新，从而保持其多能性，也可以根据培养条件分化。诱导多能干细胞（iPS）可以通过感染成人细胞产生，具有与hES细胞相似的临床潜力。原则上，hES和iPS细胞在细胞治疗和再生医学中的应用提供了巨大的潜力，因为它们天生具有分化成多种临床有用的细胞类型的能力。然而，明确的培养条件对于实现hES和iPS细胞的生物医学潜力至关重要。Matrigel是一种由小鼠肉瘤细胞分泌的凝胶状蛋白质混合物，由BD Biosciences公司上市。这种复杂的混合物含有层粘连蛋白、肠动蛋白、胶原蛋白和各种生长因子；它类似于在许多组织中发现的复杂的细胞外环境，被细胞生物学家用作细胞培养研究的模型活性底物。Matrigel在4摄氏度时是液体，但在37摄氏度时，它会形成纤维状凝胶网络。在其稀释形式下，Matrigel被用作胚胎干细胞培养的附着基质，以在没有任何喂养细胞的情况下保持其多能性和未分化状态。尽管成本高，动物来源和批间可重复性差/可变，但Matrigel仍被细胞生物学家广泛使用。然而，迫切需要一种替代的完全合成胶凝组合物，它可以可靠地用作基线材料，用于旨在最终临床应用的广泛的体外干细胞实验。此外，人们普遍认为，要将人类多能干细胞有效地转化为细胞疗法，就需要对产品的一致性、稳定性、毒性和免疫原性进行标准化测试。在这笔拨款的帮助下，我们将开发一系列新颖的、完全合成的水凝胶，这些水凝胶是基于生物相容性的甲基丙烯酸嵌段共聚物蠕虫状颗粒的自组装，这些水凝胶很容易在浓水溶液中制备。这种凝胶具有高度的生物相容性，与许多水凝胶不同的是，它可以很容易地通过冷超滤进行消毒：这是可能的，因为蠕虫在冷却到5摄氏度时转化为自由流动的球形纳米颗粒，并在返回到环境温度时重新形成蠕虫凝胶。在为期一年的非正式合作中，我们进行了概念验证研究（见a . Blanazs等人，JACS, 2012, 134, 9741），并提交了美国谢菲尔德专利申请，因此我们已经拥有强大的知识产权背景地位。然而，仍然存在许多技术挑战，现在需要协调一致的跨学科研究努力来克服这些问题。我们的新型蠕虫凝胶有望取代Matrigel（及相关动物源性材料），成为人类干细胞长期储存、操作和增殖的最方便的培养基，同时保持其多能状态。Steve Armes教授将领导这项跨学科研究的合成聚合物化学方面，而Harry Moore教授将领导干细胞研究。我们请求资助两位有经验的博士后科学家在这个项目上紧密合作。我们已经为EPSRC拨款确定了两个合适的工业合作伙伴。GEO是一家总部位于英国的特种化学品公司，将提供合成嵌段共聚物蠕虫所需的单体构建模块，协助规模化研究，并在未来商业化的情况下充当原材料供应商。Plasticell是一家总部位于英国的生物技术中小企业，专门从事干细胞技术。这家公司非常适合帮助我们评估和优化我们的蠕虫凝胶，以确保它们为干细胞生物学家提供合适的技术解决方案。这两家公司各承诺5000英镑现金，以提供EPSRC所需的1万英镑捐款。

Human embryonic stem (hES) cells are pluripotent cells that can either self-renew, thereby maintaining their pluripotency, or differentiate depending on the culture conditions. Induced pluripotent stem (iPS) cells, which offer similar clinical potential to hES cells, can be generated by infecting adult cells. In principle, the application of hES and iPS cells in cell therapy and regenerative medicine offers tremendous potential because of their innate ability to differentiate into multiple, clinically-useful cell types. However, well-defined culture conditions are essential for realising the biomedical potential of hES and iPS cells. Matrigel is a gelatinous protein mixture secreted by mouse sarcoma cells and is marketed by BD Biosciences. This complex mixture contains laminin, entactin, collagen and various growth factors; it resembles the complex extracellular environment found in many tissues and is used by cell biologists as a model active substrate for cell culture studies. Matrigel is a liquid at 4oC, but on warming to 37oC it forms a fibrillar gel network. In its diluted form, Matrigel is used as an attachment substrate for culturing embryonic stem cells to maintain their pluripotent, undifferentiated state in the absence of any feeder cells. Despite its high cost, animal origin and poor/variable batch-to-batch reproducibility, Matrigel is nevertheless widely used by cell biologists. However, an alternative wholly synthetic gelling composition that can be reliably employed as a baseline material is urgently required for a wide range of in vitro stem cell experiments aimed at eventual clinical applications. Moreover, it is widely accepted that the effective translation of human pluripotent stem cells into cell therapies will require the development of standardised tests for product consistency, stability, toxicity and immunogenicity. With the aid of this grant, we will develop a range of novel, wholly synthetic hydrogels based on the self-assembly of biocompatible methacrylic block copolymer worm-like particles, which are readily prepared in concentrated aqueous solution. Such gels are highly biocompatible and, unlike many hydrogels, can be readily sterilised simply by cold ultrafiltration: this is possible because the worms transform into free-flowing spherical nanoparticles when cooled to 5oC and reform worm gels on returning to ambient temperature. In a year-long informal collaboration, we have conducted proof-of-concept studies (see A. Blanazs et al., JACS, 2012, 134, 9741) and filed a U. Sheffield patent application, thus we already have a strong background IP position. However, there are many remaining technical challenges and a concerted inter-disciplinary research effort is now required to overcome these problems. Our new worm gels are expected to replace Matrigel (and related animal-derived materials) as the most convenient medium for the long-term storage, manipulation and proliferation of human stem cells while retaining their pluripotent state. Prof. Steve Armes will lead on the synthetic polymer chemistry aspects of this inter-disciplinary study, while Prof. Harry Moore will lead on the stem cell research. We request funding to support two experienced post-doctoral research scientists to work in close collaboration on this project. We have identified two appropriate industrial partners for this EPSRC grant. GEO is a UK-based speciality chemicals company that will provide the monomer building blocks required for the synthesis of the block copolymer worms, assist with the scale-up studies and act as a raw materials supplier in the event of future commercialisation. Plasticell is a UK-based biotech SME specialising in stem cell technologies. This company is ideally placed to help us assess and optimise our worm gels to ensure that they provide an appropriate technical solution for stem cell biologists. These two companies have each pledged £ 5 K cash to provide the £ 10 K contribution required by EPSRC.