Establishment of a cryo-bank of lineage-committed neural progenitor cells produced from engineered human pluripotent stem cells

2007年山中伸弥（Shinya Yamanaka）报道的人类诱导多能干细胞（iPSCs）可以从任何个体的血液或皮肤中提取。它们的独特之处在于它们可以在实验室中无限生长，而且它们有能力产生任何成熟的细胞类型——一种被称为多能性的特性。这些特性使多能干细胞成为生物学研究中替代动物的理想候选者。其基本原理是，ipsc衍生的心脏细胞、神经细胞、肝细胞和其他类型的细胞可以代替小鼠或其他动物用于许多实验。虽然iPSC培养物的复杂性还没有达到整个动物的水平，但在许多研究领域，替代动物已经取得了重大进展。尽管iPSCs早在10多年前就被发现了，但在学术界和工业界广泛采用iPSCs一直存在障碍，这阻碍了它们在研究中实现替代动物使用的潜力。第一个障碍是获得高质量的iPSC细胞系——现在这是常规的，全世界有数千种iPSC细胞系可用。许多实验室面临的第二个障碍是从多能干细胞中可靠和一致地生产成熟的特化细胞类型（如心脏或神经细胞）。多能干细胞固有的多能性意味着它们可以在培养皿中产生所有类型的细胞。为了诱导诱导多能干细胞产生特定的细胞类型（如神经元）而开发的方案是复杂的、冗长的和对微小扰动敏感的。此外，已发表的适用于特定iPSC品系的“分化”协议往往不容易转移到其他iPSC品系。这导致了该领域的重大挫折，并将iPSC技术限制在具有必要的专业知识和资源的选定实验室，以克服iPSC分化的挑战。该提案的主要目的是通过建立爱丁堡祖细胞库（EPB），彻底消除这一障碍，增加对iPSC技术的信心。我们已经建立了沿着细胞谱系途径分化iPSCs的方法，并在祖细胞状态下将其冷冻。“祖细胞”是介于iPSC和成熟细胞之间的过渡细胞类型。我们生产的冷冻祖细胞在解冻后被限制并承诺产生特定的成熟细胞类型，它们可以在任何实验室培养，不需要任何iPSC专业知识。EPB将为学术界和工业界的研究目的提供和支持使用谱系承诺的神经祖细胞。每一批冷冻祖细胞都经过试解冻和严格的质量控制检查，以确保受体实验室提供高质量的功能细胞。EPB将首先用于神经科学领域，但当其他冷冻ipsc衍生祖细胞(例如。包括心脏，肝脏)。EPB的目标是为不同的实验室，特别是大量使用动物的实验室，提供现成的专门的人类细胞，作为动物研究对象的可靠和有效的替代品。以这种方式提供祖细胞增加了使用这种动物替代品的能力，因为实验室不需要掌握控制干细胞分化所需的技能和知识。

Human induced pluripotent stem cells (iPSCs), reported in 2007 by Shinya Yamanaka, can be derived from blood or skin of any individual. They are unique because they can be grown indefinitely in the lab, and they have the capacity to produce any mature cell type - a property known as pluripotency. These attributes make iPSCs ideal candidates to replace animals in biological research. The rationale is that iPSC-derived heart cells, nerve cells, liver cells and other cell types can be used for many experiments instead of mice or other animals. Although the complexity of iPSC cultures is not yet approaching that of a whole animal, there is significant progress towards replacing animals in many areas of research. Despite iPSCs being discovered over 10 years ago, there have been barriers to their wide-spread adoption in academia and industry, which have prevented them from achieving their potential as an alternative to animal use in research. The first barrier was acquisition of high-quality iPSC lines - this is now routine and 1000s of iPSC lines are available world-wide. The second barrier facing many labs is reliable and consistent production of mature specialised cell types (such as cardiac or nerve cells) from iPSCs. The inherent pluripotent nature of iPSCs means they are primed to produce all cell types in a dish. The protocols developed to corral and coax iPSCs to produce specific desired cell types, such as neurons, are complicated, lengthy and sensitive to minor perturbations. Furthermore, published "differentiation" protocols that work for particular iPSC lines are often not easily transferable to other iPSC lines. This has led to a significant level of frustration in the field, and has restricted iPSC technology to selected labs with the necessary expertise and resources to overcome the challenges of iPSC differentiation. The main aim of this proposal is to completely remove this barrier and increase confidence in iPSC technology through the establishment of the Edinburgh Progenitor Cell Bank (EPB). We have established methods to differentiate iPSCs along cell lineage pathways and freeze them while in a progenitor cell state. A "progenitor cell" is a transition cell type that is half-way between an iPSC and a mature cell type. The frozen progenitor cells we produce are restricted and committed to produce a specific mature cell type after thawing, and they can be cultured by any laboratory and does not require any iPSC expertise. The EPB will supply and support the use of lineage-committed neural progenitor cells for research purposes in academia and industry. Each batch of frozen progenitor cells undergo test-thaws and vigorous quality-control checks to ensure that recipient labs are supplied with high-quality functional cells. The EPB will be for the neuroscience community in the first instance, but can be expanded to other areas of biological research when other frozen iPSC-derived progenitor cells (eg. cardiac, liver) are included. The goal of the EPB is to provide diverse labs, particularly heavy animal users, with ready-to-use specialised human cells as a reliable and effective replacement for animal research subjects. Supplying progenitor cells in this way increases the capacity for use of this animal alternative by removing the need for labs to possess the skills and knowledge necessary to control stem cell differentiation.