Validation of an in vitro humanised 3D haematopoietic system to investigate haematological malignancies.

造血干细胞（HSC）是一种特殊的细胞，具有自我更新和产生更多干细胞或成熟的能力-这一过程称为分化，产生成熟的血细胞。造血干细胞存在于骨髓（BM）的“小生境”中，这是一种支持这些过程的微环境。髓性白血病如慢性髓性白血病（CML）和急性髓性白血病（AML）的进展和发展受到自我加强的白血病干细胞（LSC）生态位的建立的鼓励，其中LSC重塑健康BM生态位以使其有利。随着时间的推移，血液学转化，特别是骨髓来源的血液学转化，通常涉及脾和肝中的髓外造血，其中LSC移出BM并驻留在这些器官中，它们在这些器官中增殖和扩增。到目前为止，已经证明很难用当前的疗法靶向LSC，也很难确切地研究LSC如何控制和改变生态位。由于缺乏重现造血系统的强大体外模型，导致大多数研究在小鼠模型中进行，因此进展受到阻碍。这些模型并不理想，缺乏肿瘤特异性和/或物种特异性微环境。该项目将创建一个创新的长期完全人源化的体外造血系统，以模拟BM和脾脏微环境来研究血液恶性肿瘤。使用组织工程，我们已经开发了一种人工造血系统，包括BM和脾龛，其中包括常驻细胞类型;间充质干细胞，HSC，LSC和脾成纤维细胞。我们的系统使用先进的纤连蛋白为基础的水凝胶设计，以配合BM的机械性能与控制刚度和蛋白酶降解模拟BM结构。通过利用这个模型系统，我们将深入了解LSC赋予的微环境变化以及这些变化如何影响驻留细胞。我们的目标是通过直接比较正常HSC和LSC的行为和变化来验证该系统，直接与实验室中已经生成的现有体内异种移植数据进行比较，使用来自CML和AML患者的相同原始样本。该技术旨在推动用于患者源性异种移植研究的动物替代，并最终帮助了解和治疗许多血液学疾病。在血液学领域内存在真实的需求，以找到替代和减少对小鼠模型的依赖的替代方案。目前，在文献中还没有完全开发的BM体外模型。我们的解决方案代表了一种强大的方法来概括BM和脾脏微环境的重要方面，同时维持细胞的存活能力，使我们能够测量微流体装置中健康和患病HSC之间的差异。如果被其他研究人员和行业采用，该系统有可能通过取代早期并大大减少对动物模型的依赖来产生实质性的3Rs影响，以便将来在白血病和其他血液疾病中进行LSC靶向疗法的临床前测试。

Haematopoietic stem cells (HSCs) are specialised cells with the ability to either renew themselves and produce more stem cells or to mature - a process called differentiation which produces mature blood cells. HSCs reside in the bone marrow (BM) 'niche', a microenvironment, which supports these processes. The progression and development of myeloid leukaemias such as chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) is encouraged by the establishment of a self-enforcing leukaemic stem cell (LSC) niche, where LSCs remodel the healthy BM niche to their advantage. Over time haematological transformation, especially of myeloid origin, often involves extramedullary haematopoiesis in the spleen and liver, where LSCs move out of the BM and reside in these organs where they proliferate and expand. To date is has proved difficult to target LSCs with current therapies and to study exactly how LSCs manage to dominate and alter the niche. Advances have been hindered by the lack of robust in vitro models that recapitulate the haematopoietic system, resulting in the majority of studies being performed in mouse models. Such models are not ideal and lack a tumour-specific and/or species-specific microenvironment. This project will create an innovative long-term fully humanised in vitro haematopoietic system to model the BM and splenic microenvironments to study haematological malignancies. Using tissue engineering we have developed an artificial haematopoietic system comprising of BM and spenic niches which includes resident cell types; mesenchymal stem cells, HSCs, LSCs and splenic fibroblasts. Our system uses advanced fibronectin-based hydrogels designed to match the mechanical properties of the BM with controlled stiffness and protease-degradability mimicking the BM structure. By utilising this model system, we will provide insight into the microenvironmental changes that LSCs confer and how these changes influence the resident cells. We aim to validate this system by directly comparing normal HSCs and LSCs behaviour and changes directly to existing in vivo xenograft data, already generated in the laboratory, using the same primary samples from CML and AML patients. This technology aims to drive the REPLACEMENT of animals used for patient-derived xenograft studies and ultimately aid knowledge and treatment of a number of haematological disorders. There is a real need within the haematology field to find an alternative to REPLACE and REDUCE reliance on mouse models. Currently there are no fully developed in vitro models of the BM in the literature. Our solution represents a robust approach to recapitulate important aspects of the BM and spleen microenvironment, whilst sustaining cell survivability, allowing us to measure differences between healthy and diseased HSCs incorporated within a microfluidic device. If adopted by other researchers and industry this system has the potential to have a substantive 3Rs impact by replacing early and substantially reducing the reliance on animal models for later pre-clinical testing of LSC-targeted therapies in leukaemia and other haematological disorders in the future.