The bone marrow supports and regulates hematopoiesis, responding to physiological requirements for blood cell production over ontogeny and during pathological challenges. Interactions between hematopoietic cells and niche components are challenging to study mechanistically in the human context, but are important to delineate in order to explore the pathobiology of blood and bone marrow disorders. Organoids are proving transformative in many research settings, but an accurate human bone marrow model incorporating multiple hematopoietic and stromal elements has been lacking. This protocol describes a method to generate three-dimensional, multilineage bone marrow organoids from human induced pluripotent stem cells (hiPSCs), detailing the steps for the directed differentiation of hiPSCs using a series of cytokine cocktails and hydrogel embedding. Over 18 days of differentiation, hiPSCs yield the key lineages that are present in central myelopoietic bone marrow, organized in a well-vascularized architecture that resembles native hematopoietic tissues. This presents a robust, in vitro system that can model healthy and perturbed hematopoiesis in a scalable three-dimensional microenvironment. Bone marrow organoids also support the growth of immortalized cell lines and primary cells from healthy donors and patients with myeloid and lymphoid cancers, including cell types that are poorly viable in standard culture systems. Moreover, we discuss assays for the characterization of organoids, including interrogation of pathogenic remodeling using recombinant TGF-ß treatment, and methods for organoid engraftment with exogenous cells. This protocol can be readily adapted to specific experimental requirements, can be easily implemented by users with tissue culture experience and does not require access to specialist equipment.

骨髓支持和调节造血功能，响应个体发育过程中和病理挑战期间血细胞生成的生理需求。造血细胞和生态位成分之间的相互作用在人类背景下进行机械研究具有挑战性，但为了探索血液和骨髓疾病的病理学而进行描述很重要。类器官在许多研究环境中被证明具有变革性，但一直缺乏包含多种造血和基质元素的准确的人类骨髓模型。该协议描述了一种从人类诱导多能干细胞 (hiPSC) 生成三维、多谱系骨髓类器官的方法，详细介绍了使用一系列细胞因子鸡尾酒和水凝胶嵌入定向分化 hiPSC 的步骤。经过 18 天的分化，hiPSC 产生了存在于中枢骨髓生成骨髓中的关键谱系，这些谱系以类似于天然造血组织的血管良好的结构组织。这提供了一个强大的体外系统，可以在可扩展的三维微环境中模拟健康和受干扰的造血作用。骨髓类器官还支持来自健康捐赠者以及骨髓癌和淋巴癌患者的永生化细胞系和原代细胞的生长，包括在标准培养系统中存活力差的细胞类型。此外，我们还讨论了类器官表征的测定方法，包括使用重组 TGF-β 治疗来探究致病性重塑，以及外源细胞移植类器官的方法。该协议可以很容易地适应特定的实验要求，可以由具有组织培养经验的用户轻松实施，并且不需要使用专业设备。