A bio-engineered hepatic niche for ex vivo expansion of HSCs

摘要/项目摘要 血液学研究新方向（SHINE-II）PAS-18-730 NIDDK 标题：在模块化人肝培养平台中扩增骨髓造血干细胞 造血干细胞（HSC）位于血细胞层级的顶部，并负责维持造血干细胞的功能。 生成造血系统的所有祖细胞和成熟细胞。HSC如何能够 产生大量多样性的造血细胞类型，同时仍然保持它们的自我更新和增殖能力， 能力是该领域积极研究的一个主题。许多研究都指出，利基作为关键的来源， 指导和支持HSC细胞命运决定的外在因素。自我平衡的主要生态位 成年哺乳动物的造血是骨髓。关于特定的细胞类型仍然存在许多争论 骨髓小生境，但普遍接受的是，在骨髓中，HSC主要是静止的， 很少被激活用于祖细胞类型扩增和分化。同样的， 尝试在组织培养塑料中使用骨髓基质细胞或细胞因子混合物离体扩增HSC 还没有成功地产生强大的和可再生的干细胞能够长期，多谱系 辐射受体的重建。我们假设，一种基于造血干细胞的个体发生的方法， 系统将允许HSC的更成功的离体扩增。以前的研究结果表明，HSC 在胚胎发育期间在胎儿肝脏中非常迅速地扩张。此外，在压力下或由于某些 在病理条件下，HSC迁移到成人肝脏，在那里它们经历髓外造血 （EmH）.根据这些生理学线索，我们建议生物工程体外共培养系统， 使用原代人肝细胞（PHH）、支持性成纤维细胞和原代人肝非 实质细胞（NPC）。我们将首先利用我们的长期微图案共培养（MPCC）系统的PHH 由支持性成纤维细胞（3 T3-J2鼠胚胎成纤维细胞和人肝门静脉）包围的集落 成纤维细胞或LPF），我们将向其引入在优化的无血清培养基中培养的成人骨髓HSC。 补充有造血细胞因子的培养基。我们会监察长者住屋计划在多功能公众诊所的扩展情况， 在体外和体内评估其功能潜力之前， 小鼠体内移植试验。接下来，我们将HSC整合到新的三维（3D）人类肝脏中， 我们已经证明，使PHH和原代人肝窦的最佳相互作用的微组织， 内皮细胞（LSEC）; PHH-LPF-LSEC微组织将进一步增加静息肝细胞 星状细胞和枯否细胞，以阐明这些细胞类型在离体调节HSC扩增中的作用。 最后，我们将在小鼠中诱导EmH以使HSC为肝脏微环境做好准备，然后收获和扩增 它们在如上增加细胞复杂性的人类肝脏平台中。总的来说，这一总体目标 聚焦SHINE II的提案是创建生理相关的、长期的和模块化的人类肝脏平台 用于成年HSC的扩增和研究及其与肝脏内关键细胞类型的相互作用。

Abstract / Project Summary New Directions in Hematology Research (SHINE-II) PAS-18-730 NIDDK Title: Expansion of bone marrow hematopoietic stem cells in modular human liver culture platforms The hematopoietic stem cell (HSC) resides at the top of the hierarchy of blood cells and is responsible for the generation of all the progenitor and mature cells of the hematopoietic system. How HSCs are capable of generating the vast diversity of hematopoietic cell types while still retaining their self-renewal and proliferative capacity is a topic of active research in the field. Many studies have pointed to the niche as the critical source of extrinsic factors that instruct and support HSC cell fate decisions. The primary niche for homeostatic hematopoiesis in adult mammals is the bone marrow. Much debate still exists regarding the specific cell types of the bone marrow niche, but it is generally accepted that in the bone marrow HSCs are primarily quiescent and infrequently activate for expansion and differentiation of progenitor cell types. Likewise, experiments that have attempted to expand HSCs ex vivo using bone marrow stromal cells or cytokine cocktails in tissue culture plastic have not been successful in generating robust and renewable stem cells capable of long-term, multi-lineage reconstitution of irradiated recipients. We hypothesize that an approach based on the ontogeny of hematopoietic system will allow for a more successful ex vivo expansion of HSCs. Previous findings have shown that HSCs expand very rapidly during embryonic development in the fetal liver. Furthermore, under stress or due to certain pathological conditions, HSCs migrate to the adult liver where they undergo extramedullary hematopoiesis (EmH). Following these physiological cues, we propose to bioengineer ex vivo co-culture systems of increasing complexities using primary human hepatocytes (PHH), supportive fibroblasts, and primary human liver non- parenchymal cells (NPC). We will first utilize our long-term micropatterned co-culture (MPCC) system of PHH colonies surrounded by supportive fibroblasts (3T3-J2 murine embryonic fibroblasts and human liver portal fibroblasts or LPF) to which we will introduce adult bone marrow HSCs cultured in an optimized serum-free culture medium supplemented with hematopoietic cytokines. We will monitor HSC expansion in MPCCs and characterize them phenotypically via flow cytometry prior to assessing their functional potential in vitro and in vivo in mouse transplantation assays. Next, we will incorporate HSCs into novel 3-dimensional (3D) human liver microtissues that we have shown to enable optimal interactions of PHHs and primary human liver sinusoidal endothelial cells (LSEC); the PHH-LPF-LSEC microtissues will be further augmented with quiescent hepatic stellate cells and Kupffer cells to elucidate the role of these cell types in modulating HSC expansion ex vivo. Lastly, we will induce EmH in mice to prime HSCs for the liver microenvironment and then harvest and expand them in human liver platforms of increasing cellular complexities as above. In conclusion, the overall goal of this focused SHINE II proposal is to create physiologically-relevant, long-term, and modular human liver platforms for the expansion and study of adult HSCs and their interactions with key cell types within the liver.