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

摘要/项目摘要 血液学研究的新方向(SIRE-II)PAS-18-730 NIDDK 标题：骨髓造血干细胞在模块化人肝培养平台中的扩增 造血干细胞(HSC)位于血细胞层次结构的顶端，负责 造血系统的所有祖细胞和成熟细胞的生成。HSC如何能够 在保持其自我更新和增殖能力的同时，产生了丰富多样的造血细胞类型 能力是该领域活跃研究的一个主题。许多研究都指出，利基市场是 指导和支持HSC细胞命运决定的外在因素。动态平衡的主要利基 成年哺乳动物的造血功能是骨髓。关于特定的细胞类型仍然存在很多争论 但人们普遍认为，在骨髓中，造血干细胞主要是静止的和 很少被激活以促进祖细胞类型的扩张和分化。同样，那些拥有 在组织培养塑料中尝试使用骨髓基质细胞或细胞因子鸡尾酒体外扩增HSCs 还没有成功地产生能够长期、多血统的健壮和可再生的干细胞 受照射受者的重建。我们假设，一种基于造血个体发育的方法 该系统将允许更成功地体外扩增造血干细胞。先前的研究结果表明，造血干细胞 在胚胎发育过程中在胎儿肝脏中迅速扩张。此外，在压力下或由于某些原因 病理条件下，造血干细胞迁移到成人肝脏，在那里它们进行髓外造血。 (EMH)。根据这些生理线索，我们提出了生物工程体外共培养系统，以增加 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PHH长期微图案化共培养(MPCC)系统 由支持性成纤维细胞(3T3-J2小鼠胚胎成纤维细胞和人肝门)包围的集落 成纤维细胞或LPF)，我们将把在优化的无血清培养中培养的成人骨髓HSCs引入 添加造血细胞因子的培养液。我们将监测MPC中HSC的扩展情况，并 在评估它们在体外和体内的功能潜力之前，通过流式细胞术来表征它们的表型 小鼠体内移植实验。接下来，我们将把造血干细胞植入新型三维(3D)人体肝脏 我们已经证明能够使PHHs和人的初级肝窦最佳相互作用的显微组织 内皮细胞(LSEC)；PHH-LPF-LSEC显微组织将随着静止的肝脏而进一步增强 星状细胞和Kupffer细胞，以阐明这些细胞类型在调节HSC体外扩增中的作用。 最后，我们将在小鼠体内诱导EMH，为肝脏微环境准备HSC，然后采集和扩增 它们在人类肝脏平台上的细胞复杂性如上所述。总而言之，这一总体目标是 Focus Sare II的建议是创建与生理相关的、长期的和模块化的人体肝脏平台 用于成人HSCs的扩增和研究，以及它们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.