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

用于 HSC 离体扩增的生物工程肝脏生态位

• 批准号: 10452482
• 负责人: Salman R Khetani
• 金额: $ 31.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-16 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452482
• 关键词: 3-Dimensional; Address; Adult; Aorta; Biological Assay; Biological Models; Biomedical Engineering; Blood Cells; Bone Marrow; Caliber; Cell Communication; Cells; Coculture Techniques; Collagen; Conditioned Culture Media; Cues; Development; Disease; Dorsal; Embryo; Embryonic Development; Encapsulated; Endothelial Cells; Extramedullary Hematopoiesis; Fetal Liver; Fibroblasts; Flow Cytometry; Generations; Genetic; Goals; Gonadal structure; Growth; Harvest; Hematologic Neoplasms; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Hemolytic Anemia; Hepatic; Hepatic Stellate Cell; Hepatocyte; Human; Human Engineering; IL3 Gene; In Vitro; Investigation; Knowledge; Kupffer Cells; Label; Literature; Liver; Liver Stem Cell; Malignant Neoplasms; Mammals; Mesonephric structure; Methods; Microfluidics; Modeling; Modification; Monitor; Mus; Myeloproliferative disease; National Institute of Diabetes and Digestive and Kidney Diseases; Neoplasm Metastasis; Pathologic; Phenotype; Physiological; Pregnancy; Process; Publishing; Research; Rodent; Role; Serum; Signal Transduction; Site; Source; Spleen; Stress; Stromal Cells; System; Translating; Transplantation; Work; base; blood formation; cell type; cytokine; experimental study; fetal; hematopoietic stem cell expansion; hematopoietic stem cell self-renewal; in vivo; mouse model; novel; progenitor; reconstitution; self-renewal; stem cell population; stem cells; success; tissue culture; transplantation therapy

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.

摘要 /项目摘要 血液学研究（Shine-II）的新方向PAS-18-730 NIDDK 标题：模块化人肝培养平台中骨髓造血干细胞的扩展 造血干细胞（HSC）位于血细胞等级的顶部，并负责 生成造血系统的所有祖细胞和成熟细胞。 HSC的能力 产生大量造血细胞类型的多样性，同时仍保持自我更新和增生 能力是该领域积极研究的话题。许多研究都将利基作为关键来源 指导和支持HSC细胞命运决策的外部因素。体内平衡的主要利基市场 成年哺乳动物中的造血是骨髓。关于特定的单元类型仍然存在很多争论 在骨髓小裂中 很少激活祖细胞类型的扩张和分化。同样，具有的实验 试图使用骨髓基质细胞或细胞因子鸡尾酒在组织培养的塑料中扩展HSC。 尚未成功产生能够长期，多条件的稳健和可再生干细胞 重组受辐照的接受者。我们假设一种基于造血的个体发育的方法 系统将允许更成功的HSC的离体扩展。以前的发现表明HSCS 在胎儿肝脏的胚胎发育过程中，扩大非常快速的扩展。此外，在压力下或由于一定 病理状况，HSC迁移到成年肝脏，在那里患有耗血外造血作用 （EMH）。遵循这些生理提示，我们建议对增加的生物工程师的共同培养系统增加 使用原发性人肝细胞（PHH），辅助成纤维细胞和原发性人肝非 - 实质细胞（NPC）。我们将首先利用PHH的长期微图案共培养（MPCC）系统 被支撑性成纤维细胞包围的菌落（3T3-J2鼠类胚胎成纤维细胞和人肝门户 成纤维细胞或LPF）我们将在其上介绍成人骨髓HSC，在无优化的无血清中培养 补充造血细胞因子的培养基。我们将监视MPCC中的HSC扩展 在评估其体外和在评估其功能电位之前，通过流式细胞仪表征表征它们 小鼠移植测定中的体内。接下来，我们将HSC纳入新颖的3维（3D）人肝 我们已经证明可以使PHH和原发性人肝正弦波的最佳相互作用的微动物 内皮细胞（LSEC）； PHH-LPF-LSEC微动物将通过静止的肝脏进一步增强 星状细胞和kupffer细胞阐明了这些细胞类型在调节HSC扩展的离体中的作用。 最后，我们将在小鼠中诱导EMH升级HSC，以进行肝微环境，然后收获和扩展 它们在人类肝平台上的增加，如上所述增加了细胞复杂性。总的来说，总体目标 专注的Shine II提案是创建与生理相关，长期和模块化的人类肝平台 为了扩展和研究成年HSC及其与肝脏中关键细胞类型的相互作用。