Regenerative Therapies for Inherited Blood Disorders-iPSC differentiation

遗传性血液疾病的再生疗法 - iPSC 分化

• 批准号: 9357246
• 负责人: Andre LaRochelle
• 金额: $ 58.72万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9357246
• 关键词: Adult; Affect; Animals; Aorta; Autologous Transplantation; Bioinformatics; Biological Assay; Blood; Bone Marrow; CD34 gene; Cell Aging; Cell Line; Cells; Child; Clinical; Collaborations; Cyclic AMP; Derivation procedure; Development; Diamond-Blackfan anemia; Disease; Dorsal; Dose; Embryo; Embryonic Development; Endothelium; Engraftment; Fanconi' s Anemia; Floods; Generations; Genes; Genetic; Growth; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Home environment; Hospitals; Human; Human body; Hypoxia; In Vitro; Inborn Genetic Diseases; Individual; Inherited; Institutes; Knowledge; Laboratories; Ligands; Lymphatic; Maintenance; Medical; Mutation; PTPRC gene; Pathway interactions; Patients; Phenotype; Play; Population; Protocols documentation; Publications; Publishing; Regulation; Reporting; Research; Role; Saint Jude Children' s Research Hospital; Signal Transduction; Signaling Molecule; Stem Cell Development; Stem cells; Suspension substance; Suspensions; System; Tissues; Transforming Growth Factor beta; Translating; Transplantation; Universities; VEGFA gene; Vascular Endothelial Growth Factors; Venous; Yolk Sac; base; bone marrow failure syndrome; cell age; comparative; individual patient; induced pluripotent stem cell; meetings; notch protein; novel; novel strategies; progenitor; programs; regenerative; regenerative therapy; research study; stem cell biology; stem cell differentiation; stem cell technology; stemness; therapeutic transgene; transcriptome sequencing

Summary 1. Objective 3.1: Understanding the ontogeny of definitive hematopoiesis During vertebrate embryonic development, multiple waves of hematopoiesis take place and are defined as either primitive or definitive. Only the intra-embryonic definitive wave provides hematopoietic stem cells (HSCs) with long-term repopulating capacity. However, most in-vitro differentiation systems developed to generate blood have successfully replicated the primitive wave, but none have been able to produce long-term repopulating HSCs. To further understand the ontogeny of definitive hematopoiesis, we have initiated basic developmental studies in collaboration with Dr. Catherine Porcher (Oxford University). Using a published in vitro differentiation system, we have identified a population of hemogenic endothelium (the precursors of HSCs). We showed that these cells do not express markers of arterial, venous, or lymphatic identity, suggesting a very early, uncommitted hemogenic endothelial population, similar to what has been reported in the primitive wave of hematopoiesis. Because hematopoietic stem cells arise within an arterial niche, and arterial endothelium contains signaling molecules important for hematopoietic and endothelial development (VEGF, Notch), we hypothesized that intra-embryonic hemogenic endothelium was different to yolk sac hemogenic endothelium due to its arterial identity. A recent publication at the single-cell level confirms that early hematopoietic progenitors in the dorsal aorta maintain the expression of arterial genes. We therefore focused on developing conditions to differentiate cells towards an arterial niche. We found that treating the cells with high levels of VEGFA led to a block in Runx1 activation in Flk-1+ hemogenic endothelium at Day 5.5 and therefore an abrogation of hematopoiesis. We also saw an increase in Dll4 ligand, an arterial marker that leads to downstream Notch signaling and further arterial differentiation. We found that the block in Runx1 activation was Notch independent. By assaying a panel of genes for arterial identity and specification, we were able to show that cells begin to acquire a more fully developed arterial program after two to three days after being replated. In FY17, we will continue developing optimal culture conditions for promoting endothelial differentiation into arterial endothelium, and for inducing the arterial endothelium towards activation of Runx1 and differentiation into definitive HSCs. Given the important role that hypoxia plays in the arterial specification of cells, as well as its critical role in the maintenance of the "stemness" of hematopoietic stem cells in the bone marrow, we will explore the role of hypoxia in the development of arterial endothelium and in the regulation of hematopoietic stem cell development as these cells arise in-vitro. Other signaling factors will be investigated, including TGF-beta, Wnt, BMP, and cAMP. 2. Objective 3.2: Development of a culture system for hematopoietic differentiation of normal human iPSCs We have established a novel system for de novo generation of easily accessible suspension human hematopoietic cells (CD45+CD34+) from iPSCs. Up to 60% of iPSC-differentiated cells have a CD45+CD34+ phenotype. These cells form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to primary CD34+ cells. However, they failed to home to the bone marrow of immuno-deficient (NSG) animals and did not result in long-term engraftment after transplantation. To understand differences in engraftment potential between bona fide HSCs and iPSC-derived HSCs, we have conducted single cell RNA Seq experiments comparing both cell populations. Bioinformatic comparative expression analysis is underway to pinpoint genes or pathways that may be deregulated in iPSC-derived hematopoietic cells. 3. Objective 3.3: Differentiation of genetically corrected iPSCs derived from patients with inherited bone marrow failure syndromes into transplantable HSCs We have obtained original and genetically corrected iPSC lines derived from individuals with inherited bone marrow failure syndromes (Fanconi Anemia and Diamond-Blackfan Anemia) from the laboratories of Dr. Juan Carlos Izpisua-Belmonte (Salk Institute) and Dr. MJ Weiss (St. Jude Childrens Research Hospital), respectively. In FY17, culture conditions for optimal growth of these lines are will be optimized and the differentiation protocol developed for normal iPSCs will be evaluated for hematopoietic differentiation of patient-derived iPSCs.

概括 1。目标3.1：了解确定造血的个体发育 在脊椎动物的胚胎发育过程中，发生多个造血作用，并定义为原始或确定性。只有胚胎内确定性波才能具有长期重塑能力的造血干细胞（HSC）。但是，大多数用于产生血液的体外分化系统成功地复制了原始波，但没有一个能够产生长期重现的HSC。为了进一步了解确定性造血的个体发育，我们与凯瑟琳·波彻（Catherine Porcher）（牛津大学）合作启动了基本发展研究。使用已发表的体外分化系统，我们已经确定了血肿内皮的群体（HSC的前体）。我们表明，这些细胞不表达动脉，静脉或淋巴认同的标记，这表明非常早，不合理的血液生成内皮人群与原始造血浪潮中报道的相似。 Because hematopoietic stem cells arise within an arterial niche, and arterial endothelium contains signaling molecules important for hematopoietic and endothelial development (VEGF, Notch), we hypothesized that intra-embryonic hemogenic endothelium was different to yolk sac hemogenic endothelium due to its arterial identity.最近在单细胞水平上的出版物证实，背主动脉的早期造血祖细胞保持动脉基因的表达。因此，我们专注于开发条件，以将细胞区分为动脉裂市场。我们发现，用高水平的VEGFA处理细胞导致第5.5天的FLK-1+血液生成内皮中Runx1激活的块，因此消除了造血。我们还看到了DLL4配体的增加，DLL4配体是一种导致下游凹口信号传导和进一步的动脉分化的动脉标记。我们发现Runx1激活中的块是独立的。通过分析用于动脉认同和规范的基因面板，我们能够证明细胞在被重新被重新测试后两到三天后开始获得更全面的动脉程序。 在2017财年，我们将继续开发最佳的培养条件，以促进内皮分化为动脉内皮，并诱导动脉内皮来激活RUNX1并分化为确定的HSC。鉴于缺氧在细胞的动脉规范中发挥的重要作用，以及其在造血干细胞在骨髓中维持“干性”中的关键作用，我们将探索缺氧在动脉内皮的发育中的作用，以及在这些细胞的调节中，如这些细胞在这些细胞的调节中，这些细胞在这些细胞的调节中均为这些细胞。将研究其他信号传导因素，包括TGF-beta，Wnt，BMP和CAMP。 2。目标3.2：开发正常人IPSC造血分化的培养系统 我们已经建立了一个新的系统，用于从IPSCS开始从头产生易于获得的易于获得的悬浮悬浮液（CD45+CD34+）。多达60％的IPSC分化细胞具有CD45+ CD34+表型。这些细胞在克隆发生祖细胞测定中形成菌落，尽管与初级CD34+细胞相比，能力降低。但是，它们未能进入免疫缺陷（NSG）动物的骨髓，并且在移植后没有导致长期植入。为了了解真正的HSC和IPSC衍生的HSC之间的植入潜力差异，我们进行了单细胞RNA SEQ实验，比较了两个细胞群体。正在进行生物信息学比较表达分析，以查明在IPSC衍生的造血细胞中可能失控的基因或途径。 3。目标3.3：遗传校正的IPSC的分化，这些IPSC源自遗传性骨髓衰竭综合征为可移植HSC的患者 我们已经从遗传性骨髓衰竭综合征（Fanconi贫血和钻石 - 黑色贫血）中得出的原始和遗传校正的IPSC系，从Juan Carlos Izpisua Izpisua Belmonte博士（Salk Institute）和MJ Weiss（St. Jude Childrens Hospital）的实验室的实验室中。在2017财年，将优化这些线路最佳生长的培养条件，并将评估针对正常IPSC的分化方案，以评估患者衍生的IPSC的造血分化。