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：了解确定性造血的个体发育 在脊椎动物胚胎发育过程中，会发生多波造血作用，并被定义为原始造血作用或确定性造血作用。只有胚胎内的定形波才能为造血干细胞（HSC）提供长期的增殖能力。然而，大多数为产生血液而开发的体外分化系统已经成功复制了原始波，但没有一个能够产生长期再生的 HSC。为了进一步了解确定性造血的个体发育，我们与 Catherine Porcher 博士（牛津大学）合作启动了基础发育研究。使用已发表的体外分化系统，我们鉴定了一群造血内皮细胞（造血干细胞的前体）。我们发现这些细胞不表达动脉、静脉或淋巴特性的标记，这表明存在非常早期的、未定型的造血内皮细胞群，类似于造血的原始波中报道的情况。 由于造血干细胞产生于动脉生态位内，并且动脉内皮含有对造血和内皮发育重要的信号分子（VEGF、Notch），因此我们假设胚胎内造血内皮由于其动脉特性而不同于卵黄囊造血内皮。最近发表的单细胞水平证实，背主动脉中的早期造血祖细胞维持动脉基因的表达。因此，我们专注于开发使细胞分化为动脉生态位的条件。我们发现，用高水平 VEGFA 处理细胞会导致第 5.5 天时 Flk-1+ 造血内皮细胞中 Runx1 的激活受阻，从而导致造血作用终止。我们还看到 Dll4 配体的增加，Dll4 配体是一种动脉标记物，可导致下游 Notch 信号传导和进一步的动脉分化。我们发现 Runx1 激活中的块与 Notch 无关。通过分析一组基因的动脉特性和规格，我们能够证明细胞在重新接种后两到三天后开始获得更充分发育的动脉程序。 在 2017 财年，我们将继续开发最佳培养条件，以促进内皮分化为动脉内皮，并诱导动脉内皮激活 Runx1 并分化为最终的 HSC。鉴于缺氧在细胞的动脉规范中发挥的重要作用，以及其在维持骨髓中造血干细胞“干性”方面的关键作用，我们将探讨缺氧在动脉内皮发育中的作用以及当这些细胞在体外出现时调节造血干细胞的发育。其他信号传导因子也将被研究，包括 TGF-β、Wnt、BMP 和 cAMP。 2. 目标 3.2：开发正常人 iPSC 造血分化的培养系统 我们建立了一种新的系统，用于从 iPSC 中从头生成易于获得的悬浮人类造血细胞 (CD45+CD34+)。高达 60% 的 iPSC 分化细胞具有 CD45+CD34+ 表型。这些细胞在克隆祖细胞测定中形成集落，尽管与原代 CD34+ 细胞相比容量有所降低。然而，它们未能归巢于免疫缺陷（NSG）动物的骨髓，并且移植后也没有实现长期植入。为了了解真正的 HSC 和 iPSC 衍生的 HSC 之间的移植潜力差异，我们进行了单细胞 RNA 测序实验来比较这两种细胞群。生物信息比较表达分析正在进行中，以查明 iPSC 衍生的造血细胞中可能失调的基因或途径。 3. 目标 3.3：来自遗传性骨髓衰竭综合征患者的基因校正 iPSC 分化为可移植的 HSC 我们从 Juan Carlos Izpisua-Belmonte 博士（索尔克研究所）和 MJ Weiss 博士（圣路易斯）的实验室获得了来自遗传性骨髓衰竭综合征（范可尼贫血和 Diamond-Blackfan 贫血）个体的原始且经过基因校正的 iPSC 系。裘德儿童研究医院），分别。 2017 财年，将优化这些细胞系最佳生长的培养条件，并将评估为正常 iPSC 开发的分化方案，以评估患者来源的 iPSC 的造血分化。