Regenerative Therapies for Inherited Blood Disorders-iPSC differentiation

总结 1.目标3.1：了解确定性造血的个体发生 在脊椎动物胚胎发育过程中，发生多波造血，并被定义为原始的或最终的。只有胚胎内的定形波才能为造血干细胞（HSCs）提供长期的再生能力。然而，大多数用于产生血液的体外分化系统已经成功地复制了原始波，但没有一个能够产生长期的再生HSC。为了进一步了解确定性造血的个体发生，我们与Catherine Porcher博士（牛津大学）合作开展了基础发育研究。使用已发表的体外分化系统，我们已经确定了造血内皮细胞（HSC的前体）的人口。我们发现，这些细胞不表达动脉，静脉或淋巴管身份的标志物，这表明一个非常早期的，未定型的造血内皮细胞群，类似于已报道的原始波造血。 由于造血干细胞出现在动脉龛内，动脉内皮含有对造血和内皮发育重要的信号分子（VEGF，Notch），我们假设胚胎内生血内皮与卵黄囊生血内皮因其动脉特性而不同。最近发表的单细胞水平证实，在背主动脉的早期造血祖细胞维持动脉基因的表达。因此，我们专注于开发条件，使细胞向动脉龛分化。我们发现，用高水平的VEGFA处理细胞导致在第5.5天Flk-1+造血内皮中Runx 1活化的阻断，因此造血作用的消除。我们还看到Dll 4配体的增加，Dll 4配体是一种导致下游Notch信号传导和进一步动脉分化的动脉标志物。我们发现Runx 1激活的阻断是Notch独立的。通过分析一组动脉特性和特化的基因，我们能够表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，以促进内皮细胞分化为动脉内皮细胞，并诱导动脉内皮细胞活化Runx 1并分化为永久性HSC。鉴于缺氧在细胞的动脉特化中发挥的重要作用，以及其在维持骨髓中造血干细胞的“干细胞性”中的关键作用，我们将探讨缺氧在动脉内皮发育中的作用，以及在造血干细胞发育的调节中，因为这些细胞在体外出现。将研究其他信号传导因子，包括TGF-β、Wnt、BMP和cAMP。 2.目的3.2：开发用于正常人iPSC造血分化的培养系统 我们已经建立了一种新的系统，用于从头生成容易获得的悬浮人造血细胞（CD 45 + CD 34+）从iPSC。高达60%的iPSC分化细胞具有CD 45 + CD 34+表型。这些细胞在克隆生成祖细胞测定中形成集落，尽管与原代CD 34+细胞相比能力降低。然而，它们未能回到免疫缺陷（NSG）动物的骨髓中，并且在移植后没有导致长期植入。为了理解真正的HSC和iPSC衍生的HSC之间的植入潜力的差异，我们进行了比较两种细胞群的单细胞RNA Seq实验。生物信息学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遗传校正的iPSC向可移植的HSC的分化 我们已经分别从Juan卡洛斯Izpisua-Belmonte博士（Salk研究所）和MJ韦斯博士（圣裘德儿童研究医院）的实验室获得了源自患有遗传性骨髓衰竭综合征（范可尼贫血和Diamond-Blackfan贫血）的个体的原始和遗传校正的iPSC系。在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，以用于患者来源的iPSC的造血分化。

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.