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: 9787984
• 负责人: Andre LaRochelle
• 金额: $ 58.15万
• 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/9787984
• 关键词: Activins; Adult; Affect; Animal Model; Aorta; Autologous; Autologous Transplantation; BMP4; Base Pairing; Blood; Blood Cells; Blood Vessels; Bone Marrow; CD34 gene; CRISPR/Cas technology; Cell Aging; Cell Cycle; Cell Differentiation process; Cells; Chemicals; Clinical; Clone Cells; Collaborations; Core Facility; DNA; DNA Damage; Data; Defect; Dependovirus; Derivation procedure; Development; Diamond-Blackfan anemia; Disease; Dorsal; Electroporation; Embryo; Endothelium; Engraftment; Erythroblasts; Erythroid; FGF2 gene; Fanconi Anemia Complementation Group A Protein; Fanconi' s Anemia; Fibroblasts; Floods; Functional disorder; Gene Delivery; Gene Targeting; Generations; Genes; Genetic; Genetic Diseases; Genetic Transcription; Guide RNA; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hereditary Disease; Human; Human body; Hypoxia; In Vitro; Inherited; Interleukin-3; Kinetics; Knowledge; Laboratories; Lead; Ligands; Maintenance; Mediating; Medical; Methodology; Mononuclear; Mus; Mutation; National Heart, Lung, and Blood Institute; Nodal; PTPRC gene; Pathway interactions; Patients; Pattern; Periodicity; Plasmid Cloning Vector; Play; Point Mutation; Population; Process; Production; Protocols documentation; Reporting; Repression; Sendai virus; Serotyping; Signal Transduction; Stem cells; Stress; Stromal Cells; System; TP53 gene; Technology; Therapeutic; Time; Tissues; Translating; Transplantation; VEGFA gene; Vascular Endothelial Growth Factors; Work; base; bone marrow failure syndrome; c-myc Genes; clinical translation; clinically relevant; cytotoxicity; gene therapy; genetic approach; genome editing; improved; in vitro Model; in vivo; individual patient; induced pluripotent stem cell; inhibitor/antagonist; jagged1 protein; monolayer; morphogens; mouse model; notch protein; novel; novel strategies; nuclease; programs; regenerative; regenerative therapy; repaired; response; small hairpin RNA; small molecule; small molecule inhibitor; stem cell biology; stem cell differentiation; stem cell technology; therapeutic transgene; transcription factor; trend

Objective 1: Fibroblast- or mononuclear cell (MNC)-differentiated erythroblasts from DBA and FA patient have been successfully reprogrammed in collaboration with the NHLBI iPSC core facility. Reprogramming FA cells has proven to be challenging and efforts in the past year have centered on enhancing efficiency of reprogramming of these cells. Progressive loss of HSPCs in FA patients has been attributed in part to a heightened p53/p21 axis that restricts the cell cycle to G0/G1 in response to accumulation of unresolved DNA damage and replicative stress. Silencing of p53 was previously shown to rescue HSPC defects in FA models in vitro and in vivo. A recent study reported low-efficiency generation of integration-free FA-iPSCs by electroporation of fibroblasts with episomal plasmid vectors expressing both reprogramming factors and p53 shRNA. Here we sought to develop an improved and streamlined reprogramming strategy for FA by transduction of readily accessible FA MNCs with commercially available non-integrating reprogramming Sendai Viruses (SeV2), combined with a more adaptable inhibition of the p53 pathway by addition of variable concentrations of a small molecule during reprogramming. Approximately 100,000 MNCs from an FA patient with confirmed biallelic mutations in FANCA were cultured for 10 days under hypoxic conditions (5% O2) favoring erythroid expansion. Erythroid-expanded FA cells were transduced with SeV2 harboring four standard reprogramming transcription factors (SOX2, OCT4, KLF4 and c-MYC). Cells were subsequently cultured under hypoxia for at least 30 days in a reprogramming cocktail supplemented or not with 0.3uM, 0.6M or 1.2M cyclic pifthrin- (cPFT), a reversible small molecule inhibitor of p53-dependent gene transcription. In the absence of cPFT, no FA-iPSC clones were obtained, consistent with reported reprogramming barriers in FA cells. In contrast, several iPSC colonies were generated with cPFT-treated FA cells, albeit at much slower kinetics (30 days) compared to MNCs without FA mutations (20 days). The overall efficiency of reprogramming was 0.01%, with a trend toward enhanced efficiencies in cultures containing the highest concentration of cPFT (1.2M). To investigate the need for p53 inhibition beyond the reprogramming period, half of the FA-iPSC clones were passaged and further cultured in 5% O2 with cPFT 1.2M, whereas the remaining clones were similarly treated but without p53 inhibitor. None of the FA-iPSC clones that were maintained in cPFT-containing cultures following reprogramming survived further passaging. In contrast, when cPFT was removed after reprogramming, all iPSC clones could stably undergo repeated passages provided that hypoxia (5% O2) was maintained. Overall, this work provides a practical approach for successful reprogramming of FA peripheral blood cells based on commercially available non-integrating SeV2 reprogramming strategies supplemented with p53 inhibition. Objective 2: All selected DBA and FA patients have a known mutation and include single DNA base pair changes, small or large deletions. Novel CRISPR/Cas9-based genome editing approaches are used for genetic correction. Two general homology-directed repair (HDR) approaches are investigated, including targeted gene addition at the endogenous gene locus and correction of point mutations or deletions. With current approaches, only low levels (<1%) of targeted integration via HDR have been reported in human iPSCs, necessitating a time-consuming selection process to identify the few corrected iPSCs clones. Moreover, targeted gene addition and correction of larger mutations (>50bp) rely on electroporation of bulky exogenous homologous DNA donor template which results in pronounced cytotoxicity to iPSCs. Therefore, new strategies to advance the current state of CRISPR/Cas9-mediated targeted gene delivery are being explored. Specifically, delivery of gRNA, Cas9 nuclease and DNA templates for HDR using adeno-associated virus (AAV) are investigated. Multiple novel AAV serotypes have been generated in our laboratory and are investigated for optimization of transduction of human iPSCs and efficiency of genome editing. Objective 3: Efficient and clinically relevant methodologies to derive transplantable autologous HSPCs from human iPSCs ex vivo remain unavailable. To facilitate the development of functional HSPCs from human iPSCs, we previously developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, mesodermal specification is induced using morphogens (bFGF, BMP4, VEGF 10ng/mL) and, for the subsequent 18 days, cells are further differentiated into HSPCs with the addition of hematopoietic cytokines. As previously described by our group, this differentiation system recapitulates the successive waves of hematopoiesis during development and leads to robust production of immunophenotypic HSC-like cells (CD34+CD38-CD90+CD45RA-CD49f+). However, these cells do not result in efficient, long-term engraftment in immunodeficient (NSG) mouse models. To identify possible causes for the lack of durable repopulating potential of iPSC-derived HSPCs in this system, we characterized the supportive monolayer from which HSPCs arise during vitro differentiation. Observations in the developing embryo indicate that definitive HSPCs arise in the dorsal aorta from hemogenic endothelium (HE) in close association with an arterial vascular endothelial niche. The Notch pathway plays a key role in arterial and HSC differentiation; in the embryo, only HE populations found in arterial regions with active Notch signaling through Delta-like ligand 4 (Dll4) and Jagged 1 (Jag1) lead to HSPCs with repopulating potential. Recent studies have also shown that modulation of mesodermal patterning through repression and activation of Activin/Nodal and Wnt/-catenin pathways, respectively, promotes arterial programs and definitive hematopoiesis. In contrast to observations in embryos, we found that the supportive monolayer in the STEMdiffTM in vitro differentiation system has limited percentages of arterial HE (CD43-CD45-CD34hiCD144+CD73-Dll4+) and arterial endothelium (CD43-CD45-CD34hiCD144+CD73midCD184+), and overabundance of stromal cells (CD43-CD45-CD34-CD144-). This provides a possible explanation for the lack of engraftment potential of iPSC-derived HSPCs in this system. To restrict stromal development and further promote differentiation and maintenance of a supportive arterial endothelial niche, we modified the standard differentiation protocol by addition of CHIR99021 (CHIR) and SB431542 (SB) during the mesodermal stage of differentiation (days 2-3) to activate Wnt/-catenin and block of Activin/Nodal signaling, respectively. Given that VEGF acts upstream of the Notch pathway during arterial endothelial differentiation, we also increased the concentration of VEGFA 20-fold throughout differentiation (200ng/mL). Our results showed that mesodermal patterning alone was insufficient to repress stromal production and maintain an endothelial niche. However, increased VEGF concentrations, alone or in combination with CHIR/SB, markedly reduced stromal differentiation and enhanced arterial endothelium formation compared to the standard system. Importantly, combination treatments also led to significantly higher percentages of arterial HE at days 5 and 7. Current assessment of these treatments on the hematopoietic potential of the system is ongoing, and include NSG mouse transplantations. Overall, our data indicate that commercially available technologies can be further modified and improved to move closer to chemically-defined and scalable HSPC differentiation protocols.

目标1： 与 NHLBI iPSC 核心设施合作，来自 DBA 和 FA 患者的成纤维细胞或单核细胞 (MNC) 分化的成红细胞已成功重新编程。事实证明，对 FA 细胞进行重编程具有挑战性，过去一年的努力集中在提高这些细胞的重编程效率上。 FA 患者中 HSPC 的逐渐丧失部分归因于 p53/p21 轴升高，该轴将细胞周期限制为 G0/G1，以应对未解决的 DNA 损伤和复制应激的累积。此前研究表明，p53 沉默可在体外和体内挽救 FA 模型中的 HSPC 缺陷。最近的一项研究报告了通过使用表达重编程因子和 p53 shRNA 的附加型质粒载体对成纤维细胞进行电穿孔来低效生成无整合的 FA-iPSC。 在这里，我们试图开发一种改进和简化的 FA 重编程策略，通过使用市售的非整合重编程仙台病毒 (SeV2) 转导易于获得的 FA MNC，并通过添加不同浓度的 p53 途径来抑制 p53 途径。重编程过程中的小分子。来自 FA 患者的约 100,000 个 MNC 在 FANCA 中确认有双等位基因突变，在有利于红细胞扩张的缺氧条件 (5% O2) 下培养 10 天。用含有四种标准重编程转录因子（SOX2、OCT4、KLF4 和 c-MYC）的 SeV2 转导红系扩增的 FA 细胞。随后将细胞在缺氧条件下在补充或不补充0.3uM、0.6M或1.2M环pifthrin-(cPFT)（一种p53依赖性基因转录的可逆小分子抑制剂）的重编程混合物中培养至少30天。在没有 cPFT 的情况下，没有获得 FA-iPSC 克隆，这与报道的 FA 细胞重编程障碍一致。相比之下，用 cPFT 处理的 FA 细胞产生了几个 iPSC 集落，尽管与没有 FA 突变的 MNC（20 天）相比，动力学慢得多（30 天）。重编程的总体效率为 0.01%，在含有最高浓度 cPFT (1.2M) 的培养物中，效率有提高的趋势。为了研究重编程期后 p53 抑制的需要，将一半 FA-iPSC 克隆传代并在 5% O2 和 cPFT 1.2M 中进一步培养，而其余克隆进行类似处理，但不含 p53 抑制剂。重编程后在含有 cPFT 的培养物中维持的 FA-iPSC 克隆没有一个能在进一步传代中存活。相比之下，当重编程后去除 cPFT 时，只要维持缺氧 (5% O2)，所有 iPSC 克隆都可以稳定地重复传代。总体而言，这项工作提供了一种基于市售非整合 SeV2 重编程策略并辅以 p53 抑制的成功重编程 FA 外周血细胞的实用方法。 目标 2： 所有选定的 DBA 和 FA 患者均具有已知突变，包括单个 DNA 碱基对变化、小或大缺失。基于 CRISPR/Cas9 的新型基因组编辑方法用于基因校正。研究了两种通用的同源定向修复（HDR）方法，包括在内源基因位点添加靶向基因以及点突变或缺失的校正。使用目前的方法，在人类 iPSC 中仅报告了低水平（<1%）的通过 HDR 进行的靶向整合，因此需要耗时的选择过程来识别少数经过校正的 iPSC 克隆。此外，靶向基因的添加和较大突变（> 50bp）的校正依赖于庞大的外源同源DNA供体模板的电穿孔，这会导致对iPSC的明显细胞毒性。因此，正在探索新的策略来推进 CRISPR/Cas9 介导的靶向基因递送的现状。具体来说，研究了使用腺相关病毒 (AAV) 递送用于 HDR 的 gRNA、Cas9 核酸酶和 DNA 模板。我们的实验室已产生多种新型 AAV 血清型，并对其进行研究以优化人类 iPSC 的转导和基因组编辑的效率。 目标 3： 从人类 iPSC 离体中衍生出可移植的自体 HSPC 的有效且临床相关的方法仍然不可用。为了促进从人类 iPSC 中开发出功能性 HSPC，我们之前开发了一种简单的、基于单层的、化学定义的、可扩展的分化方案，无需重新铺板或胚状体 (EB) 形成（市售产品为 STEMdiffTM 造血试剂盒、Stem Cell Technologies ）。在前 3 天，使用形态发生素（bFGF、BMP4、VEGF 10ng/mL）诱导中胚层规范，在接下来的 18 天，通过添加造血细胞因子，细胞进一步分化为 HSPC。正如我们小组之前所描述的，这种分化系统再现了发育过程中连续的造血波，并导致免疫表型 HSC 样细胞 (CD34+CD38-CD90+CD45RA-CD49f+) 的大量产生。然而，这些细胞并不能在免疫缺陷（NSG）小鼠模型中实现有效、长期的移植。 为了确定该系统中 iPSC 衍生的 HSPC 缺乏持久再增殖潜力的可能原因，我们对体外分化过程中产生 HSPC 的支持性单层进行了表征。对发育胚胎的观察表明，确定性 HSPC 在背主动脉中由与动脉血管内皮生态位密切相关的造血内皮 (HE) 产生。 Notch 通路在动脉和 HSC 分化中发挥关键作用；在胚胎中，只有在动脉区域发现的 HE 群体通过 Delta 样配体 4 (Dll4) 和 Jagged 1 (Jag1) 具有活跃的 Notch 信号传导，才能产生具有增殖潜力的 HSPC。最近的研究还表明，通过分别抑制和激活 Activin/Nodal 和 Wnt/-catenin 途径来调节中胚层模式，可促进动脉程序和最终造血。与胚胎中的观察结果相反，我们发现STEMdiffTM体外分化系统中的支持单层具有有限的动脉HE（CD43-CD45-CD34hiCD144+CD73-Dll4+）和动脉内皮（CD43-CD45-CD34hiCD144+CD73midCD184+）百分比，和基质细胞过多(CD43-CD45-CD34-CD144-)。这为该系统中 iPSC 衍生的 HSPC 缺乏植入潜力提供了可能的解释。 为了限制基质发育并进一步促进支持性动脉内皮微环境的分化和维持，我们修改了标准分化方案，在中胚层分化阶段（第 2-3 天）添加 CHIR99021 (CHIR) 和 SB431542 (SB) 以激活 Wnt分别是β-连环蛋白和激活素/Nodal 信号传导阻断。鉴于 VEGF 在动脉内皮分化过程中作用于 Notch 通路上游，我们还在整个分化过程中将 VEGFA 的浓度增加了 20 倍 (200ng/mL)。我们的结果表明，仅中胚层模式不足以抑制基质产生并维持内皮生态位。然而，与标准系统相比，单独或与 CHIR/SB 组合增加 VEGF 浓度，显着减少基质分化并增强动脉内皮形成。重要的是，联合治疗还导致第 5 天和第 7 天的动脉 HE 百分比显着升高。目前正在对这些治疗对系统造血潜力的评估正在进行，其中包括 NSG 小鼠移植。总体而言，我们的数据表明，商业可用的技术可以进一步修改和改进，以更接近化学定义和可扩展的 HSPC 分化协议。