Investigating thrombocytopenia absent radius syndrome during primitive and definitive hematopoiesis using an induced pluripotent stem cell model

使用诱导多能干细胞模型研究原始造血和确定性造血过程中无桡骨综合征的血小板减少症

• 批准号: 10802107
• 负责人: Catriana Nations
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10802107
• 关键词: Ablation; Affect; Agonist; Alleles; Apoptosis; Apoptotic; Axon; Biological Assay; Biology; Blood; Blood Cells; Blood Platelets; Blood specimen; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell Line; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Cessation of life; Characteristics; Complex; Congenital Disorders; Defect; Development; Disease model; Embryo; Erythroid; Etiology; Exhibits; Feedback; Flow Cytometry; Generations; Genes; Genetic; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Impairment; In Vitro; Knockout Mice; Life; Megakaryocytes; Megakaryocytopoieses; Messenger RNA; Mitotic; Modeling; Mutation; Myelogenous; Organism; Pathogenesis; Pathway interactions; Patients; Pattern; Penetrance; Phenotype; Platelet Count measurement; Ploidies; Polyploid Cells; Population; Predisposition; Process; Proliferating; Proteins; RNA; RNA Processing; RNA Splicing; Radial; Regulation; Role; Sampling; Single Nucleotide Polymorphism; Specificity; Surface; Syndrome; System; TP53 gene; Testing; Therapeutic; Thrombocytopenia; Tissue-Specific Gene Expression; Tissues; Umbilical Cord Blood; Untranslated RNA; Validation; Yolk Sac; cell type; disease phenotype; experimental study; gene correction; human disease; improved; in vitro Model; in vivo; induced pluripotent stem cell; insight; mRNA Expression; mouse model; novel; postnatal; premature; progenitor; programs; promoter; response; skeletal; small hairpin RNA; stem cell model; stem cells; transcriptome sequencing

Thrombocytopenia absent radius (TAR) syndrome is a rare congenital disorder that causes absence of the radii, reduced numbers of mature megakaryocytes (MKs), and thrombocytopenia. TAR is caused by mutations in the RBMBA gene, resulting in reduced mRNA expression of RBMBA and levels of its encoded protein, Y14, in patient platelets. Since Y14 has no known roles in MK biology, it is currently not understood how deficiencies in this protein contributes to a MK phenotype without affecting other hematopoietic lineages. Previous studies of Y14 depletion have identified a role for Y14 in apoptosis and cell cycle regulation, but it is unclear whether this is the mechanism responsible. Both the postnatal emergence of the thrombocytopenia in TAR and the known differences in MKs derived from primitive or definitive progenitor cells suggest that definitive MKs may present a more severe phenotype and thus be a more insightful model. By modeling this disease in vitro using patient-derived induced pluripotent stem cells (iPSCs) and isogenic corrected lines, we can assess the effects of TAR on pure cell populations to observe lineage- and developmental stage-specific changes without influence from the compensatory feedback mechanisms that regulate blood cell generation in vivo. Overall, we hypothesize that Y14 depletion in TAR syndrome impairs maturation of definitive MKs more severely than primitive MKs through altered cell cycle and apoptosis regulation, and it does not affect the development of other blood lineages. Aim 1 of this proposal will determine the specific characteristics of MKs that is altered due to Y14 depletion during primitive and definitive differentiation. Aim 1A will evaluate aspects of MK maturation and functionality to determine the specific MK phenotype, and Aim 1 B will determine if reduced Y14 alters apoptosis and cell cycle progression in MKs as a potential mechanism for this phenotype. Using RNA-seq, we will detect differential expression of genes related to these pathways or identify any novel targets with the potential to contribute to the MK defect. Aim 2 will address the MK specificity of TAR by comparing consequences of Y14 depletion in MK differentiation to erythroid and myeloid differentiations. Aim 2A will discern whether the hematopoietic lineages regulate Y14 RNA or protein levels differently. Aim 28 will use cell proliferation and lineage-specific surface marker expression to detect potential defects in erythroid or myeloid development. We will also determine whether cell cycle and apoptosis regulation are altered in these other lineages as well as any additional pathways that are identified in Aim 1 B. This will be the first study to directly compare the regulation of cell cycle, apoptosis, and MK maturation during primitive and definitive hematopoiesis and test whether these models have the potential for divergent disease phenotypes. The results of this study will not only elucidate the mechanism of TAR syndrome in MKs, but its insight into MK biology at different stages of development will have important implications for improving current in vitro disease models and tailoring therapeutics to distinct tissue systems to reduce human disease.

血小板减少性桡骨缺如（TAR）综合征是一种罕见的先天性疾病， 半径、成熟巨核细胞（MK）数量减少和血小板减少。TAR是由以下原因引起的： RBMBA基因突变，导致RBMBA的mRNA表达降低及其编码的 患者血小板中的Y14蛋白。由于Y14在MK生物学中没有已知的作用，因此目前尚不清楚 这种蛋白质的缺陷如何导致MK表型而不影响其他造血谱系。 以前关于Y14缺失的研究已经确定了Y14在细胞凋亡和细胞周期调节中的作用，但它在细胞凋亡和细胞周期调节中的作用并不明显。 不清楚这是否是负责的机制。出生后出现的血小板减少症， TAR和来源于原始或定形祖细胞的MK的已知差异表明， 永久性MK可能呈现更严重的表型，因此是更有洞察力的模型。通过模拟这个 使用患者来源的诱导多能干细胞（iPSC）和等基因校正系体外治疗疾病，我们 可以评估TAR对纯细胞群的影响，以观察谱系和发育阶段特异性 不受调节血细胞生成的补偿反馈机制影响的变化， vivo.总的来说，我们假设TAR综合征中Y14的缺失损害了永久性MK的成熟， 通过改变细胞周期和凋亡调节，它比原始MK严重，并且不影响细胞周期。 其他血系的发展。本提案的目标1将确定MK的具体特性 其在原始和最终分化期间由于Y14耗尽而改变。目标1A将评估各个方面 MK成熟和功能性，以确定特定的MK表型，目标1 B将确定是否 减少的Y14改变MK中的细胞凋亡和细胞周期进程，作为该表型的潜在机制。 使用RNA-seq，我们将检测与这些途径相关的基因的差异表达，或鉴定任何新的 有可能导致MK缺陷的目标。目标2将通过以下方式解决TAR的MK特异性： 比较MK分化中Y14缺失与红系和髓系分化的结果。目的 图2A将辨别造血谱系是否不同地调节Y14 RNA或蛋白质水平。目标28将 使用细胞增殖和谱系特异性表面标志物表达来检测红系细胞中的潜在缺陷，或 骨髓发育我们还将确定在这些细胞中细胞周期和凋亡调节是否改变， 其他谱系以及目标1 B中鉴定的任何其他途径。这将是第一项研究， 直接比较原始和永久性细胞周期、凋亡和MK成熟的调节， 造血并测试这些模型是否具有不同疾病表型的潜力。结果 这项研究的结果不仅将阐明MK中TAR综合征的机制，而且将有助于深入了解MK生物学， 不同的发展阶段将对改善目前的体外疾病模型具有重要意义 以及针对不同的组织系统定制治疗剂以减少人类疾病。