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.

摘要无半径血小板减少症(TAR)是一种罕见的先天性疾病，它会导致半径缺乏。 半径、成熟巨核细胞(MK)数量减少和血小板减少。焦油是由 RBMBA基因突变导致RBMBA的mRNA表达及其编码水平降低 患者血小板中的蛋白质，Y14。由于Y14在MK生物学中没有已知的作用，目前还不清楚 这种蛋白的缺乏如何在不影响其他造血系的情况下促进MK表型。 之前对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，但它是 不清楚这是否是负责的机制。无论是出生后出现的血小板减少症 TAR和来源于原始或最终祖细胞的MK的已知差异表明 明确的MK可能表现出更严重的表型，因此是一个更有洞察力的模型。通过对此进行建模 使用患者来源的诱导多能干细胞(IPSCs)和等基因校正系在体外治疗疾病，我们 可以评估TAR对纯细胞群体的影响，以观察特定的谱系和发育阶段 不受调节血细胞生成的代偿反馈机制影响的变化 活着。总体而言，我们假设TAR综合征中的Y14缺失更多地损害了确定的MK的成熟。 通过改变细胞周期和细胞凋亡的调控，而不影响原始的MKs 其他血统的发展。本提案的目标1将确定MK的具体特征 这是由于Y14在原始和最终分化过程中的枯竭而改变的。目标1A将评估各方面 以确定特定的MK表型，目标1 B将确定是否 Y14的减少改变了MKs的细胞凋亡和细胞周期进程，这是该表型的一个潜在机制。 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识别任何新的。 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后果。目标 2a将辨别造血系是否以不同方式调节Y14 RNA或蛋白质水平。AIM 28将 使用细胞增殖和谱系特异性表面标记表达来检测红系或红系的潜在缺陷 髓系发育。我们还将确定这些细胞的细胞周期和凋亡调控是否发生了变化 目标1B中确定的其他血统以及任何其他途径。这将是第一个研究 直接比较原始期和终止期细胞周期、凋亡和MK成熟的调节 并测试这些模型是否具有不同疾病表型的潜力。结果是 这项研究不仅将阐明MKs中TAR综合征的机制，而且它对MK生物学的洞察 不同的发展阶段将对改进当前的体外疾病模型具有重要意义 并针对不同的组织系统量身定做治疗方法，以减少人类疾病。