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.

桡骨缺失血小板减少症 (TAR) 综合征是一种罕见的先天性疾病，会导致桡骨缺失 半径、成熟巨核细胞（MK）数量减少和血小板减少。 TAR 的原因是 RBMBA 基因突变，导致 RBMBA 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，目前尚不清楚 该蛋白的缺陷如何在不影响其他造血谱系的情况下导致 MK 表型。 先前对 Y14 耗竭的研究已经确定了 Y14 在细胞凋亡和细胞周期调节中的作用，但它是 尚不清楚这是否是负责的机制。产后出现血小板减少症 TAR 和源自原始或定形祖细胞的 MK 的已知差异表明 明确的 MK 可能呈现出更严重的表型，因此是一个更有洞察力的模型。通过建模这个 使用患者来源的诱导多能干细胞 (iPSC) 和等基因校正细胞系在体外研究疾病，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 变化不受调节血细胞生成的补偿反馈机制的影响 体内。总体而言，我们假设 TAR 综合征中 Y14 缺失更会损害最终 MK 的成熟 通过改变细胞周期和凋亡调节，比原始 MK 更严重，并且它不影响 其他血统的发展。该提案的目标 1 将确定 MK 的具体特征 这是由于原始分化和最终分化过程中 Y14 耗尽而改变的。目标 1A 将评估各个方面 MK 成熟和功能以确定特定 MK 表型，Aim 1 B 将确定是否 Y14 减少会改变 MK 中的细胞凋亡和细胞周期进程，这是这种表型的潜在机制。 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识别任何新的 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 分化为红细胞和骨髓细胞分化过程中 Y14 缺失的后果。目的 图2A将辨别造血谱系是否以不同方式调节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记表达来检测红细胞或细胞中的潜在缺陷 骨髓发育。我们还将确定这些细胞周期和细胞凋亡调节是否发生改变 其他谱系以及目标 1 B 中确定的任何其他途径。这将是第一项研究 直接比较原始期和终期期细胞周期、细胞凋亡和 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 这项研究不仅将阐明 MK 中 TAR 综合征的机制，还将深入了解 MK 生物学 不同的发展阶段将对改善当前的体外疾病模型产生重要影响 并针对不同的组织系统定制治疗方法以减少人类疾病。