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.

桡骨缺失性血小板减少症（TAR）综合征是一种罕见的先天性疾病，导致桡骨缺失