Role of pH on Lineage Fate during Pluripotent Stem Cell Differentiation

pH 对多能干细胞分化过程中谱系命运的作用

• 批准号: 10158520
• 负责人: Vivian Lu
• 金额: $ 3.78万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2022-05-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158520
• 关键词: 3-Dimensional; Adult; Affect; Applications Grants; Automobile Driving; Carbonic Acid; Cell Lineage; Cell Therapy; Cells; Chromatin Structure; Clinical; Culture Media; Data; Development; Disease; Disease model; Drug Screening; Ectoderm; Ectoderm Cell; Embryo; Embryonic Development; Endoderm; Endoderm Cell; Epigenetic Process; Epithelial; Event; Exhibits; Feasibility Studies; G-Protein-Coupled Receptors; GPR4 gene; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic; Germ; Germ Layers; Glycolysis; Goals; Growth Factor; Health; Histidine; Histones; Human Development; Hypoxia; In Vitro; Ions; Lactate Dehydrogenase; Lactic acid; Lead; Link; Malignant Neoplasms; Mediating; Mesenchymal; Mesoderm; Mesoderm Cell; Metabolic; Metabolism; Methodology; Modeling; Oxidative Phosphorylation; Pathogenicity; Pathway interactions; Phenotype; Physiological; Pluripotent Stem Cells; Process; Protein Isoforms; Protocols documentation; Protons; Regulatory Pathway; Residual state; Role; Signal Transduction; Somatic Cell; Stimulus; System; Testing; Training; Vascular Endothelial Growth Factors; cancer cell; cell type; chemokine; cost; cytokine; differentiation protocol; embryonic stem cell; epigenetic memory; epigenetic regulation; epigenome; epithelial to mesenchymal transition; extracellular; human embryonic stem cell; human pluripotent stem cell; improved; in vitro Model; in vivo; induced pluripotent stem cell; novel strategies; pluripotency; process optimization; progenitor; programs; protonation; reconstitution; sensor; stem; stem cell differentiation; success; transcriptomics

ABSTRACT Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), complete a tightly controlled sequence of events during differentiation to recapitulate functional cells from our bodies. This in vitro maturation process initiates by induction of mesoderm, ectoderm, and endoderm lineage progenitors, and provides unique opportunities for disease modeling, drug screening, cell- based therapies, and uncovering mechanisms in cell development. Yet, key obstacles remain and include (1) inefficient PSC differentiation, (2) costly, labor-intensive protocols that promote genetic aberrations, and (3) developmental immaturity and reduced functionality of terminal cells. Mechanistically, failed erasure of epigenetic memory leaves residual marks that affects chromatin structure and gene expression from a hiPSC's somatic cell type of origin. Thus, it is imperative to understand and implement optimized in vitro hPSC differentiation protocols that reconstitute gene regulation and epigenome configuration programs matching in vivo cell counterparts to achieve superior cell types for all applications that employ this remarkable system. The role of cytokines, growth factors, and chemokines in driving hPSC differentiation is firmly established in cell fate determination. Additionally, the facilitating role of metabolic flux and metabolite levels in reconfiguring the epigenome to improve hPSC differentiation has recently been uncovered. Yet, a factor generated by metabolic activity—acidification—has not been examined as a microenvironment stimulus controlling hPSC differentiation despite its known roles in driving de- and re-differentiation during pathogenic and physiological development. We recently discovered both differential metabolic programs and extracellular acidification rates during ectoderm and mesoderm lineage differentiation. This confirms a direct connection between metabolism and acidification, and further suggests a role for lowered pH in early lineage fate acquisition. The overall study goal here is to uncover pH-dependent mechanisms in lineage-specific cell fate, and to ultimately exploit these processes for optimized hPSC differentiation. To test the hypothesis that pH-sensitive mechanisms control early cell fate, we will embark on 3 specific aims: (1) To determine the importance of pH in lineage partitioning under spontaneous PSC differentiation in an embryoid body model. (2) To examine remodeling of epigenome configuration and resulting lineage-specific gene expression to identify pH-sensitive regulators governing early differentiation under low pH using transcriptomic and epigenetic screens. And (3) to study the feasibility of pH modulation to enrich for functional mesoderm derivatives compared to current hPSC differentiation methodologies. Success in these studies will open new pathways through pH manipulations for generating superior in vitro models of early human development for numerous promising applications in health and disease.

抽象的 人类多能干细胞（hPSC），包括胚胎干细胞（hESC）和诱导多能干细胞 细胞（hiPSC），在分化过程中完成一系列严格控制的事件序列，以重现功能 这种体外成熟过程是通过诱导中胚层、外胚层和 内胚层谱系祖细胞，并为疾病建模、药物筛选、细胞- 然而，关键的障碍仍然存在，包括 (1) PSC 分化效率低下，(2) 成本高昂、劳动密集型的方案会促进遗传畸变，以及 (3) 从机制上讲，终末细胞的发育不成熟和功能降低，表观遗传的消除失败。 记忆留下的残留标记会影响 hiPSC 体细胞的染色质结构和基因表达 因此，必须了解并实施优化的体外 hPSC 分化方案。 重建与体内细胞对相匹配的基因调控和表观基因组配置程序 为采用这一卓越系统的所有应用实现卓越的细胞类型。 细胞因子、生长因子和趋化因子在驱动 hPSC 分化中的作用在细胞中已得到牢固确立 此外，代谢通量和代谢水平在重构中的促进作用。 最近发现了一个由代谢产生的因素来改善 hPSC 分化。 活性——酸化——尚未被检查为控制 hPSC 分化的微环境刺激 尽管它在致病和生理发育过程中驱动去分化和再分化方面发挥着已知的作用。 我们最近发现了外胚层期间的差异代谢程序和细胞外酸化率 和中胚层谱系分化，这证实了新陈代谢和酸化之间的直接联系， 并进一步表明降低 pH 在早期谱系命运获得中的作用。 揭示谱系特异性细胞命运中 pH 依赖性机制，并最终利用这些过程 为了验证 pH 敏感机制控制早期细胞命运的假设，我们进行了优化 hPSC 分化。 将着手实现 3 个具体目标：(1) 确定 pH 在自发谱系分配中的重要性 (2) 检查表观基因组构型的重塑和 由此产生的谱系特异性基因表达来识别控制早期分化的 pH 敏感调节因子 (3) 研究 pH 调节的可行性。 与目前的 hPSC 分化方法相比，丰富了功能性中胚层衍生物。 这些研究将通过 pH 值操纵开辟新的途径，以生成早期早期的卓越体外模型。 人类发展在健康和疾病方面有许多有前景的应用。