Identifying Markers of Induced Pluripotent Stem Cell-Derived Cardiomyocyte (iPSC-CM) Maturity

鉴定诱导多能干细胞来源的心肌细胞 (iPSC-CM) 成熟的标志物

• 批准号: 9555819
• 负责人: Edward Lau
• 金额: $ 3.3万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-28 至 2019-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9555819
• 关键词: Adrenergic Agents; Adult; Advisory Committees; Analytical Chemistry; Birds; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Line; Cell Size; Cell Surface Proteins; Cell surface; Cells; Characteristics; Complement; Data; Data Science; Derivation procedure; Development; Disease; Disease model; Drug Screening; Drug Targeting; Engineering; Fellowship; Flow Cytometry; Funding; Future; Genes; Goals; Gold; Heart; Heart Diseases; Human; In Vitro; K-Series Research Career Programs; Laboratories; Lead; Maps; Mass Spectrum Analysis; Measurement; Medicine; Membrane Proteins; Methods; Modeling; Molecular; Muscle Cells; Natriuretic Peptides; Patients; Peptide Receptor; Pharmaceutical Preparations; Phenotype; Production; Proteins; Proteomics; Protocols documentation; Public Health; Regenerative Medicine; Reproducibility; Research; Research Training; Resolution; Resources; Study models; Techniques; Testing; Time; Tissues; Training; Transcript; United States National Institutes of Health; Universities; base; candidate marker; combat; disease phenotype; experimental study; genetic signature; improved; individual response; induced pluripotent stem cell; molecular marker; protein biomarkers; protein expression; response; single cell analysis; stem cell biology; stem cell differentiation; tandem mass spectrometry; training opportunity; transcriptome sequencing; treatment response

PROJECT SUMMARY Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are now widely employed to dis- cover the mechanisms of heart diseases and identify potential drug targets. A challenge for current iPSC- CM applications, however, is how to identify and promote functionally mature myocytes that can more faith- fully recapitulate human adult cardiomyocyte characteristics. To propel the next stage of discoveries, there is a critical need for methods that can derive mature iPSC-CM that can accurately model adult heart dis- ease phenotypes, but current efforts are hampered by a dearth of molecular markers that can serve as sur- rogate readouts of iPSC-CM functional maturity. Accordingly, the goal of the present F32 fellowship proposal is identify protein markers that can re- flect the status of in vitro functional maturation of human iPSC-CMs. iPSC-CMs gradually acquire functional- ly mature characteristics following prolonged periods in culture. We recently discovered 190 membrane- protein-encoding genes that are significantly induced at the transcript level in iPSC-CMs after prolonged (30-90 days) of culturing in vitro. Here I will test the hypothesize that a subset of these prolonged culture signatures (PCS) represent bona fide maturity markers of human cardiomyocytes and thus may be har- nessed to isolate functionally mature iPSC-CMs. To achieve this goal, I propose two specific aims: In Aim 1 I will employ high-resolution mass spectrometry to determine genes which are enriched in culture and in adult hearts at the protein-level, and which can potentially distinguish and isolate functionally mature sub- populations. In Aim 2 I will verify protein expression of the candidate markers at the single-cell level, and further evaluate the functional characteristics of iPSC-CMs isolated using protein markers, to compare the functional maturity and homogeneity of the acquired iPSC-CMs against current standards. The anticipated payoff of the proposed experiments will be an improved molecular understanding of iPSC-CM functional maturity in culture, which may lead to methods to isolate more mature iPSC-CM popu- lations that can be used for disease modeling studies. These goals are significant in my opinion because they have the potential to greatly improve current iPSC-CMs applications and open doors to development of engineering approaches to further enhance iPSC-CM production. At the same time, the proposed research training plan will also provide valuable training opportunities in stem cell biology (with Sponsor Dr. Joseph Wu) and single-cell analysis (with Co-Sponsor Dr. Garry Nolan), which will complement my existing exper- tise in proteomics and aid me in my future goal of setting up an independent research group in cardiovascu- lar medicine.

项目总结 人诱导多能干细胞来源的心肌细胞(IPSC-CMS)目前被广泛用于分离培养心肌细胞。 涵盖心脏病的机制，并确定潜在的药物靶点。当前IPSC面临的挑战- 然而，CM的应用是如何识别和促进功能成熟的心肌细胞，这些细胞可以更多地相信- 全面概括了人类成年心肌细胞的特征。为了推动下一阶段的发现，那里 迫切需要能够衍生出成熟的IPSC-CM的方法，能够准确地模拟成人心脏疾病。 易表型，但目前的努力受到缺乏分子标记的阻碍，这些分子标记可以作为表型的基础。 罗盖特的IPSC-CM功能成熟度读数。 因此，目前的F32奖学金提案的目标是识别可以重新定位的蛋白质标记。 反映人IPSC-CMS体外功能成熟状况。IPSC-CMS逐渐获得功能性- 随着培养时间的延长，LY的成熟特征。我们最近发现了190个膜- IPSC-CMS延长后在转录水平显著诱导的蛋白质编码基因 (30-90天)体外培养。在这里，我将测试这样一种假设，即这些长期文化的子集 信号(PCs)代表了人心肌细胞真正的成熟标志，因此可能是人心肌细胞成熟的标志 目的是分离功能成熟的IPSC-CMS。为了实现这一目标，我提出两个具体目标：目标1 我将使用高分辨率质谱仪来确定哪些基因在培养中和在 成人心脏在蛋白质水平上，并且可能区分和分离功能成熟的亚细胞 人口。在目标2中，我将在单细胞水平上验证候选标记的蛋白质表达，以及 用蛋白质标记进一步评价分离的IPSC-CMS的功能特性，比较 收购的IPSC-CMS相对于当前标准的功能成熟度和同质性。 拟议中的实验的预期回报将是提高对 IPSC-CM在培养中的功能成熟度，这可能导致分离更成熟的IPSC-CM细胞的方法。 可用于疾病模型研究的激光。在我看来，这些目标意义重大，因为 它们有可能极大地改进当前的IPSC-CMS应用，并为开发 进一步提高IPSC-CM生产的工程方法。同时，提出了研究建议 培训计划还将提供宝贵的干细胞生物学培训机会(由约瑟夫博士赞助 和单细胞分析(与加里·诺兰博士共同赞助)，这将补充我现有的经验- 致力于蛋白质组学，并帮助我实现未来在心血管疾病上建立一个独立研究小组的目标- 拉尔医学。

项目成果

NextGen VOICES: Research resolutions.

下一代声音：研究决议。

• DOI: 10.1126/science.aar7504
• 发表时间: 2018
• 期刊: Science (New York, N.Y.)
• 作者: Olmeta-Schult,Felicia;Segal,LaurenMassa;Tyner,Sam;Moon,TwilaAlexandra;Chow,RyanDz-Wei;Chakrabarty,Prosanta;Pacesa,Martin;Podgornaia,AnnaIgorevna;Chen,Jennifer;Singh,Bipin;Cao,Bo;Sidhu,RishiRajSingh;Tan,BryceWQ;Sood,Prash
• 通讯作者: Sood,Prash