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

项目摘要 人诱导多能干细胞衍生的心肌细胞（iPSC-CM）现在被广泛用于显示心肌细胞的功能。 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，可以更可靠地- 完全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， 是对可以获得成熟iPSC-CM的方法的迫切需要，所述成熟iPSC-CM可以准确地模拟成人心脏病。 容易表型，但目前的努力受到缺乏分子标记，可以作为表面， 对iPSC-CM功能成熟度进行全面的读数。 因此，目前F32奖学金提案的目标是确定可以重新定位的蛋白质标记物。 反映人iPSC-CM的体外功能成熟状态。iPSC-CM逐渐获得功能- 在长期培养后具有完全成熟的特征。我们最近发现了190个膜- 在iPSC-CM中，蛋白质编码基因在转录水平上被显著诱导， （30 - 90天）体外培养。在这里，我将测试的假设，这些长期培养的一个子集， 签名（PCS）代表人类心肌细胞的真正成熟标志物，因此可能是有害的。 用于分离功能成熟的iPSC-CM。为了实现这一目标，我提出了两个具体目标： 我将使用高分辨率质谱仪来确定在培养物中富集的基因， 成年心脏在蛋白质水平，并有可能区分和分离功能成熟的亚， 人口。在目标2中，我将在单细胞水平上验证候选标志物的蛋白质表达， 进一步评估使用蛋白质标记物分离的iPSC-CM的功能特征，以比较 所收购iPSC-CM相对于现行标准的功能成熟度和同质性。 所提出的实验的预期回报将是对以下分子的更好的理解： 培养中iPSC-CM功能成熟，这可能导致分离更成熟iPSC-CM群体的方法。 可以用于疾病建模研究的模型。这些目标在我看来意义重大，因为 它们有可能极大地改善当前iPSC-CM的应用，并为开发 工程方法以进一步增强iPSC-CM生产。同时，建议研究 培训计划还将提供有价值的干细胞生物学培训机会（与申办者Joseph博士 吴）和单细胞分析（与共同赞助商加里诺兰博士），这将补充我现有的实验， 我在蛋白质组学方面很有天赋，并帮助我实现了建立一个独立的心血管研究小组的未来目标， 最大的药

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.