Cell Surface Phenotyping Human Primary Cells

 心力衰竭是一种流行性综合征，影响570万美国人，其中虚弱的心脏无法向身体提供足够的血流量。这些患者中约有10%对药物治疗无效，并逐渐恶化，发展为晚期心力衰竭，唯一的确定性治疗是心脏移植。由于合适的供体心脏的供应限制在每年约2000个， 在美国，晚期心力衰竭患者的当前和未来护理需要替代治疗。对于一些患者，植入式泵装置形式的机械循环支持可以提供短期或长期支持，并且在1-2%的接受者中，心脏改善到可以移除泵的程度，称为心肌恢复。目前，无法预测哪些心力衰竭患者将对单独的药物治疗有反应，哪些将受益于器械支持，哪些没有恢复的潜力。对于器械接受者，量化心肌恢复以告知是否以及何时取出器械需要进行侵入性测试。因此，我们的长期目标是开发新的策略来识别具有恢复潜力的患者，并开发非侵入性方法来更精确地监测疾病进展和恢复。在短期内，我们的重点是开发新的细胞表面标志物来实现这些目标。我们先前对培养中的人类细胞的研究已经成功地应用质谱方法来捕获和鉴定细胞表面糖蛋白的胞外结构域，称为细胞表面捕获（CSC）。拟议的工作扩展了这些努力，并侧重于实施创新方法来改善CSC，以识别和定量来自活患者细胞的细胞表面蛋白， 对于许多人类疾病和综合征，如晚期心力衰竭，不可能在体外模拟疾病表型，并且获得原代细胞受到严重限制。目前的CSC需要1亿个细胞。本申请中概述的研究旨在开发“微型CSC（µCSC）”，以将所需的起始材料量减少10倍。本申请的具体目标是使用新的生物缀合试剂和创新的处理管道来开发µCSC，并扩大其范围以捕获具有更大序列覆盖率的更广泛类别的细胞表面蛋白，以改善定量（目标1）;在从多能干细胞体外分化心肌细胞期间以及在从人类心脏分离的细胞上应用µCSC（目标2）;并将µCSC应用于心肌恢复程度不同的患者的原代心脏细胞（目标3）。新的标记物的鉴定和用于鉴定具有不同发育表型的人心肌细胞的试剂的产生将使未来的机制研究能够确定适合修复的细胞的阶段和分子特征。一旦在来自临床样本的原代心脏细胞上得到验证，拟议研究所产生的技术进步将广泛适用于从干细胞生物学到癌症和患者诊断等广泛领域的研究人员。

 DESCRIPTION (provided by applicant): Heart failure is an epidemic syndrome affecting 5.7 million Americans in which the weakened heart is unable to supply sufficient blood flow to the body. Approximately 10% of these patients will fail to respond to medical therapy and progressively worsen to develop advanced heart failure, for which the only definitive therapy is cardiac transplantation. As the supply of suitable donor hearts is limited to ~2000 per year in the US, the current and future care of advanced heart failure patients requires therapeutic alternatives. For some patients, mechanical circulatory support in the form of an implantable pump device can provide short- or long-term support, and in 1-2% of recipients, the heart improves to the point where the pump can be removed, termed myocardial recovery. Currently, it is not possible to predict which heart failure patients will respond to medical therapy alone, which will benefit from device support, and which have no potential for recovery. For device recipients, quantifying myocardial recovery to inform if, and when, to explant the device requires invasive testing. Thus, our long range goals are to develop novel strategies to identify patients with potential for recovery and develop non-invasive methods to monitor disease progression and recovery more precisely. In the short-term, our focus is on the development of novel cell surface markers to address these goals. Our previous studies of human cells in culture have successfully applied a mass spectrometric approach to capture and identify extracellular domains of cell surface glycoproteins, termed Cell Surface Capture (CSC). The proposed work extends these efforts and focuses on the implementation of innovative approaches to improve the CSC to enable identification and quantitation of cell surface proteins from live patient cells, as for many human diseases and syndromes like advanced heart failure, it is not possible to model the disease phenotype in vitro, and access to primary cells is severely limited. The current CSC requires 100 million cells. The studies outlined in this application are designed to develop a "microscale-CSC (µCSC)" to reduce the amount of starting material required by 10-fold. The specific goals of this application are to use new bioconjugation reagents and an innovative processing pipeline to develop µCSC and to expand its scope to capture a broader class of cell surface proteins with greater sequence coverage for improved quantitation (Aim 1); apply µCSC during in vitro cardiomyocyte differentiation from pluripotent stem cells and on cells isolated from human hearts (Aim 2); and apply µCSC to primary heart cells from patients with varying degrees of myocardial recovery (Aim 3). The identification of new markers and generation of reagents for identifying human cardiomyocytes with distinct developmental phenotypes will enable future mechanistic studies to determine the stages and molecular characteristics of cells amenable to repair. Once validated on primary heart cells derived from clinical samples, the technological advances resulting from the proposed studies will be widely applicable to researchers across a wide range of fields, from stem cell biology to cancer and patient diagnostics.