Electrophysiological characterization of human pluripotent stem cell derived cardiomyocytes and their application as biological pacemakers

摘要 背景：从人类干细胞第一次成功分化为心肌细胞开始，干细胞就具有 在心脏再生领域很有希望这些细胞提供了一个不确定的来源， 心肌细胞和潜在的无限组织用于人类移植。然而哺乳动物的心脏是一个复杂的 器官，由四个腔室，高度协调的电传导系统和异质混合物组成 细胞类型，这可能不匹配体外产生的分化组织。尽管努力集中在 增加心肌细胞分化产量，在分化功能方面取得的进展相对较小， 特定的心肌细胞，如起搏细胞。淋巴结起搏细胞的分化将提供一种新的 用于心脏生物起搏的细胞源。最近，视黄酸（Ra）信号已被证明在细胞凋亡中起作用。 在心房和起搏器细胞的分化中起关键作用，模仿天然起搏器中发现的细胞。 我推测，具有Ra的起搏细胞的特异性分化将提供上级生物学特性。 与传统的、异质性干细胞衍生的 人口。拟议的工作旨在充分表征遗传概况，功能电生理 性质和生物起搏器的潜力，在体外和体内，Ra衍生的心肌细胞。 方法：为了获得起搏器，人类诱导多能干细胞（hiPSC）将分化为 单层在14天的协议与和没有Ra。我们的初步数据表明Ra治疗 使分子和基因表达谱丰富到天然起搏细胞的水平。本工作的目标1是 将我们的Ra分化方案应用于来源于脐带血的6个不同hiPSC系（一半雄性/一半雌性）， 脐带血因此，允许我们检查这种方法对儿科患者的适用性。在目标2中，hiPSC- 衍生的细胞将聚集成球状体、起搏单元。这些起搏装置的尺寸将进行优化， 最大程度的自发搏动和最小程度的细胞死亡。这些球体的起搏器功能将使用 我们的体外移植模型在目标3中，将优化的起搏单元植入大鼠心室肌 以记录由hiPSC起搏单元诱导的自发搏动。主要读数是i）RT-qPCR 基因表达分析，ii）来自膜片钳的单细胞细胞内电位记录，iii）宏观尺度， 场电位的多电极阵列测量，iv）单分子层的高分辨率光学映射和 具有电压敏感染料的体内全心脏，v）体内ECG的24/7遥测生物电势记录，和vi） 超声心动图测量。这一项目的成功完成将导致第一次深入研究 Ra衍生的心肌细胞的生物起搏电位，根据优化的分化，动态剂量 范围、安全范围和体内治疗效果。拟议的工作将在监督下进行 来自埃默里大学和格鲁吉亚理工学院的赵熙哲博士。

ABSTRACT Background: From the first successful differentiation of human stem cells to cardiomyocytes, stem cells have held promise in the field of cardiac regeneration. These cells provide an indefinite source of de novo cardiomyocytes and potentially unlimited tissue for human transplantation. Yet the mammalian heart is a complex organ, comprised of four chambers, a highly coordinated electrical conduction system, and a heterogeneous mix of cell types, which may not match the differentiated tissues produced in vitro. Despite efforts focused on increasing cardiomyocyte differentiation yield, relatively little progress has been made in differentiating function- specific cardiomyocytes, such as pacemaker cells. Differentiation of nodal pacemaker cells would provide a new cell source for biological pacing of the heart. Recently, retinoic acid (Ra) signaling has been shown to play a critical role in the differentiation of atrial and pacemaker cells, mimicking the cells found in the native pacemaker. I hypothesize that specific differentiation of pacemaker cells with Ra would provide superior biological pacemaking function both in vitro and in vivo, compared to conventional, heterogeneous stem cell-derived populations. The proposed work seeks to fully characterize the genetic profile, functional electrophysiological properties, and biological pacemaker potential, both in vitro and in vivo, of Ra-derived cardiomyocytes. Approach: To derive pacemakers, human induced pluripotent stem cells (hiPSC) will be differentiated as monolayers over a 14-day protocol with and without Ra. Our preliminary data indicates that Ra treatment enriches molecular and genetic expression profiles to that of native pacemaker cells. Aim 1 of this work seeks apply our Ra differentiation protocol on 6 different hiPSC lines (half male / half female) derived from umbilical cord blood. Thus, allowing us to examine the applicability of this method for pediatric patients. In Aim 2, hiPSC- derived cells will be aggregated into spheroids, pacing units. The size of these pacing units will be optimized for maximum spontaneous beating with minimal cell death. Pacemaker function of these spheres will be tested with our in vitro engraftment model. In Aim 3, optimized pacing units will be engrafted to rat ventricular myocardium in vivo to record spontaneous beating induced by the hiPSC-pacing units. The major readouts are i) RT-qPCR analysis of gene expression, ii) single-cell intracellular potential recordings from patch-clamp, iii) macro-scale, multi-electrode array measurements of field potentials, iv) high-resolution optical mapping of monolayers and ex vivo whole hearts with a voltage-sensitive dye, v) 24/7 telemetry biopotential recordings of ECG in vivo, and vi) echocardiographic measurements. Successful completion of this project will lead to the first in-depth study of the biological pacemaker potential of Ra-derived cardiomyocytes, in terms of optimized differentiation, dynamic dose range, safety margin, and therapeutic effect in vivo. The proposed work will be conducted under the supervision of Dr. Hee Cheol Cho at Emory University & Georgia Tech.