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

摘要 背景：自人类干细胞首次成功分化为心肌细胞以来，干细胞 在心脏再生领域有希望。这些细胞提供了不确定的从头开始的来源 用于人类移植的心肌细胞和潜在的无限组织。然而，哺乳动物的心脏是一个复杂的 管风琴，由四个腔室、一个高度协调的导电系统和一个不同种类的混合物组成 细胞类型，这可能与体外产生的分化组织不匹配。尽管努力将重点放在 提高心肌细胞分化产率，在分化功能方面取得的进展相对较少。 特定的心肌细胞，如起搏细胞。结节起搏细胞的分化将提供一种新的 心脏生物起搏的细胞来源。最近，维甲酸(RA)信号已被证明在一种 在心房和起搏器细胞的分化中起关键作用，模仿在天然起搏器中发现的细胞。 我假设起搏细胞与RA的特异性分化将提供更好的生物学特性 体外和体内的起搏功能与传统的、异种干细胞来源的比较 人口。这项拟议的工作旨在全面描述基因图谱、功能性电生理 体外和体内的RA来源的心肌细胞的特性和生物起搏潜力。 方法：为了获得起搏器，人诱导多能干细胞(HiPSC)将分化为 在14天的方案中使用和不使用Ra的单层。我们的初步数据表明，Ra治疗 丰富了天然起搏细胞的分子和遗传表达谱。这项工作的目标1寻求 将我们的Ra分化方案应用于6个不同的来自脐带血的HiPSC系(一半男性/一半女性) 脐带血。因此，我们可以检查这种方法在儿科患者中的适用性。在目标2中，HiPSC- 衍生的细胞将聚集成球体，即起搏单位。这些起搏单元的大小将优化为 最大的自发搏动和最小的细胞死亡。这些球体的起搏器功能将通过 我们的体外植入模型。在目标3中，优化的起搏单元将被植入大鼠的心室肌 在活体内记录由HiPSC起搏单位引起的自发搏动。主要读数为：1)RT-qPCR 基因表达分析，ii)来自膜片钳的单细胞内电位记录，iii)宏观尺度， 场电位的多电极阵列测量，IV)单分子膜和EX的高分辨率光学标绘 使用电压敏感染料的活体完整心脏，v)24/7 24/7遥测在体心电生物电势记录，以及vi) 超声心动图测量。该项目的成功完成将导致首次深入研究 Ra来源的心肌细胞的生物起搏潜力，在优化分化、动态剂量方面 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 来自埃默里大学和佐治亚理工学院的Hee Cheol Cho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.