Background and Objectives Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. Methods and Results We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring- mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. Conclusions To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

中文翻译：

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异种物质对人诱导多能干细胞来源的心肌细胞的聚糖谱和电生理特性的影响。

背景和目的人诱导多能干细胞 (hiPSC) 衍生的心肌细胞 (CM) 作为 CM 的细胞来源，有望用于缺血性心脏病心脏功能的恢复。然而，在 hiPSC-CM 的生物制造过程中使用动物源性异种物质可能会诱导无意的免疫反应或慢性炎症，进而导致致瘤性。在本研究中，我们旨在揭示异种物质在分化过程中对 hiPSC-CM 功能特性和潜在免疫原性的影响，证明基于 hiPSC 的细胞疗法的质量和安全性。方法和结果 我们在存在和不存在异种物质（分别为含异种物质 (XC) 和无异种物质 (XF) 条件）的情况下成功生成了 hiPSC-CM，并比较了它们的特性，包括收缩功能和聚糖谱。通过 MEA 和 RT-qPCR 检测，与 XC-hiPSC-CM 相比，XF-hiPSC-CM 显示心肌细胞收缩性搏动和成熟开始较早，并且心脏谱系特异性基因（ACTC1、TNNT2 和 RYR2）高表达。我们使用间接酶联免疫吸附测定和液相色谱-多反应监测-质谱法对 hiPSC-CM 中的 N-乙醇酰神经氨酸 (Neu5Gc)（一种异种唾液酸）进行了定量。Neu5Gc 在含有异种的分化过程中被整合到 hiPSC-CM 的聚糖中，而在 XF-hiPSC-CM 中几乎检测不到。结论 据我们所知，这是第一项表明 hiPSC-CM 的电生理功能和聚糖谱在分化和成熟过程中可能受到异种物质存在影响的研究。为了确保基于 hiPSC 的细胞疗法的质量控制和安全性，异种物质应排除在生物制造过程之外。