Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.
Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands.
J Mol Cell Cardiol. 2015 May;82:228-37. doi: 10.1016/j.yjmcc.2015.02.016. Epub 2015 Mar 25.
Cardiac progenitor cells (CPCs) have been isolated from adult and developing hearts using an anti-mouse Sca-1 antibody. However, the absence of a human Sca-1 homologue has hampered the clinical application of the CPCs. Therefore, we generated novel monoclonal antibodies (mAbs) specifically raised against surface markers expressed by resident human CPCs. Here, we explored the suitability of one of these mAbs, mAb C19, for the identification, isolation and characterization of CPCs from fetal heart tissue and differentiating cultures of human embryonic stem cells (hESCs).
METHODS & RESULTS: Using whole-cell immunization, mAbs were raised against Sca-1+ CPCs and screened for reactivity to various CPC lines by flow cytometry. mAb C19 was found to be specific for Sca-1+ CPCs, with high cell surface binding capabilities. mAb C19 stained small stem-like cells in cardiac tissue sections. Moreover, during differentiation of hESCs towards cardiomyocytes, a transient population of cells with mAb C19 reactivity was identified and isolated using magnetic-activated cell sorting. Their cell fate was tracked and found to improve cardiomyocyte purity from hESC-derived cultures. mAb C19+ CPCs, from both hESC differentiation and fetal heart tissues, were maintained and expanded in culture, while retaining their CPC-like characteristics and their ability to further differentiate into cardiomyocytes by stimulation with TGFβ1. Finally, gene expression profiling of these mAb C19+ CPCs suggested a highly angiogenic nature, which was further validated by cell-based angiogenesis assays.
mAb C19 is a new surface marker for the isolation of multipotent CPCs from both human heart tissues and differentiating hESCs.
已使用抗鼠 Sca-1 抗体从成体和发育中的心脏中分离出心脏祖细胞（CPCs）。然而，由于缺乏人类 Sca-1 同源物，这阻碍了 CPCs 的临床应用。因此，我们生成了专门针对常驻人类 CPCs 表面标志物的新型单克隆抗体（mAb）。在此，我们探讨了其中一种 mAb，mAb C19，用于鉴定、分离和表征胎儿心脏组织和人胚胎干细胞（hESC）分化培养物中的 CPCs 的适用性。
通过全细胞免疫，针对 Sca-1+CPCs 产生 mAb，并通过流式细胞术筛选其对各种 CPC 系的反应性。发现 mAb C19 特异性针对 Sca-1+CPCs，具有高细胞表面结合能力。mAb C19 可染色心脏组织切片中的小干细胞样细胞。此外，在 hESC 向心肌细胞分化过程中，通过磁激活细胞分选鉴定并分离出具有 mAb C19 反应性的短暂细胞群。跟踪它们的细胞命运，发现它们可以提高 hESC 衍生培养物中的心肌细胞纯度。来自 hESC 分化和胎儿心脏组织的 mAb C19+CPCs 在培养中得以维持和扩增，同时保持其 CPC 样特征及其在受到 TGFβ1 刺激后进一步分化为心肌细胞的能力。最后，这些 mAb C19+CPCs 的基因表达谱分析表明其具有高度的血管生成特性，通过基于细胞的血管生成测定进一步验证了这一点。
mAb C19 是一种从人类心脏组织和分化的 hESC 中分离多能性 CPCs 的新型表面标志物。
