National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark.
Department for Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
Stem Cells Dev. 2021 Apr 1;30(7):374-385. doi: 10.1089/scd.2020.0184.
The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers [platelet-derived growth factor receptor, alpha polypeptide (PDGFR-α), fetal liver kinase 1 (FLK1), ISL1, NK2 homeobox 5 (NKX2.5), cardiac troponin T (CTNT), connexin43 (CX43), and myosin heavy chain 7 (MYHC-B)] in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22, and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and hiPSC, with an earlier onset in the hiPSC compared with reported murine expression. Markers of immature cardiomyocytes, CTNT, and MYHC-B were consistently expressed throughout E16-E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, while this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm and ISL1/NKX2.5 for cardiac progenitors. Furthermore, a combination of two or more of the following, CTNT/MYHC-B/ISL1 could mark immature cardiomyocytes and CTNT/ISL1 mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo-like cardiac tissue in the future.
多能干细胞向心肌细胞和中间细胞类型的分化过程，可使用不同发育阶段的分子标志物来描述。这些标志物主要来自于对小鼠的研究，以及数量有限的人类研究。然而，在分子水平上，尚不清楚小鼠的心脏发生与人类的心脏发生有多大程度的相似。我们通过分析和比较发育中的猪心在胚胎期（E）15、E16、E18、E20、E22 和 E24 以及从人类诱导多能干细胞（hiPSC）分化的心肌细胞中常见的心肌发生标志物[血小板衍生生长因子受体，α多肽（PDGFR-α）、胎肝激酶 1（FLK1）、ISL1、NK2 同源盒 5（NKX2.5）、心肌肌钙蛋白 T（CTNT）、连接蛋白 43（CX43）和肌球蛋白重链 7（MYHC-B）]的表达情况，来解决这个问题。我们发现，猪的中胚层标志物 FLK1 和心脏祖细胞标志物 ISL1 的表达与我们分化的 hiPSC 和报道的鼠类表达一致。心脏谱系标志物 NKX2.5 在猪和 hiPSC 中的几乎所有阶段都有表达，与报道的鼠类表达相比，在 hiPSC 中的起始时间更早。不成熟的心肌细胞标志物 CTNT 和 MYHC-B 在猪中的表达贯穿 E16-E70，与鼠类发育相当，而在 hiPSC 中，这些标志物随时间增加。然而，常用的成熟心肌细胞标志物 CX43 的使用应谨慎，因为它也在猪的中胚层以及 hiPSC 的不成熟心肌细胞中表达，而这在鼠类中尚未报道。基于我们在不同物种中的观察，我们建议使用 FLK1/PDGFR-α 来鉴定心脏中胚层，使用 ISL1/NKX2.5 来鉴定心脏祖细胞。此外，以下两种或两种以上标志物的组合，如 CTNT/MYHC-B/ISL1 可标记不成熟的心肌细胞，而 CTNT/ISL1 可标记成熟的心肌细胞。CX43 应与肌节蛋白一起使用。这些知识可能有助于未来将 hiPSC 分化为更类似于体内的心脏组织。
