Ireland Jake, Kilian Kristopher A
School of Chemistry, UNSW Sydney, Sydney, New South Wales, Australia.
School of Materials Science and Engineering, UNSW Sydney, Sydney, New South Wales, Australia.
Matrix Biol Plus. 2024 Aug 20;24:100160. doi: 10.1016/j.mbplus.2024.100160. eCollection 2024 Dec.
Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are a promising source of cardiac cells for disease modelling and regenerative medicine. However, current protocols invariably lead to mixed population of cardiac cell types and often generate cells that resemble embryonic phenotypes. Here we developed a combinatorial approach to assess the importance of extracellular matrix proteins (ECMP) in directing the differentiation of cardiomyocytes from human embryonic stem cells (hESC). We did this by focusing on combinations of ECMP commonly found in the developing heart with a broad goal of identifying combinations that promote maturation and influence chamber specific differentiation. We formulated 63 unique ECMP combinations fabricated from collagen 1, collagen 3, collagen 4, fibronectin, laminin, and vitronectin, presented alone and in combinations, leading to the identification of specific ECMP combinations that promote hESC proliferation, pluripotency, and germ layer specification. When hESC were subjected to a differentiation protocol on the ECMP combinations, it revealed precise protein combinations that enhance differentiation as determined by the expression of cardiac progenitor markers kinase insert domain receptor (KDR) and mesoderm posterior transcription factor 1 (MESP1). High expression of cardiac troponin (cTnT) and the relative expression of myosin light chain isoforms (MLC2a and MLC2v) led to the identification of three surfaces that promote a mature cardiomyocyte phenotype. Action potential morphology was used to assess chamber specificity, which led to the identification of matrices that promote chamber-specific cardiomyocytes. This study provides a matrix-based approach to improve control over cardiomyocyte phenotypes during differentiation, with the scope for translation to cardiac laboratory models and for the generation of functional chamber specific cardiomyocytes for regenerative therapies.
人胚胎干细胞衍生的心肌细胞（hESC-CM）是用于疾病建模和再生医学的有前景的心脏细胞来源。然而，目前的方案总是导致心脏细胞类型的混合群体，并且常常产生类似于胚胎表型的细胞。在这里，我们开发了一种组合方法来评估细胞外基质蛋白（ECMP）在指导人胚胎干细胞（hESC）分化为心肌细胞中的重要性。我们通过关注发育中心脏中常见的ECMP组合来做到这一点，其广泛目标是识别促进成熟并影响腔室特异性分化的组合。我们配制了由胶原蛋白1、胶原蛋白3、胶原蛋白4、纤连蛋白、层粘连蛋白和玻连蛋白单独或组合制成的63种独特的ECMP组合，从而鉴定出促进hESC增殖、多能性和胚层特化的特定ECMP组合。当hESC在这些ECMP组合上进行分化方案时，发现了由心脏祖细胞标志物激酶插入结构域受体（KDR）和中胚层后转录因子1（MESP1）的表达所确定的增强分化的精确蛋白质组合。心肌肌钙蛋白（cTnT）的高表达和肌球蛋白轻链同工型（MLC2a和MLC2v）的相对表达导致鉴定出三种促进成熟心肌细胞表型的表面。动作电位形态用于评估腔室特异性，从而鉴定出促进腔室特异性心肌细胞的基质。这项研究提供了一种基于基质的方法，以改善分化过程中对心肌细胞表型的控制，具有转化为心脏实验室模型以及为再生疗法生成功能性腔室特异性心肌细胞的潜力。
