Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA; Sanford Consortium for Regenerative Medicine, La Jolla, CA, USA.
Biomedical Sciences Program, University of California, San Diego, La Jolla, CA 92093, USA; Laboratory of Gene Regulation, Ludwig Institute for Cancer Research, La Jolla, CA, USA.
J Mol Cell Cardiol. 2022 Mar;164:58-68. doi: 10.1016/j.yjmcc.2021.11.011. Epub 2021 Nov 24.
Since the initial isolation of human embryonic stem cells and subsequent discovery of reprogramming methods for somatic cells, thousands of protocols have been developed to create each of the hundreds of cell types found in-vivo with significant focus on disease-prone systems, e.g., cardiovascular. Robust protocols exist for many of these cell types, except for cardiac fibroblasts (CF). Very recently, several competing methods have been developed to generate these cells through a developmentally conserved epicardial pathway. Such methods generate epicardial cells, but here we report that prolonged exposure to growth factors such as bFGF induces fibroblast spindle-like morphology and similar chromatin architecture to primary CFs. Media conditions for growth and assays are provided, as well as suggestions for seeding densities and timepoints for protein harvest of extracellular matrix. We demonstrate marker expression and matrix competency of resultant cells as shown next to primary human cardiac fibroblasts. These methods provide additional guidance to the original protocol and result in an increasingly stable phenotype.
自人类胚胎干细胞的最初分离和随后发现体细胞的重编程方法以来，已经开发了数千种方案来创建体内存在的数百种细胞类型中的每一种，并且非常关注易患病系统，例如心血管系统。已经存在许多针对这些细胞类型的稳健方案，除了心脏成纤维细胞（CF）。直到最近，已经开发了几种有竞争力的方法来通过发育上保守的心外膜途径生成这些细胞。这些方法可生成心外膜细胞，但在这里我们报告说，长时间暴露于 bFGF 等生长因子会诱导成纤维细胞呈纺锤形形态，并且与原代 CF 具有相似的染色质结构。提供了用于生长和测定的培养基条件，以及用于收获细胞外基质的接种密度和时间点的建议。我们展示了标记物的表达和所得细胞的基质能力，这些细胞与原代人心肌成纤维细胞相邻。这些方法为原始方案提供了更多指导，并导致表型越来越稳定。
