Scaffold-Mediated Developmental Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Are Preserved After External Support Removal

概要

〔目的(Purpose)〕
Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The present study aimed to investigate the extent to which topographic substrates actively influence hiPS-CMs.

〔方法ならびに成績(Methods/Results)〕
Anisotropic patterned culture techniques (aligned patterning) have been extensively used to induce hiPS-CMs maturation. However, their efficiency has been varying among previous studies, probably due to technical differences, such as patterning materials and/or culture protocols. Therefore, firstly, to confirm if the aligned cell arrangement can induce maturation of iPS-CMs, hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression.
Subsequently, to investigated the memory of iPS-CMs for the aligned pattern-induced facilitation of maturity, the cells were harvested from the different substrates and were then replated on the non-scaffold (flat) pattern for an additional week. As a result, the improvements in morphological and functional properties diminished, however, the hiPS-CMs pre-cultured on the aligned pattern somehow retained the memory of the beneficial effects from topographic induced maturation and maintained during development even without the fiber matrix.
To obtain insights into the underlying molecular pathways/signaling involved in the facilitation of hiPS-CM maturation driven by specific topographic stimuli, we performed RNA-seq on hiPS*-CM samples after different patterns culturing. The RNA-seq data revealed that several cardiac maturation markers, such as MYH7 and TNNI3, were significantly upregulated in the align group, which is consistent with the QPCR results. Upon cross analysis of the upregulated genes in align group compared lo the flat or random group, six genes were found to be specifically upregulated in the align pattern (including F13A1, XIRP2. TTC29, PTPRH, TULP1, and LINC00702). Based on our findings, the aligned topographic cell arrangement may be related to the regulation of CM maturation via modulation of the cell cycle.

〔総括(Conclusion)〕
Our results suggested that the anisotropic fiber substrate can induce the formation of geometrical mimic-oriented heart tissue in a short time. Although the morphological and electrophysiological properties of hiPS-CMs obtained via facilitated maturation somehow rely on the existence of an exterior scaffold, the molecular developmental features were preserved even in the absence of external support, which might persist throughout hiPS-CM development.