Ishioka Mina, Nihashi Yuma, Sunagawa Yoichi, Umezawa Koji, Shimosato Takeshi, Kagami Hiroshi, Morimoto Tatsuya, Takaya Tomohide
Department of Agriculture, Graduate School of Science and Technology, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan.
Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Central 5-41, 1-1-1 Higashi, Tsukuba 305-8565, Ibaraki, Japan.
Int J Mol Sci. 2023 Sep 21;24(18):14380. doi: 10.3390/ijms241814380.
An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts as an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 inhibited the proliferation of ESCs and iPSCs but did not affect the expression of pluripotent markers. In the differentiating condition, iSN04 treatment of ESCs/iPSCs from day 5 onward dramatically induced differentiation into beating cardiomyocytes with upregulation of , , and , whereas iSN04 treatment from earlier stages completely inhibited cardiomyogenesis. RNA sequencing revealed that iSN04 treatment from day 5 onward contributes to the generation of cardiac progenitors by modulating the Wnt signaling pathway. Immunostaining showed that iSN04 suppressed the cytoplasmic translocation of nucleolin and restricted it to the nucleoli. These results demonstrate that nucleolin inhibition by iSN04 facilitates the terminal differentiation of cardiac mesoderm into cardiomyocytes but interferes with the differentiation of early mesoderm into the cardiac lineage. This is the first report on the generation of cardiomyocytes from pluripotent stem cells using a DNA aptamer. Since iSN04 did not induce hypertrophic responses in primary-cultured cardiomyocytes, iSN04 would be useful and safe for the regenerative therapy of heart failure using stem cell-derived cardiomyocytes.
一条18个碱基的肌源性寡脱氧核苷酸（myoDN），即iSN04，作为一种抗核仁素适体，可诱导骨骼肌成肌细胞发生肌源性分化。本研究调查了iSN04对小鼠胚胎干细胞（ESC）和诱导多能干细胞（iPSC）的影响。在未分化状态下，iSN04抑制ESC和iPSC的增殖，但不影响多能性标志物的表达。在分化条件下，从第5天起用iSN04处理ESC/iPSC可显著诱导其分化为跳动的心肌细胞，同时 、 和 的表达上调，而从更早阶段开始用iSN04处理则完全抑制心肌发生。RNA测序显示，从第5天起用iSN04处理可通过调节Wnt信号通路促进心脏祖细胞的产生。免疫染色显示，iSN04抑制核仁素的细胞质转位并将其限制在核仁中。这些结果表明，iSN04对核仁素的抑制作用促进了心脏中胚层向心肌细胞的终末分化，但干扰了早期中胚层向心脏谱系的分化。这是关于使用DNA适体从多能干细胞生成心肌细胞的首次报道。由于iSN04在原代培养的心肌细胞中未诱导肥大反应，因此iSN04对于使用干细胞衍生的心肌细胞进行心力衰竭的再生治疗将是有用且安全的。
