Department of Regenerative Science and Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
Division of Cardiovascular Surgery, Department of Surgery, Shinshu University School of Medicine, Matsumoto, 390-8621, Japan.
Stem Cell Res Ther. 2023 Sep 7;14(1):240. doi: 10.1186/s13287-023-03468-4.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat heart diseases; however, the optimal maturity of hiPSC-CMs for effective regenerative medicine remains unclear. We aimed to investigate the benefits of long-term cultured mature hiPSC-CMs in injured rat hearts.
Cardiomyocytes were differentiated from hiPSCs via monolayer culturing, and the cells were harvested on day 28 or 56 (D28-CMs or D56-CMs, respectively) after differentiation. We transplanted D28-CMs or D56-CMs into the hearts of rat myocardial infarction models and examined cell retention and engraftment via in vivo bioluminescence imaging and histological analysis. We performed transcriptomic sequencing analysis to elucidate the genetic profiles before and after hiPSC-CM transplantation.
Upregulated expression of mature sarcomere genes in vitro was observed in D56-CMs compared with D28-CMs. In vivo bioluminescence imaging studies revealed increased bioluminescence intensity of D56-CMs at 8 and 12 weeks post-transplantation. Histological and immunohistochemical analyses showed that D56-CMs promoted engraftment and maturation in the graft area at 12 weeks post-transplantation. Notably, D56-CMs consistently promoted microvessel formation in the graft area from 1 to 12 weeks post-transplantation. Transcriptomic sequencing analysis revealed that compared with the engrafted D28-CMs, the engrafted D56-CMs enriched genes related to blood vessel regulation at 12 weeks post-transplantation. As shown by transcriptomic and western blot analyses, the expression of a small heat shock protein, alpha-B crystallin (CRYAB), was significantly upregulated in D56-CMs compared with D28-CMs. Endothelial cell migration was inhibited by small interfering RNA-mediated knockdown of CRYAB when co-cultured with D56-CMs in vitro. Furthermore, CRYAB overexpression enhanced angiogenesis in the D28-CM grafts at 4 weeks post-transplantation.
Long-term cultured mature hiPSC-CMs promoted engraftment, maturation and angiogenesis post-transplantation in infarcted rat hearts. CRYAB, which was highly expressed in D56-CMs, was identified as an angiogenic factor from mature hiPSC-CMs. This study revealed the benefits of long-term culture, which may enhance the therapeutic potential of hiPSC-CMs.
人类诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）可用于治疗心脏病；然而，用于有效再生医学的 hiPSC-CMs 的最佳成熟度尚不清楚。我们旨在研究长期培养的成熟 hiPSC-CMs 在损伤大鼠心脏中的益处。
通过单层培养从 hiPSC 中分化出心肌细胞，并在分化后第 28 天或第 56 天（分别为 D28-CMs 或 D56-CMs）收获细胞。我们将 D28-CMs 或 D56-CMs 移植到大鼠心肌梗死模型的心脏中，并通过体内生物发光成像和组织学分析来检测细胞保留和植入情况。我们进行了转录组测序分析，以阐明 hiPSC-CM 移植前后的基因谱。
体外观察到 D56-CMs 中成熟肌节基因的表达上调，与 D28-CMs 相比。体内生物发光成像研究显示，移植后 8 周和 12 周时 D56-CMs 的生物发光强度增加。组织学和免疫组织化学分析显示，移植后 12 周时，D56-CMs 促进了移植物区域的植入和成熟。值得注意的是，从移植后 1 周到 12 周，D56-CMs 始终促进移植物区域微血管形成。转录组测序分析显示，与植入的 D28-CMs 相比，植入的 D56-CMs 在移植后 12 周时富含与血管调节相关的基因。正如转录组和 Western blot 分析所示，与 D28-CMs 相比，D56-CMs 中α-B 晶体蛋白（CRYAB）的小热休克蛋白表达显著上调。体外与 D56-CMs 共培养时，CRYAB 的小干扰 RNA 介导的敲低抑制了内皮细胞迁移。此外，CRYAB 过表达增强了移植后 4 周时 D28-CM 移植物的血管生成。
长期培养的成熟 hiPSC-CMs 促进了梗死大鼠心脏中移植后的植入、成熟和血管生成。在 D56-CMs 中高度表达的 CRYAB 被鉴定为成熟 hiPSC-CMs 中的血管生成因子。本研究揭示了长期培养的益处，这可能增强 hiPSC-CMs 的治疗潜力。
