Human iPSC-derived cardiomyocytes under hypoxia/normoxia, treated with bone marrow or cardiac stromal cell extracellular vesicles

RNA-seq was performed on cultured human induced pluripotent cell derived cardiomyocytes (iPSC-CMs). There are four groups, with three samples per group: H1,H2,H3. Negative control. Healthy, normoxic iPSC-CMs D1,D2,D3. Positive control. iPSCs cultured under hypoxic (0-1% O2) for 48h with 2% v/v PBS as vehicle control EV1,EV2,EV3. Treatment 1. iPSCs cultured under hypoxic (0-1% O2) for 48h, with cardiac stromal cell derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) EV21, EV23, EV24. Treatment 2 iPSCs cultured under hypoxic (0-1% O2) for 48h, with bone marrow mesenchymal stromal cell (BM-MSC) derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) All EVs were isolated from cultured human cells using sequential centrifugation methods. Cells were cultured using commercial EV-depleted FBS to avoid contamination of bovine EVs. EVs were validated for CD81, CD9, ALIX positivity, and visualised by cryoEM. Particle to protein ratios were not different between cardiac and bone marrow EV isolates. Therefore EV doses were standardised so that the same dose by protein (67ng/µl) and EV number (~2,000 EVs per iPSC-CM) were added.

本研究对体外培养的人诱导多能干细胞衍生心肌细胞（iPSC-CMs）进行了RNA测序（RNA-seq）。实验共设置4组，每组包含3个生物学重复样本：
1. 阴性对照组：常氧培养的健康人iPSC-CMs，样本编号为H1、H2、H3；
2. 阳性对照（低氧载体对照组）：将iPSCs置于低氧（0~1% O₂）环境中培养48小时，以2%体积分数的磷酸盐缓冲液（PBS）作为载体对照，样本编号为D1、D2、D3；
3. 处理组1：将iPSCs置于低氧（0~1% O₂）环境中培养48小时，加入心脏基质细胞衍生的细胞外囊泡（extracellular vesicles，EVs），给药剂量为67 ng/µL（2%体积分数），样本编号为EV21、EV23、EV24；
4. 处理组2：将iPSCs置于低氧（0~1% O₂）环境中培养48小时，加入骨髓间充质基质细胞（bone marrow mesenchymal stromal cell，BM-MSC）衍生的细胞外囊泡，给药剂量为67 ng/µL（2%体积分数）。

所有细胞外囊泡均通过连续离心法从体外培养的人源细胞中分离得到。实验采用商用的去除细胞外囊泡的胎牛血清（EV-depleted FBS）培养细胞，以避免牛源细胞外囊泡的污染。

我们通过检测CD81、CD9、ALIX的阳性表达对分离得到的细胞外囊泡进行了验证，并通过冷冻电镜（cryoEM）对其进行了可视化观察。心脏来源与骨髓来源的细胞外囊泡的颗粒数与蛋白质量比值无显著差异，因此我们对细胞外囊泡的给药剂量进行了标准化，确保每组均采用相同的蛋白质量剂量（67 ng/µL）及细胞外囊泡数量（约每iPSC-CM对应2000个细胞外囊泡）进行处理。