Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria.
Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria.
Int J Mol Sci. 2021 Apr 9;22(8):3890. doi: 10.3390/ijms22083890.
Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.
干细胞分泌旁分泌因子，包括细胞外囊泡（EVs），这些因子可以介导细胞间通讯并支持受损组织的再生。作为发育和再生的关键调节剂的低氧（缺氧）可能会通过 EV 影响细胞间通讯。我们询问了在人诱导多能干细胞（iPSC）培养过程中进行低氧处理是否会影响其 EV 的数量、质量或基于 EV 的血管生成潜能。我们使用切向流过滤（TFF）从 1%、5%和 18%的空气氧气培养条件下的大规模培养条件培养基中产生 iPSC-EVs，或者在不进行超离心（TUCF）浓缩的情况下进行。通过可调电阻脉冲感应（TRPS）定量 EV，根据 MISEV2018 指南进行特征描述，并分析其血管生成潜能。与 TUCF 相比，TFF 观察到 EV 回收率更高。我们通过 HIF-1α稳定和 pimonidazole 低氧探针证实了缺氧的效果。在不同的氧气条件下，EV 的数量没有显著差异。与在较高氧气条件下或相应的 EV 耗尽的可溶性因子部分获得的 EV 相比，通过 TFF 或 TUCF 从 1%氧气培养中获得的 iPSC-EVs 表现出显著提高的血管生成潜能。因此，数据表明细胞培养氧气条件和 EV 制备方式会影响 iPSC-EV 的功能。我们得出结论，选择合适的方案将进一步提高特别有效的 iPSC-EV 基治疗药物的产量。
