Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs.

中文翻译：

---

人多能干细胞来源的小细胞外囊泡特异性标记物的鉴定

多能干细胞衍生的小细胞外囊泡（PSC-sEV）已在多种与衰老相关的退行性疾病中表现出巨大的临床转化潜力。 PSC-sEV 的表征对其临床应用至关重要。然而，PSC-sEV 的具体标记模式仍然未知。在这里，使用液相色谱串联质谱法 (LC-MS/MS) 进行蛋白质组分析，对源自两种典型 PSC 类型（包括诱导多能干细胞 (iPSC-sEV) 和胚胎干细胞 (ESC-sEV)）的 sEV 进行了分析，以及通过纳米颗粒流式细胞术 (NanoFCM) 进行表面标记表型分析。通过 LC-MS/MS 发现一组多能性相关蛋白在 PSC-sEV 中富集，然后通过蛋白质印迹分析进行验证。为了研究这些蛋白是否在 PSC-sEV 中特异性表达，采用源自七种非 PSC（非 PSC-sEV）的 sEV 进行分析。结果表明，PODXL、OCT4、Dnmt3a 和 LIN28A 在 PSC-sEV 中特异性富集，但在非 PSC-sEV 中则没有。然后，通过 NanoFCM 以单颗粒分辨率测量用于 PSC 识别的常用表面抗原（SSEA4、Tra-1-60 和 Tra-1-81）和 PODXL，以进行表面标记物识别。结果显示，PSC-sEVs中PODXL（>50%）和SSEA4（>70%）的阳性率远高于非PSC-sEVs（<10%）。这些结果通过密度梯度超速离心纯化的样品得到进一步验证。综上所述，本研究首次鉴定了一组 PSC-sEV 的特异性标记物，其中 PODXL、OCT4、Dnmt3a 和 LIN28A 可以通过 Western Blot 分析检测，PODXL 和 SSEA4 可以通过 NanoFCM 分析检测。应用这些特定标记进行 PSC-sEV 鉴定可能会促进 PSC-sEV 的临床转化。