{"title":"Letter to editor on ‘isolation and characterization of human Wharton's jelly mesenchymal stem cell-derived extracellular vesicles’","authors":"Saravanan Sampoornam Pape Reddy, Delfin Lovelina Francis, Vishal Kulkarni, Sukhbir Singh Chopra","doi":"10.1111/jcmm.70168","DOIUrl":null,"url":null,"abstract":"<p>We have carefully reviewed the methodology described in the study titled ‘Isolation and Characterization of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles’ by Torabi et al.,<span><sup>1</sup></span> published in the <i>Journal of Cellular and Molecular Medicine</i>. This letter concerns the isolation, characterization, and functional assessment of extracellular vesicles (EVs) derived from Wharton's Jelly mesenchymal stem cells (WJ-MSCs) and their effects on macrophages and stellate cells. While the study presents a comprehensive framework, there are several methodological concerns that warrant further clarification or may pose potential conflicts regarding the robustness and reproducibility of the results. Although the authors provide a comprehensive overview of the isolation and characterization WJ-MSC derived EVs, the experimental design and interpretation of the data merit further scrutiny.</p><p>The authors detail a multistep ultracentrifugation process to isolate EVs, with the final fractions labelled as EV20K and EV110K. However, the reported methods lack adequate controls to confirm the purity of these EV populations. Specifically, there is no mention of assessing the presence of other potential contaminants, such as protein aggregates or apoptotic bodies, which could confound the results. The absence of additional characterization techniques, such as nanoparticle tracking analysis (NTA) or density gradient purification raises concerns about the reliability of the size distribution data obtained via dynamic light scattering (DLS). Furthermore, a more stringent debris removal step, such as a lower-speed centrifugation or filtration, would enhance the purity of the isolated EV populations.<span><sup>2</sup></span> Given that EV populations differ greatly in size and content, the omission of such steps could influence the downstream biological assays, particularly when assessing functional outcomes.</p><p>The authors utilized flow cytometry to measure the uptake of calcein-labelled EVs by UCB-derived monocytes. However, the experimental conditions surrounding the labelling and washing steps may inadvertently affect the quantification of EV uptake. It is critical that the authors provide a more detailed methodology regarding how they ensured the complete removal of unbound calcein dye and how this might affect the interpretation of their flow cytometry results. Although Western blotting for EV markers (CD63, CD81) and the absence of calnexin were performed to validate EV identity, it would be beneficial to include NTA in addition to DLS for more accurate size distribution and concentration profiling. The use of calcein AM to label EVs for the uptake study introduces potential issues; calcein dye can leak from vesicles and be taken up by cells, which may inflate the reported uptake rates. A more vesicle-specific dye (such as DiD or PKH dyes) could reduce this confounding factor, providing a clearer understanding of EV internalization.<span><sup>3</sup></span></p><p>The concentration of WJ-EVs (10 and 50 μg/mL) and the addition of M-CSF as a positive control are consistent with polarization assays. While the authors included controls (M-CSF and WJ-EV-depleted media), it would strengthen their findings to discuss the rationale behind the chosen concentrations and how they correlate with physiological conditions. Furthermore, the cytokine profile evaluated could benefit from additional inflammatory mediators to paint a more comprehensive picture of macrophage polarization dynamics. However, the inclusion of human platelet lysate (hPL) in the culture medium is a confounding factor, as it can introduce undefined growth factors and EVs from platelets that might have specific effect of WJ-EVs on monocyte differentiation.<span><sup>4</sup></span></p><p>The investigation into the anti-fibrotic effects of macrophage-conditioned media on LX2 stellate cells introduces interesting insights. However, the conclusions drawn regarding the therapeutic potential of the EVs should be approached with caution. The authors could have enhanced their discussion by considering the complexities of the in vivo environment, where the interactions among various cell types and soluble factors can differ significantly from in-vitro findings.</p><p>In conclusion, while the study offers valuable insights, addressing the aforementioned concerns could significantly improve the clarity and reproducibility of the results. Further refinement in EV isolation, characterization and culture conditions would help resolve potential methodological conflicts and yield more robust conclusions regarding the biological roles of WJ-EVs.</p><p>Thank you for considering this perspective. We look forward to your response.</p><p><b>Saravanan Sampoornam Pape Reddy:</b> Conceptualization (equal); data curation (equal); formal analysis (equal); writing – original draft (equal). <b>Delfin Lovelina Francis:</b> Formal analysis (equal); investigation (equal); methodology (equal). <b>Vishal Kulkarni:</b> Investigation (equal); methodology (equal); project administration (equal); resources (equal). <b>Sukhbir Singh Chopra:</b> Supervision (equal); validation (equal); visualization (equal).</p><p>There is no funding support received in any form for this letter to editor.</p><p>None of the authors have potential or actual conflicts of interest.</p>","PeriodicalId":101321,"journal":{"name":"JOURNAL OF CELLULAR AND MOLECULAR MEDICINE","volume":null,"pages":null},"PeriodicalIF":5.3000,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11495967/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"JOURNAL OF CELLULAR AND MOLECULAR MEDICINE","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/jcmm.70168","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
We have carefully reviewed the methodology described in the study titled ‘Isolation and Characterization of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles’ by Torabi et al.,1 published in the Journal of Cellular and Molecular Medicine. This letter concerns the isolation, characterization, and functional assessment of extracellular vesicles (EVs) derived from Wharton's Jelly mesenchymal stem cells (WJ-MSCs) and their effects on macrophages and stellate cells. While the study presents a comprehensive framework, there are several methodological concerns that warrant further clarification or may pose potential conflicts regarding the robustness and reproducibility of the results. Although the authors provide a comprehensive overview of the isolation and characterization WJ-MSC derived EVs, the experimental design and interpretation of the data merit further scrutiny.
The authors detail a multistep ultracentrifugation process to isolate EVs, with the final fractions labelled as EV20K and EV110K. However, the reported methods lack adequate controls to confirm the purity of these EV populations. Specifically, there is no mention of assessing the presence of other potential contaminants, such as protein aggregates or apoptotic bodies, which could confound the results. The absence of additional characterization techniques, such as nanoparticle tracking analysis (NTA) or density gradient purification raises concerns about the reliability of the size distribution data obtained via dynamic light scattering (DLS). Furthermore, a more stringent debris removal step, such as a lower-speed centrifugation or filtration, would enhance the purity of the isolated EV populations.2 Given that EV populations differ greatly in size and content, the omission of such steps could influence the downstream biological assays, particularly when assessing functional outcomes.
The authors utilized flow cytometry to measure the uptake of calcein-labelled EVs by UCB-derived monocytes. However, the experimental conditions surrounding the labelling and washing steps may inadvertently affect the quantification of EV uptake. It is critical that the authors provide a more detailed methodology regarding how they ensured the complete removal of unbound calcein dye and how this might affect the interpretation of their flow cytometry results. Although Western blotting for EV markers (CD63, CD81) and the absence of calnexin were performed to validate EV identity, it would be beneficial to include NTA in addition to DLS for more accurate size distribution and concentration profiling. The use of calcein AM to label EVs for the uptake study introduces potential issues; calcein dye can leak from vesicles and be taken up by cells, which may inflate the reported uptake rates. A more vesicle-specific dye (such as DiD or PKH dyes) could reduce this confounding factor, providing a clearer understanding of EV internalization.3
The concentration of WJ-EVs (10 and 50 μg/mL) and the addition of M-CSF as a positive control are consistent with polarization assays. While the authors included controls (M-CSF and WJ-EV-depleted media), it would strengthen their findings to discuss the rationale behind the chosen concentrations and how they correlate with physiological conditions. Furthermore, the cytokine profile evaluated could benefit from additional inflammatory mediators to paint a more comprehensive picture of macrophage polarization dynamics. However, the inclusion of human platelet lysate (hPL) in the culture medium is a confounding factor, as it can introduce undefined growth factors and EVs from platelets that might have specific effect of WJ-EVs on monocyte differentiation.4
The investigation into the anti-fibrotic effects of macrophage-conditioned media on LX2 stellate cells introduces interesting insights. However, the conclusions drawn regarding the therapeutic potential of the EVs should be approached with caution. The authors could have enhanced their discussion by considering the complexities of the in vivo environment, where the interactions among various cell types and soluble factors can differ significantly from in-vitro findings.
In conclusion, while the study offers valuable insights, addressing the aforementioned concerns could significantly improve the clarity and reproducibility of the results. Further refinement in EV isolation, characterization and culture conditions would help resolve potential methodological conflicts and yield more robust conclusions regarding the biological roles of WJ-EVs.
Thank you for considering this perspective. We look forward to your response.
我们仔细审阅了 Torabi 等人1 发表在《细胞与分子医学杂志》(Journal of Cellular and Molecular Medicine)上的题为《人沃顿果冻间充质干细胞衍生胞外小泡的分离与表征》(Isolation and Characterization of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles)的研究中所描述的方法。这封信涉及沃顿果冻间充质干细胞(WJ-MSCs)衍生的细胞外囊泡(EVs)的分离、表征和功能评估,以及它们对巨噬细胞和星状细胞的影响。虽然该研究提出了一个全面的框架,但有几个方法上的问题需要进一步澄清,或可能对结果的稳健性和可重复性造成潜在冲突。虽然作者全面概述了WJ-间充质干细胞衍生EVs的分离和表征,但实验设计和数据解释值得进一步仔细研究。作者详细介绍了分离EVs的多步超速离心过程,最终馏分被标记为EV20K和EV110K。然而,报告中的方法缺乏足够的控制措施来确认这些 EV 群体的纯度。具体来说,报告中没有提到对其他潜在污染物(如蛋白质聚集体或凋亡体)的存在进行评估,这些污染物可能会混淆结果。由于缺乏额外的表征技术,如纳米颗粒跟踪分析(NTA)或密度梯度纯化,人们对通过动态光散射(DLS)获得的粒度分布数据的可靠性产生了担忧。此外,更严格的碎片去除步骤,如低速离心或过滤,会提高分离出的 EV 群体的纯度。2 鉴于 EV 群体在大小和含量上差别很大,省略这些步骤可能会影响下游生物检测,尤其是在评估功能结果时。作者利用流式细胞术测量了 UCB 衍生单核细胞对钙黄素标记 EV 的摄取。然而,围绕标记和洗涤步骤的实验条件可能会无意中影响 EV 吸收的量化。作者必须提供更详细的方法,说明他们如何确保完全去除未结合的钙黄绿素染料,以及这可能会如何影响流式细胞仪结果的解释。虽然对 EV 标记(CD63、CD81)进行了 Western 印迹检测,并且没有检测到钙黄绿素,以验证 EV 的身份,但除了 DLS 外,最好还加入 NTA,以进行更准确的粒度分布和浓度分析。在摄取研究中使用钙黄绿素 AM 标记 EV 会带来潜在的问题;钙黄绿素染料会从囊泡中渗出并被细胞摄取,这可能会夸大报告的摄取率。3 WJ-EVs的浓度(10 和 50 μg/mL)以及添加 M-CSF 作为阳性对照与极化测定一致。虽然作者加入了对照组(M-CSF 和 WJ-EV 贫化培养基),但如果能讨论所选浓度背后的原理以及这些浓度与生理条件的相关性,将能加强他们的研究结果。此外,评估的细胞因子图谱可能会受益于更多的炎症介质,以更全面地描绘巨噬细胞极化动态。然而,在培养基中加入人血小板裂解液(hPL)是一个干扰因素,因为它可能会引入未定义的生长因子和血小板中的 EVs,而这些 EVs 可能会对单核细胞分化产生 WJ-EVs 的特定影响。然而,有关 EVs 治疗潜力的结论应谨慎对待。作者本可以通过考虑体内环境的复杂性来加强讨论,因为在体内环境中,各种细胞类型和可溶性因子之间的相互作用可能与体外研究结果大相径庭。总之,虽然这项研究提供了有价值的见解,但解决上述问题可以大大提高结果的清晰度和可重复性。进一步完善 EV 的分离、表征和培养条件将有助于解决潜在的方法论冲突,并就 WJ-EV 的生物学作用得出更可靠的结论。Saravanan Sampoornam Pape Reddy:构思(相同);数据整理(相同);正式分析(相同);撰写-原稿(相同)。Delfin Lovelina Francis:正式分析(相同);调查(相同);方法(相同)。Vishal Kulkarni:调查(相同);方法(相同);项目管理(相同);资源(相同)。
期刊介绍:
The Journal of Cellular and Molecular Medicine serves as a bridge between physiology and cellular medicine, as well as molecular biology and molecular therapeutics. With a 20-year history, the journal adopts an interdisciplinary approach to showcase innovative discoveries.
It publishes research aimed at advancing the collective understanding of the cellular and molecular mechanisms underlying diseases. The journal emphasizes translational studies that translate this knowledge into therapeutic strategies. Being fully open access, the journal is accessible to all readers.