Robert and Arlene Kogod Center on Aging and Division of Endocrinology, Mayo Clinic College of Medicine, Rochester, MN 55905, United States.
Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science, Rochester, MN 55905, United States.
J Bone Miner Res. 2024 Oct 29;39(11):1633-1643. doi: 10.1093/jbmr/zjae135.
Extracellular vesicles (EVs) are key mediators of cell-cell communication and are involved in transferring specific biomolecular cargo to recipient cells to regulate their physiological functions. A major challenge in the understanding of EV function in vivo is the difficulty ascertaining the origin of the EV particles. The recent development of the "Snorkel-tag," which includes EV-membrane-targeted CD81 fused to a series of extra-vesicular protein tags, can be used to mark EVs originating from a specific source for subsequent isolation and characterization. We developed an in vivo mouse model, termed "CAGS-Snorkel," which expresses the Snorkel-tag under the control of the Cre-lox system, and crossed this mouse with either Prx1-Cre (mesenchymal progenitors) or Ocn-Cre (osteoblasts/osteocytes) and isolated Snorkel-tagged EVs from the mouse bone marrow plasma using a magnetic bead affinity column. miRNA-sequencing was performed on the isolated EVs, and although similar profiles were observed, a few key miRNAs involved in bone metabolism (miR-106b-5p, miRs-19b-3p, and miRs-219a-5p) were enriched in the Ocn-derived relative to the Prx1-derived EV subpopulations. To characterize the effects of these small EVs on a bone cell target, cultured mouse bone marrow stromal cells were treated with Prx1 or Ocn EVs, and mRNA-sequencing was performed. Pathways involved in ossification, bone development, and extracellular matrix interactions were regulated by both EV subpopulations, whereas a few pathways including advanced glycation end-products signaling were uniquely regulated in the Ocn EV subpopulation, underlying important biological effects of specific EV subpopulations within the bone marrow microenvironment. These data demonstrate that EV isolation in vivo using the CAGS-Snorkel mouse model is a useful tool in characterizing the cargo and understanding the biology of tissue-specific EVs. Moreover, while bone mesenchymal cell populations share a common EV secretory profile, we uncover key differences based on the stage of osteoblastic differentiation that may have important biological consequences.
细胞外囊泡 (EVs) 是细胞间通讯的关键介质，参与将特定的生物分子货物传递到受体细胞，以调节其生理功能。理解 EV 在体内的功能的一个主要挑战是难以确定 EV 颗粒的来源。最近开发的“Snorkel-tag”，其中包括与一系列囊外蛋白标签融合的 EV 膜靶向 CD81，可以用于标记源自特定来源的 EV，以便随后进行分离和表征。我们开发了一种体内小鼠模型，称为“CAGS-Snorkel”，它在 Cre-lox 系统的控制下表达 Snorkel-tag，然后将这种小鼠与 Prx1-Cre（间充质祖细胞）或 Ocn-Cre（成骨细胞/破骨细胞）杂交，并使用磁珠亲和柱从小鼠骨髓血浆中分离 Snorkel 标记的 EV。对分离的 EV 进行 miRNA 测序，尽管观察到相似的图谱，但一些参与骨代谢的关键 miRNA（miR-106b-5p、miRs-19b-3p 和 miRs-219a-5p）在 Ocn 衍生的 EV 亚群中富集，而在 Prx1 衍生的 EV 亚群中则相对较少。为了表征这些小 EV 对骨细胞靶标的影响，用 Prx1 或 Ocn EV 处理培养的小鼠骨髓基质细胞，并进行 mRNA 测序。成骨、骨发育和细胞外基质相互作用相关的途径受到两种 EV 亚群的调节，而一些途径，包括晚期糖基化终产物信号通路，仅在 Ocn EV 亚群中受到调节，这突显了骨髓微环境中特定 EV 亚群的重要生物学效应。这些数据表明，使用 CAGS-Snorkel 小鼠模型在体内分离 EV 是一种有用的工具，可用于表征货物并理解组织特异性 EV 的生物学。此外，尽管骨间充质细胞群体具有共同的 EV 分泌特征，但我们根据成骨细胞分化的阶段揭示了关键差异，这些差异可能具有重要的生物学后果。
