Al-Sowayan Balta, Keogh Rosemary J, Abumaree Mohammed, Georgiou Harry M, Kalionis Bill
Pregnancy Research Centre, Department of Maternal-Fetal Medicine, Royal Women's Hospital, Parkville, Victoria 3052, Australia.
University of Melbourne Department of Obstetrics and Gynaecology, Royal Women's Hospital, Parkville, Victoria 3052, Australia.
Stem Cell Investig. 2019 Jan 2;6:2. doi: 10.21037/sci.2018.12.03. eCollection 2019.
To initiate tissue repair, mesenchymal stem/stromal cells (MSCs) must enter the blood stream, migrate to the targeted area, cross the endothelial barrier and home to the damaged tissue. This process is not yet fully understood in humans and thus, the aim of this study was to develop an placental vessel perfusion method to examine human MSC movement from a blood vessel into human tissue. This will provide a better understanding of MSC migration, movement through the endothelial barrier and engraftment into target tissue, in a setting that more closely represents the state, compared with conventional human cell culture models. Moreover, important similarities and differences to animal experimental model systems may be revealed by this method.
Human placental hTERT transformed MSC lines were labelled with live-cell fluorescence dyes, and then perfused into term human placental blood vessel. After labelled MSCs were perfused into the vessel, the vessel was dissected from the placenta and incubated at cell growth conditions. Following incubation, the vessel was washed thoroughly to remove unattached, labelled MSCs and then snap frozen for sectioning. After sectioning, immunofluorescence staining of the endothelium was carried out to detect if labelled MSCs crossed the endothelial barrier.
Twelve placental vessel perfusions were successfully completed. In eight of the twelve perfused vessels, qualitative assessment of immunofluorescence in sections (n=20, 5 µm sections/vessel) revealed labelled MSCs had crossed the endothelial barrier.
The human placental vessel perfusion method could be used to assess human MSC migration into human tissue. Cells of the MSC lines were able to adhere and transmigrate through the endothelial barrier in a manner similar to that of leukocytes. Notably, cells that transmigrated remained in close proximity to the endothelium, which is consistent with the reported MSC vascular niche in placental blood vessels.
为启动组织修复，间充质干/基质细胞（MSC）必须进入血流，迁移至目标区域，穿过内皮屏障并归巢至受损组织。此过程在人类中尚未完全明晰，因此，本研究旨在开发一种胎盘血管灌注方法，以检测人类MSC从血管进入人体组织的运动情况。相较于传统的人类细胞培养模型，这将在更接近真实状态的环境中，让人更好地理解MSC的迁移、穿过内皮屏障以及植入靶组织的过程。此外，该方法可能揭示与动物实验模型系统的重要异同点。
将人胎盘hTERT转化的MSC系用活细胞荧光染料标记，然后灌注到足月人胎盘血管中。在标记的MSC灌注到血管后，将血管从胎盘中分离出来，并在细胞生长条件下孵育。孵育后，将血管彻底冲洗以去除未附着的、标记的MSC，然后速冻以进行切片。切片后，对内皮进行免疫荧光染色，以检测标记的MSC是否穿过内皮屏障。
成功完成了12次胎盘血管灌注。在12个灌注血管中的8个中，对切片（每个血管20个5μm切片）进行的免疫荧光定性评估显示，标记的MSC已穿过内皮屏障。
人胎盘血管灌注方法可用于评估人类MSC向人体组织的迁移。MSC系的细胞能够以类似于白细胞的方式黏附并穿过内皮屏障。值得注意的是，迁移的细胞仍与内皮紧密相邻，这与胎盘血管中报道的MSC血管微环境一致。
