O'Brien Institute Department, St Vincent's Institute of Medical Research, Melbourne, Australia.
Department of Surgery, St Vincent's Hospital, University of Melbourne, Melbourne, Australia.
Angiogenesis. 2018 Aug;21(3):581-597. doi: 10.1007/s10456-018-9610-0. Epub 2018 Mar 26.
The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes.
In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1/CD32b/FactorVIII/CD31. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1/CD31 vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31 blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks.
Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.
肝窦内皮细胞在肝再生中起着关键作用，这表明它们可能有益于肝生物工程。本研究从成年 C57BL/6 小鼠肝脏中分离出肝窦内皮细胞（LSEC），并确定其在体外和体内形成毛细血管网络的能力，以用于肝组织工程。
从成年 C57BL/6 小鼠肝脏中分离出体外 LSEC。免疫荧光标记表明其为 LYVE-1/CD32b/FactorVIII/CD31。LSEC 的扫描电子显微镜显示，在第 2 天存在特征性的筛板。LSEC 在管状形成测定中形成管和芽，类似于人微血管内皮细胞（HMEC）；并且在体外植入多孔胶原支架中形成具有管腔的毛细血管。LSEC 能够形成球体，并且在球体凝胶三明治测定中，与 HMEC 球体相比，在 24 小时内产生明显更多数量的毛细血管样芽（p=0.0011）。LSEC 球体的上清液显示血管内皮生长因子-A 和 C（VEGF-A、VEGF-C）和肝细胞生长因子（HGF）水平明显高于 LSEC 单层（p=0.0167；p=0.0017；p<0.0001），分别在第 2 天，并且在第 4 天 HGF（p=0.0017）和 VEGF-A（p=0.0051）仍保持较高水平。体内分离的小鼠 LSEC 被制备为 500,000 个细胞的单细胞悬液，或为 5000 个细胞的球体（100 个球体），并植入 SCID 小鼠双侧血管化组织工程室中 2 周。免疫组织化学鉴定植入的 LSEC 形成 LYVE-1/CD31 血管。在 LSEC 植入的构建体中，总体淋巴管生长增加（不显著），而宿主来源的 CD31 血管生长显著增加（p=0.0127）与未植入对照相比。在植入前用荧光标记物 DiI 标记的 LSEC 在体内形成毛细血管，并在 2 周内保持 LYVE-1 和 CD32b 标记物。
分离的小鼠 LSEC 表达一系列与血管相关的细胞标记物，并在体外和体内显示出大量的血管毛细血管形成能力。它们产生的肝生长因子 VEGF-A、VEGF-C 和 HGF 使这些细胞在移植后能够对体内宿主来源的毛细血管床施加生长刺激，从而增强它们在肝组织工程研究中的促再生能力。
