Department of Radiology, University Hospitals Case Medical Center, Cleveland, Ohio 44016, USA.
J Orthop Res. 2013 Jun;31(6):871-9. doi: 10.1002/jor.22328. Epub 2013 Feb 25.
Stem cells, such as mesenchymal stem cells (MSCs), contribute to bone fracture repair if they are delivered to the injury site. However, it is difficult to assess the retention and differentiation of these cells after implantation. Current options for non-invasively tracking the transplanted stem cells are limited. Cell-based therapies using MSCs would benefit greatly through the use of an imaging methodology that allows cells to be tracked in vivo and in a timely fashion. In this study, we implemented an in vivo imaging methodology to specifically track early events such as differentiation of implanted human MSCs (hMSCs). This system uses the collagen type 1 (Col1α1) promoter to drive expression of firefly luciferase (luc) in addition to a constitutively active promoter to drive the expression of green fluorescent protein (GFP). The resulting dual-promoter reporter gene system provides the opportunity for osteogenic differentiation-specific luc expression for in vivo imaging and constitutive expression of GFP for cell sorting. The function of this dual-promoter reporter gene was validated both in vitro and in vivo. In addition, the ability of this dual-promoter reporter system to image an early event of osteogenic differentiation of hMSCs was demonstrated in a murine segmental bone defect model in which reporter-labeled hMSCs were seeded into an alginate hydrogel scaffold and implanted directly into the defect. Bioluminescence imaging (BLI) was performed to visualize the turn-on of Col1α1 upon osteogenic differentiation and followed by X-ray imaging to assess the healing process for correlation with histological analyses.
干细胞，如间充质干细胞（MSCs），如果被递送到损伤部位，有助于骨骨折修复。然而，评估这些细胞在植入后的保留和分化是很困难的。目前，非侵入性跟踪移植干细胞的选择有限。使用 MSCs 的基于细胞的疗法将通过使用允许在体内和及时跟踪细胞的成像方法大大受益。在这项研究中，我们实施了一种体内成像方法，专门跟踪植入的人 MSCs（hMSCs）的早期分化等事件。该系统使用胶原类型 1（Col1α1）启动子来驱动萤火虫荧光素酶（luc）的表达，除了组成性激活启动子来驱动绿色荧光蛋白（GFP）的表达。由此产生的双启动子报告基因系统为体内成像提供了骨向分化特异性 luc 表达的机会，并为细胞分选提供了 GFP 的组成性表达。该双启动子报告基因的功能在体外和体内都得到了验证。此外，该双启动子报告系统在鼠节段性骨缺损模型中成像 hMSCs 早期成骨分化的能力得到了证明，其中报告基因标记的 hMSCs 被播种到藻酸盐水凝胶支架中，并直接植入缺损部位。进行生物发光成像（BLI）以可视化 Col1α1 在成骨分化时的开启，并随后进行 X 射线成像以评估愈合过程，与组织学分析相关联。
