Real-time in vivo monitoring of viable stem cells implanted on biocompatible scaffolds
Real-time in vivo monitoring of viable stem cells implanted on biocompatible scaffolds
复制标题
DOI：
10.1007/s00259-008-0751-z
复制
发表时间：
2008-10-01
影响因子：
9.1
通讯作者：
Lee, Dong Soo
中科院分区：
文献类型：
--
作者：
Hwang, Do Won;Jang, Sung June;Lee, Dong Soo
目的三维纤维支架为提高移植干细胞在体内的存活提供了一个环境，并有助于对其在体内的定位、活性和生长进行成像。为了在体内评价生物相容性聚合物支架上移植的干细胞的活性，我们建立了体内成像系统，用于评估使用荧光素酶或钠/碘转运体(NIS)基因在支架上植入的存活神经干细胞(NSC)和间充质干细胞(MSC)。方法建立稳定表达-C6细胞的萤火虫荧光素酶(C6-Fluc)。将携带荧光素酶基因的腺病毒载体(F3-Fluc)感染人神经干细胞F3，经慢病毒载体处理2周，建立了由泛素C启动子调控的表达NIS的慢病毒载体(MSC-NIS)。用壳聚糖和聚L-乳酸(PLLA)支架进行体内成像。分别用生物发光法和99m-高铁酸酯伽玛照相技术检测荧光素酶和人NIS的体内表达。将细胞/支架复合体植入BALB/C裸鼠皮下或腹部。结果随着细胞数量的增加，C6-Fluc荧光素酶活性逐渐增强。C6-Fluc/壳聚糖复合体植入裸鼠皮下后34天仍显示出纵向生物发光图像。腹部注射C6-Fluc/PLLA复合体的荧光素酶图像仅在14天内就达到饱和，显示出丰富的营养物质使细胞生长迅速。扫描电子显微镜显示F3细胞与纤维状壳聚糖支架结合良好。感染Ad-Fluc的F3的荧光素酶活性是F3的100倍。以壳聚糖为载体的F3-Fluc荧光素酶活性随细胞数的增加而增加。腹部注射后，F3-Fluc掺入壳聚糖的生物发光图像清晰可见，最长可达11天。放射性核素成像显示MSC-NIS在PLLA支架上的摄取高于未种植在支架上的MSC-NIS。定量数据显示，植入后72 h，MSC-NIS在PLLA支架上的存活率明显高于未植入支架的细胞，这与组织学结果一致。结论这些结果表明，通过连续活体成像可以长期监测NSC-Fluc和MSC-NIS细胞在聚合物支架上的情况。我们相信，基于支架的生物兼容成像系统可以用来以非侵入性的方式评估干细胞的活性，以帮助再生疗法的发展。
Purpose Three-dimensional fibrous scaffolds provide an environment that enhances transplanted stem cell survival in vivo and facilitates imaging their localization, viability, and growth in vivo. To assess transplanted stem cell viability on biocompatible polymer scaffolds in vivo, we developed in vivo imaging systems for evaluation of implanted viable neural stem cells (NSC) and mesenchymal stem cells (MSC) on scaffolds using luciferase or sodium/iodide symporter (NIS) genes.Methods Firefly luciferase stably expressing-C6 cell was established (C6-Fluc). The human neural stem cell, F3, was infected with adenoviral vector carrying luciferase gene (F3-Fluc) and MSC expressing NIS controlled by ubiquitin C promoter using lentiviral vector was established by treating blasticidine for 2 weeks (MSC-NIS). Chitosan and poly L-lactic acid (PLLA) scaffolds were used for in vivo image. In vivo expression of luciferase and human NIS was examined by bioluminescence image or Tc-99m-pertechnetate gamma camera image, respectively. The cell/scaffold complex was implanted into subcutaneous or abdominal area of BALB/C nude mouse. For quantitative evaluation of cell viability, regions of interest were drawn on Tc-99m-pertechnetate scintigraphy by manual.Results The gradual increase of luciferase activity was observed in C6-Fluc seeded with chitosan according to the increase in the number of cells. C6-Fluc/chitosan complex subcutaneously implanted into nude mice showed longitudinal bioluminescence image until 34 days. Luciferase image of abdominal-injected C6-Fluc/PLLA complex was saturated in only 14 days, showing great cell growth due to abundant nutrients. F3 cells showed well-incorporated pattern with fibrous chitosan scaffold using scanning electron microscopy. F3 infected with Ad-Fluc showed > 100-fold higher luciferase activity than luciferase activity in F3. Cell-number-dependent increase of luciferase activity was shown in F3-Fluc seeded on chitosan. F3-Fluc incorporation into chitosan after abdominal injection was clearly visible on bioluminescence image up to 11 days. Radionuclide imaging showed higher uptake by MSC-NIS on PLLA scaffolds than by MSC-NIS not seeded on a scaffold. Quantitative data showed significantly better survival of MSC-NIS on PLLA scaffolds than without scaffold at 72 h post-implantation, which concurred with histologic findings.Conclusion These results suggest that NSC-Fluc and MSC-NIS cells incorporated within polymer scaffolds can be monitored on a long-term basis by serial in vivo imaging. We believe that a biocompatible scaffold-based imaging system could be used to assess stem cell viabilities in a non-invasive way to aid the development of regenerative therapeutics.