Adipose derived stem cells: efficiency, toxicity, stability of BrdU labeling and effects on self-renewal and adipose differentiation
Adipose derived stem cells: efficiency, toxicity, stability of BrdU labeling and effects on self-renewal and adipose differentiation
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DOI：
10.1007/s11010-011-0712-x
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发表时间：
2011-05-01
影响因子：
4.3
通讯作者：
Brown, Spencer A.
中科院分区：
文献类型：
--
作者：
Lequeux, Charlotte;Oni, Georgette;Brown, Spencer A.
5-溴-2-脱氧尿苷（BrdU）可用作体外预标记分离的干细胞后，在受体宿主内进行后续细胞跟踪的体内研究的方法学工具。本研究的目的是通过检查BrdU毒性、BrdU细胞内稳定性和对脂肪干细胞（ASC）分化的潜在影响来确定BrdU作为脂肪干细胞（ASC）的标记方式有多有用。用5或10 μ M的BrdU标记猪和人ASC（分别为pASC和hASC）2、6、24和48小时。BrdU毒性和稳定性随着时间的推移，在单层培养，在3-D胶原支架植入到猪模型和解冻后，从长期储存进行了评估，MTT测定和免疫组化。油红O染色评价ASC分化。BrdU在所有试验浓度和孵育时间下均无细胞毒性。每个细胞内的BrdU颜色强度和用BrdU标记的ASC的数量作为孵育时间和BrdU浓度的函数而降低。标记强度随着时间的推移而降低，并且在pASC传6代和hASC传4代后无法检测到。在3-D支架中，BrdU标记的ASC在体外培养90天后和在猪模型中培养30天后可被识别。BrdU没有阻止前脂肪细胞分化，并且在ASC长期储存后随后解冻后仍然可以检测到BrdU标记。BrdU是标记和追踪ASC的优秀候选试剂，其将允许区分BrdU标记的供体细胞和宿主细胞。这些数据为使用BrdU标记的ASC进行未来的组织工程项目提供了基础。
5-bromo-2-deoxyurudine (BrdU) can be used as a methodological tool for in vivo investigations following in vitro prelabeling of isolated stem cells for subsequent cell tracking within the recipient host. The objective of this study was to determine how useful BrdU may be as a labeling modality for adipose derived stem cells (ASC) by examining BrdU toxicity, BrdU intracellular stability, and potential effects on ASC differentiation. Porcine and human ASC (pASC and hASC, respectively) were labeled with BrdU at 5 or 10 mu M for 2, 6, 24, and 48 h. BrdU toxicity and stability over time in monolayer cultures, in 3-D collagen scaffolds implanted to a porcine model and after thawing from long-term storage were evaluated by MTT assays and immunohistochemistry. ASC differentiation was evaluated by Oil Red O staining. BrdU was not cytotoxic at all tested concentrations and incubation times. BrdU color intensity within each cell and the number of ASC labeled with BrdU decreased as a function of both incubation time and BrdU concentrations. Labeling intensities decreased over time and were undetectable after 6 passages for pASC and 4 passages for hASC. In 3-D scaffolds, BrdU-labeled ASC were identifiable after 90 days of in vitro cultures and for 30 days in a porcine model. BrdU did not prevent preadipocyte differentiation and BrdU labeling was still detectable after subsequent thawing after long-term storage of ASC. BrdU is an excellent candidate reagent to label and track ASC that will allow distinction between BrdU-labeled donor cells and host cells. The data provides a foundation for conducting future tissue engineering projects using BrdU-labeled ASC.