Unexpected artifacts raise new caution in stem cell culture research.
Unexpected artifacts raise new caution in stem cell culture research.
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意外的假象在干细胞培养研究中引起了新的注意。
DOI：
10.1002/hep.20013
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Sell,Stewart
中科院分区：
文献类型：
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作者：
Leffert,HyamL;Sell,Stewart
通过这封信，我们希望对结果的解释提出警告，因为当细胞系通过在细胞衍生的饲养层上培养而衍生时，可能会出现人为因素。我们最近报道了明显的衍生大鼠肝祖细胞系的肝细胞富集门静脉周围的卵圆细胞与辐照小鼠STO（SIM [山德士近交系瑞士小鼠]）成纤维细胞饲养层的肝细胞共培养后，烯丙醇损伤。1这些报道的细胞系现在正在我们的实验室进行深入的再研究，结果表明这些细胞实际上来自STO饲养层。在一系列第一阶段RT-PCR和PCR中，旨在扩展组成型和诱导型分化的关键原始发现，我们无法在11个报告的细胞株和系中的任何一个中检测到大鼠基因或大鼠基因产物，包括克隆“大鼠”系3（8）# 21及其克隆衍生物3（8）# 21-EGFP，一种用增强型绿色荧光蛋白转导的系。相反，在所有情况下仅检测到小鼠基因。这些结果是用在STO饲养层上或离STO饲养层生长的纯化和克隆的推定“大鼠肝祖细胞”（在UCSD辐照），以及用来自奥尔巴尼医学院非常早期传代的未篡改小瓶获得的原始冷冻“大鼠”细胞获得的。这些和其他意想不到的观察，这往往消除细胞融合的来源，这种假象，详细描述了其他地方。2对这些细胞系中肝细胞功能的持续观察表明，原始报告的某些方面可能是正确的。例如，最佳表征的细胞系3（8）#21-EGFP在体外和移植后表达几种肝细胞特性（手稿在准备中）。总之，这种令人惊讶的和以前未报道的类型的细胞培养伪影应考虑培养的干细胞系统，需要与有丝分裂“抑制”饲养层细胞系共培养的所有研究者。
With this letter, we wish to raise caution about interpretations of results owing to artifacts that may occur when cell lines are derived by culturing on cell-derived feeder layers. We recently reported the apparent derivation of rat liver progenitor cell lines by co-culture of liver cells enriched in periportal oval cells after allyl alcohol injury with-irradiated mouse STO (SIM [Sandoz inbred Swiss mouse]) fibroblast feeder layers. 1 These reported lines are now undergoing intensive reinvestigation in our laboratories and the results indicate that the cells were actually derived from the STO feeder layers. In a series of first stage RT-PCR and PCR, aimed at extending the key original findings of constitutive and inducible differentiation, we were unable to detect rat genes or rat gene products in any of the 11 reported cell strains and lines, including the clonal “rat” line 3 (8)# 21, and its clonal derivative 3 (8)# 21-EGFP, a line transduced with enhanced green fluorescent protein. Instead, only mouse genes were detected in all instances. These results were obtained both with purified and cloned putative “rat liver progenitor” cells grown on or off STO feeder layers (-irradiated at UCSD), and with the original frozen “rat” cells obtained from untampered-with vials derived from very early passages at the Albany Medical College. These and other unexpected observations, which tend to eliminate cell fusion as the source of this artifact, are described in detail elsewhere. 2 Ongoing observations of hepatocytic functions in these lines suggest that some aspects of the original report may yet be correct. For example, the line best characterized, 3 (8)# 21-EGFP, expresses several hepatocyte properties in vitro and following transplantation (manuscript in preparation). In conclusion, this surprising and previously unreported type of cell culture artifact should be considered by all investigators of cultured stem cell systems which require co-culture with mitotically “inhibited” feeder layer cell lines.