Jozefczuk Justyna, Drews Katharina, Adjaye James
Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics.
J Vis Exp. 2012 Jun 21(64):3854. doi: 10.3791/3854.
In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)(1) can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial. The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium(2). Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs(3). Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)(4). Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions(5-7). Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established(8). To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells(9), adult skin cells(10), foreskin fibroblasts(11-12), amniotic mesenchymal cells(13)). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A(14). Obviously, there is an evident difference in growth factor production by mouse and human feeder cells. Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgfβ1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs(15-16). For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs. Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.
一般来说，人类胚胎干细胞（hESCs）和人类诱导多能干细胞（hiPSCs）（1）可以在多种条件下培养。然而，建立一个有效的培养这些细胞的体系并不容易。由于培养条件会影响赋予hESCs和hiPSCs多能性的基因表达，因此培养方法的优化和标准化至关重要。hESC系的建立最初是通过使用小鼠胚胎成纤维细胞（MEFs）作为饲养层细胞和含胎牛血清（FBS）的培养基（2）来描述的。接下来，FBS被敲除血清替代品（KSR）和FGF2所取代，后者可增强hESCs的增殖（3）。最后，无饲养层培养系统能够在涂有基质胶的平板上，在含KSR的条件培养基（由MEFs条件化的培养基）中培养细胞（4）。随后，hESCs的培养条件已朝着在化学成分确定的条件下进行无饲养层培养发展（5 - 7）。此外，为避免病原体和动物蛋白的潜在污染，已建立了使用无动物源成分的培养方法（8）。为了获得更好的条件，小鼠饲养细胞已被人类细胞系所取代（例如胎儿肌肉和皮肤细胞（9）、成人皮肤细胞（10）、包皮成纤维细胞（11 - 12）、羊膜间充质细胞（13））。然而，由于激活素A的分泌水平较低，使用人包皮成纤维细胞衍生的饲养层维持未分化hESCs的效率不如小鼠饲养细胞（14）。显然，小鼠和人类饲养细胞在生长因子产生方面存在明显差异。对小鼠和人类饲养细胞转录组的分析揭示了支持性细胞和非支持性细胞之间的显著差异。外源性FGF2对于维持hESCs和hiPSCs的自我更新至关重要，并且已被确定为调节饲养层细胞中Tgfβ1、激活素A和Gremlin（一种BMP拮抗剂）表达的关键因子。激活素A已被证明可诱导hESCs中OCT4、SOX2和NANOG的表达（15 - 16）。为了进行长期培养，hESCs和hiPSCs可以在有丝分裂失活的MEFs上生长，或者在无饲养层条件下，在涂有基质胶的平板上的MEF - CM（MEF条件培养基）中生长，以维持其未分化状态。两种培养条件的成功完全取决于饲养层细胞的质量，因为它们直接影响hESCs的生长。在这里，我们提出了一种优化的方法，用于分离和培养小鼠胚胎成纤维细胞（MEFs）、制备条件培养基（CM）以及酶联免疫吸附测定（ELISA），以评估培养基中激活素A的水平。
