Slager H G, Freund E, Buiting A M, Feijen A, Mummery C L
Hubrecht Laboratory, Netherlands Institute of Developmental Biology, Utrecht.
J Cell Physiol. 1993 Aug;156(2):247-56. doi: 10.1002/jcp.1041560205.
Murine embryonic stem (ES) cells are maintained in an undifferentiated state when cultured in medium conditioned by Buffalo rat liver (BRL) cells. BRL conditioned medium (CM) contains a differentiation inhibitory activity (DIA) that is synonymous with leukemia inhibitory factor (LIF). ES cells in monolayer culture can be induced to differentiate by addition of all-trans retinoic acid (RA) to the BRL CM, when they mainly form cells resembling parietal endoderm, or by culture in medium not conditioned by BRL cells. ES cells thus deprived of LIF/DIA differentiate spontaneously to a cell type that expresses Brachyury (T), a marker of early mesoderm. Northern blot analyses have shown previously that transcripts for transforming growth factor beta 1 (TGF-beta 1) are detected in undifferentiated cells while transcripts for TGF-beta 2 and TGF-beta 3 only become detectable after differentiation. We have now determined levels of TGF-beta protein in CM and in the extracellular matrix (ECM) and have used neutralizing antibodies specific for TGF-beta 1 and TGF-beta 2 that do not react with recombinant human TGF-beta 3 to determine the isoform secreted. Using the growth inhibition of mink lung CCL64 cells as a bioassay for TGF-beta activity, we demonstrate that undifferentiated ES cells secrete latent TGF-beta 1 into the medium but no activity is found in their ECM. Cells induced to differentiate with RA contain TGF-beta 2 in both active and latent forms in their CM. Likewise their ECM contains TGF-beta 2 as the sole isoform. ES cells deprived of LIF/DIA secrete both TGF-beta 1 and TGF-beta 2 isoforms in their CM but TGF-beta-like activity remains after addition of neutralizing antibodies for TGF-beta 1 and TGF-beta 2. This active TGF beta is the major component of the TGF-beta activity in this CM. By contrast, ECM from LIF/DIA deprived cells contains only the TGF-beta 1 and beta 2 isoforms. The remaining activity in CM correlates with high expression of TGF-beta 3 by Northern blot analysis in these cells. We speculate that TGF-beta 3 is secreted by these cells and may be activated more efficiently and/or in a different manner to TGF-beta 1 and TGF-beta 2, since it is present in CM only in its active form.
当在经水牛大鼠肝（BRL）细胞条件培养的培养基中培养时，小鼠胚胎干细胞（ES细胞）维持在未分化状态。BRL条件培养基（CM）含有一种与白血病抑制因子（LIF）同义的分化抑制活性（DIA）。单层培养的ES细胞，当向BRL CM中添加全反式视黄酸（RA）时可被诱导分化，此时它们主要形成类似壁内胚层的细胞，或者通过在未经BRL细胞条件培养的培养基中培养来诱导分化。如此剥夺LIF/DIA的ES细胞会自发分化为表达短尾相关转录因子（T）的细胞类型，T是早期中胚层的标志物。Northern印迹分析先前已表明，在未分化细胞中可检测到转化生长因子β1（TGF-β1）的转录本，而TGF-β2和TGF-β3的转录本仅在分化后才可检测到。我们现已测定了CM和细胞外基质（ECM）中TGF-β蛋白的水平，并使用对TGF-β1和TGF-β2特异且不与重组人TGF-β3反应的中和抗体来确定分泌的异构体。利用貂肺CCL64细胞的生长抑制作为TGF-β活性的生物测定方法，我们证明未分化的ES细胞向培养基中分泌潜伏性TGF-β1，但在其ECM中未发现活性。用RA诱导分化的细胞在其CM中含有活性和潜伏形式的TGF-β2。同样，它们的ECM中含有TGF-β2作为唯一的异构体。剥夺LIF/DIA的ES细胞在其CM中分泌TGF-β1和TGF-β2两种异构体，但在添加针对TGF-β1和TGF-β2的中和抗体后仍保留TGF-β样活性。这种活性TGF-β是该CM中TGF-β活性的主要成分。相比之下，来自剥夺LIF/DIA细胞的ECM仅含有TGF-β1和β2异构体。通过Northern印迹分析，CM中剩余的活性与这些细胞中TGF-β3的高表达相关。我们推测TGF-β3由这些细胞分泌，并且可能比TGF-β1和TGF-β2更有效地被激活和/或以不同方式被激活，因为它仅以活性形式存在于CM中。
