SECRETION OF TRANSFORMING GROWTH-FACTOR-BETA ISOFORMS BY EMBRYONIC STEM-CELLS - ISOFORM AND LATENCY ARE DEPENDENT ON DIRECTION OF DIFFERENTIATION

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-beta1) are detected in undifferentiated cells while transcripts for TGF-beta2 and TGF-beta3 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 TCF-beta1 and TGF-beta2 that do not react with recombinant human TGF-beta3 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-beta1 into the medium but no activity is found in their ECM. Cells induced to differentiate with RA contain TGF-beta2 in both active and latent forms in their CM. Likewise their ECM contains TGF-beta2 as the sole isoform. ES cells deprived of LIF/DIA secrete both TGF-beta1 and TGF-beta2 isoforms in their CM but TGF-beta-like activity remains after addition of neutralizing antibodies for TGF-beta1 and TGF-beta2. This active TGFbeta is the major component of the TGF-beta activity in this CM. By contrast, ECM from LIF/DIA deprived cells contains only the TGF-beta1 and beta2 isoforms. The remaining activity in CM correlates with high expression of TGF-beta3 by Northern blot analysis in these cells. We speculate that TGF-beta3 is secreted by these cells and may be activated more efficiently and/or in a different manner to TGF-beta1 and TGF-beta2, since it is present in CM only in its active form. (C) 1993 Wiley-Liss, Inc.