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Inhibition of Caspase-mediated Anoikis Is Critical for Basic Fibroblast Growth Factor-sustained Culture of Human Pluripotent Stem Cells

2009; Elsevier BV; Volume: 284; Issue: 49 Linguagem: Inglês

10.1074/jbc.m109.052290

1083-351X

Xiaofang Wang, Ge Lin, Kristen Martins‐Taylor, Hui Zeng, Ren‐He Xu,

CRISPR and Genetic Engineering

Apoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix. By using the defined mTeSR1 medium and collecting freshly detached cells, we found here that human pluripotent stem (PS) cells including embryonic stem (ES) cells and induced pluripotent stem cells are subject to constant anoikis in culture, which is escalated in the absence of basic fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and differentiation of the remaining adherent cells without affecting their cell cycle progression. Insulin-like growth factor 2 (IGF2) has previously been reported to act downstream of FGF signaling to support self-renewal of human ES cells. However, we found that IGF2 cannot substitute bFGF in the TeSR1-supported culture, although endogenous IGF signaling is required to sustain self-renewal of human ES cells. On the other hand, all of the bFGF withdrawal effects observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We further demonstrated that the bFGF-repressed anoikis is dependent on activation of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of pre-detachment apoptosis and differentiation of the cells. Because previous studies of human PS cells have been focused on attached cells, our findings revealed a neglected role of bFGF in sustaining self-renewal of human PS cells: preventing them from anoikis via inhibition of caspase activation. Apoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix. By using the defined mTeSR1 medium and collecting freshly detached cells, we found here that human pluripotent stem (PS) cells including embryonic stem (ES) cells and induced pluripotent stem cells are subject to constant anoikis in culture, which is escalated in the absence of basic fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and differentiation of the remaining adherent cells without affecting their cell cycle progression. Insulin-like growth factor 2 (IGF2) has previously been reported to act downstream of FGF signaling to support self-renewal of human ES cells. However, we found that IGF2 cannot substitute bFGF in the TeSR1-supported culture, although endogenous IGF signaling is required to sustain self-renewal of human ES cells. On the other hand, all of the bFGF withdrawal effects observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We further demonstrated that the bFGF-repressed anoikis is dependent on activation of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of pre-detachment apoptosis and differentiation of the cells. Because previous studies of human PS cells have been focused on attached cells, our findings revealed a neglected role of bFGF in sustaining self-renewal of human PS cells: preventing them from anoikis via inhibition of caspase activation. IntroductionFibroblast growth factor (FGF) 2The abbreviations used are: FGFfibroblast growth factorbFGFbasic FGFPS cellspluripotent stem cellsES cellsembryonic stem cellsiPS cellsinduced PS cellsIGFinsulin-like growth factorROCKRho-associated kinaseT1mTeSR1and T1/F0T1 without bFGFz-VAD-FMKbenzyloxycarbonyl-valyl-alanyl-aspartyl (O-methyl)-fluoromethyl ketoneTGFtransforming growth factorERKextracellular signal-regulated kinaseMEKmitogen-activated protein kinase/ERK kinaseFACSfluorescence-activated cell sorter7-AAD7-amino-actinomycin DBrdUrd5-bromo2′-deoxy-uridineF-Actinfilamentous actinMLCmyosin light chainDMSOdimethyl sulfoxideBIMBcl-2-interacting mediator of cell death. signaling plays important roles in the regulation of early embryogenesis as well as in embryonic stem (ES) cell self-renewal and differentiation. It supports the self-renewal of human ES cells but is required for differentiation of mouse ES cells into a number of lineages (1.Rossant J. Cell. 2008; 132: 527-531Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 2.McDevitt T.C. Palecek S.P. Curr. Opin. Biotechnol. 2008; 19: 527-533Crossref PubMed Scopus (64) Google Scholar). Basic FGF (bFGF or FGF2), at 4 ng/ml, was first used to supplement the medium used to culture human ES cells on mouse embryonic fibroblast feeder cells (3.Amit M. Carpenter M.K. Inokuma M.S. Chiu C.P. Harris C.P. Waknitz M.A. Itskovitz-Eldor J. Thomson J.A. Dev. Biol. 2000; 227: 271-278Crossref PubMed Scopus (1208) Google Scholar) and then was used to supplement medium conditioned on mouse embryonic fibroblasts for the feeder-free culture of human ES cells on Matrigel (BD Biosciences, San Jose, CA) (4.Xu C. Inokuma M.S. Denham J. Golds K. Kundu P. Gold J.D. Carpenter M.K. Nat. Biotechnol. 2001; 19: 971-974Crossref PubMed Scopus (1573) Google Scholar). We have previously found that high dose (40 ng/ml) bFGF can synergize with Noggin, an antagonist of bone morphogenetic proteins, to maintain human ES cell culture without the need for feeders or feeder-conditioned medium (5.Xu R.H. Peck R.M. Li D.S. Feng X. Ludwig T. Thomson J.A. Nat. Methods. 2005; 2: 185-190Crossref PubMed Scopus (827) Google Scholar). Bone morphogenetic proteins belong to the transforming growth factor β (TGFβ) superfamily and can induce human ES cell differentiation to trophoblast (6.Xu R.H. Chen X. Li D.S. Li R. Addicks G.C. Glennon C. Zwaka T.P. Thomson J.A. Nat. Biotechnol. 2002; 20: 1261-1264Crossref PubMed Scopus (866) Google Scholar) or primitive endoderm (7.Pera M.F. Andrade J. Houssami S. Reubinoff B. Trounson A. Stanley E.G. Ward-van Oostwaard D. Mummery C. J. Cell Sci. 2004; 117: 1269-1280Crossref PubMed Scopus (404) Google Scholar), depending on the culture contexts. Noggin is no longer necessary when the bFGF concentration is increased to 100 ng/ml to compensate for the degradation of bFGF in medium (8.Levenstein M.E. Ludwig T.E. Xu R.H. Llanas R.A. VanDenHeuvel-Kramer K. Manning D. Thomson J.A. Stem Cells. 2006; 24: 568-574Crossref PubMed Scopus (363) Google Scholar). FGF signaling also works concertedly with TGFβ signaling to inhibit bone morphogenetic protein signaling (9.Xu R.H. Sampsell-Barron T.L. Gu F. Root S. Peck R.M. Pan G. Yu J. Antosiewicz-Bourget J. Tian S. Stewart R. Thomson J.A. Cell Stem Cell. 2008; 3: 196-206Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) and synergizes with TGFβ and WNT signaling to support human ES cell culture (1.Rossant J. Cell. 2008; 132: 527-531Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 2.McDevitt T.C. Palecek S.P. Curr. Opin. Biotechnol. 2008; 19: 527-533Crossref PubMed Scopus (64) Google Scholar). The defined medium TeSR1 was formulated as serum-free, animal-free medium that supports feeder-free culture of human ES cells, which contains bFGF (100 ng/ml), TGFβ1, and lithium chloride (an activator of WNT signaling) (10.Ludwig T.E. Levenstein M.E. Jones J.M. Berggren W.T. Mitchen E.R. Frane J.L. Crandall L.J. Daigh C.A. Conard K.R. Piekarczyk M.S. Llanas R.A. Thomson J.A. Nat. Biotechnol. 2006; 24: 185-187Crossref PubMed Scopus (892) Google Scholar). It was later commercialized as mTeSR1 with bovine serum albumin to replace human serum albumin (11.Ludwig T.E. Bergendahl V. Levenstein M.E. Yu J. Probasco M.D. Thomson J.A. Nat. Methods. 2006; 3: 637-646Crossref PubMed Scopus (502) Google Scholar) (Stem Cell Technologies, Inc., Vancouver, Canada). Other defined media have included bFGF, as well, to support human ES cell culture (12.Yao S. Chen S. Clark J. Hao E. Beattie G.M. Hayek A. Ding S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6907-6912Crossref PubMed Scopus (375) Google Scholar, 13.Lu J. Hou R. Booth C.J. Yang S.H. Snyder M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5688-5693Crossref PubMed Scopus (181) Google Scholar, 14.Vallier L. Alexander M. Pedersen R.A. J. Cell Sci. 2005; 118: 4495-4509Crossref PubMed Scopus (744) Google Scholar). bFGF-supplemented media have also been used to derive and culture human induced pluripotent stem (iPS) cells (15.Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14662) Google Scholar, 16.Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar).Extensive studies have been carried out to explore the mechanism whereby FGF signaling acts on human ES cells. Many FGF receptors and ligands are expressed in human ES cells (17.Sperger J.M. Chen X. Draper J.S. Antosiewicz J.E. Chon C.H. Jones S.B. Brooks J.D. Andrews P.W. Brown P.O. Thomson J.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13350-13355Crossref PubMed Scopus (566) Google Scholar, 18.Kang H.B. Kim J.S. Kwon H.J. Nam K.H. Youn H.S. Sok D.E. Lee Y. Stem Cells Dev. 2005; 14: 395-401Crossref PubMed Scopus (84) Google Scholar) with FGFR1 (19.Dvorak P. Hampl A. Folia Histochem. Cytobiol. 2005; 43: 203-208PubMed Google Scholar) and FGF4 (20.Mayshar Y. Rom E. Chumakov I. Kronman A. Yayon A. Benvenisty N. Stem Cells. 2008; 26: 767-774Crossref PubMed Scopus (56) Google Scholar) being the most abundant species. Expression of endogenous bFGF decreases during human ES cell differentiation (21.Eiselleova L. Matulka K. Kriz V. Kunova M. Schmidtova Z. Neradil J. Tichy B. Dvorakova D. Pospisilova S. Hampl A. Dvorak P. Stem Cells. 2009; 27: 1847-1857Crossref PubMed Scopus (167) Google Scholar). Inhibition of FGF receptors with SU5402 decreases phosphorylation/activation of ERK in human ES cells and induces differentiation (22.Greber B. Lehrach H. Adjaye J. BMC Dev. Biol. 2007; 7: 46Crossref PubMed Scopus (61) Google Scholar), whereas exogenous bFGF increases phosphorylation/activation of ERK in the cells (18.Kang H.B. Kim J.S. Kwon H.J. Nam K.H. Youn H.S. Sok D.E. Lee Y. Stem Cells Dev. 2005; 14: 395-401Crossref PubMed Scopus (84) Google Scholar, 19.Dvorak P. Hampl A. Folia Histochem. Cytobiol. 2005; 43: 203-208PubMed Google Scholar). MEK/ERK cascade cooperates with phosphatidylinositol 3-kinase/AKT cascade (also downstream of FGF receptor signaling) to maintain self-renewal of the cells, and inhibition of MEK/ERK activity causes a loss of the self-renewal capacity of human ES cells (23.Li J. Wang G. Wang C. Zhao Y. Zhang H. Tan Z. Song Z. Ding M. Deng H. Differentiation. 2007; 75: 299-307Crossref PubMed Scopus (199) Google Scholar). Moreover, it has been shown that bFGF may also work by stimulating differentiated human ES cells to produce IGF2, which then activates IGF receptors on adjacent undifferentiated ES cells to sustain their self-renewal, the so-called paracrine mechanism (24.Bendall S.C. Stewart M.H. Menendez P. George D. Vijayaragavan K. Werbowetski-Ogilvie T. Ramos-Mejia V. Rouleau A. Yang J. Bossé M. Lajoie G. Bhatia M. Nature. 2007; 448: 1015-1021Crossref PubMed Scopus (499) Google Scholar).We have previously found that withdrawal of bFGF from TeSR1 medium causes a rapid decline of human ES cell proliferation and a slow reduction in the expression of pluripotency genes (9.Xu R.H. Sampsell-Barron T.L. Gu F. Root S. Peck R.M. Pan G. Yu J. Antosiewicz-Bourget J. Tian S. Stewart R. Thomson J.A. Cell Stem Cell. 2008; 3: 196-206Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). However, the detailed mechanism by which bFGF maintains self-renewal of human ES cells and promotes their proliferation remains elusive. We hypothesized that bFGF may also act on human ES cells through another mechanism irrelevant to regulation of the pluripotency genes. With this in mind, we studied cell cycle progression and apoptosis including cell death that is caused by the detachment (anoikis) of human ES and iPS cells. Our results suggest that these human pluripotent stem (PS) cells are subject to constant anoikis in culture, which is inhibited by bFGF via repression of caspase activities, and bFGF withdrawal-induced differentiation of the remaining adherent cells is also mediated by caspase.DISCUSSIONApoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by the loss of cell-matrix interaction, playing a critical role in many biological processes including development, tissue homeostasis, and cancer metastasis (32.Chiarugi P. Giannoni E. Biochem. Pharmacol. 2008; 76: 1352-1364Crossref PubMed Scopus (380) Google Scholar). Because of the adherent nature of human PS cells, previous studies have been focused on attached cells with detached cells removed and ignored. By using the defined mTeSR1 medium and collecting both the attached and freshly detached cells at various times, we have demonstrated here that human PS cells undergo constant anoikis, a process probably used by the cells to maintain balanced cell densities by expelling extra (maybe also differentiated or abnormal) cells. The caspase-ROCK1-myosin signaling appears to be associated with the anoikis, which is under the tight control of bFGF via activation of ERK and AKT, decrease of BIM, and suppression of the caspase activities. Withdrawal of bFGF removes the control and causes extra loss of the cells and hence failure of human PS cell maintenance.Inhibition of the caspase activity also enables bFGF to prevent the remaining attached human PS cells from apoptosis, which also contributes to maintenance of the homeostasis and increase of the cell number, although its impact on the cell proliferation is much less than the anti-anoikis effect of bFGF. It has been shown that bFGF prevents apoptosis in many other cell types (27.Peluso J.J. Biochem. Pharmacol. 2003; 66: 1363-1369Crossref PubMed Scopus (27) Google Scholar, 28.Mason I.J. Cell. 1994; 78: 547-552Abstract Full Text PDF PubMed Scopus (525) Google Scholar). Recently, Eiselleova et al. (21.Eiselleova L. Matulka K. Kriz V. Kunova M. Schmidtova Z. Neradil J. Tichy B. Dvorakova D. Pospisilova S. Hampl A. Dvorak P. Stem Cells. 2009; 27: 1847-1857Crossref PubMed Scopus (167) Google Scholar) reported that bFGF antagonizes irradiation- or hydrogen peroxide-induced apoptosis of human ES cells and increases the cloning efficiency of the cells by ∼20% by promoting adhesion of newly passaged cells (which also reduces the number of unattached cells) as shown previously (3.Amit M. Carpenter M.K. Inokuma M.S. Chiu C.P. Harris C.P. Waknitz M.A. Itskovitz-Eldor J. Thomson J.A. Dev. Biol. 2000; 227: 271-278Crossref PubMed Scopus (1208) Google Scholar). However, they did not address the inhibitory effect of bFGF on anoikis (cell detachment after adhesion) and apoptosis that spontaneously occurs among human ES cells, which is the key role of bFGF in sustaining human PS cell culture as revealed in this study. In the undefined medium used by Eiselleova et al., the proprietary serum replacement may contain components that favor cell attachment, thus masking the anti-anoikis effect of bFGF and resulting in a very mild increase of attached cell number in their experiments, as also observed in the report by Filipczyk et al. (34.Filipczyk A.A. Laslett A.L. Mummery C. Pera M.F. Stem Cell Res. 2007; 1: 45-60Crossref PubMed Scopus (92) Google Scholar). The absence of the masking components in the defined mTeSR1 medium and analysis of the freshly detached cells have enabled us to identify the anti-anoikis effect of bFGF reflected by remarkable increase of attached cell number (10.Ludwig T.E. Levenstein M.E. Jones J.M. Berggren W.T. Mitchen E.R. Frane J.L. Crandall L.J. Daigh C.A. Conard K.R. Piekarczyk M.S. Llanas R.A. Thomson J.A. Nat. Biotechnol. 2006; 24: 185-187Crossref PubMed Scopus (892) Google Scholar) (Fig. 1A) and decline of floating cells (Fig. 1C). We also confirmed that this effect is only obvious in mTeSR1 but not the undefined medium (data not shown).Further, we provided evidence that the NANOG protein level decreases upon bFGF withdrawal even in the presence of a translation inhibitor and that the reduction of both the NANOG protein level and OCT4+ cell ratio in human PS cells cultured in the absence of bFGF can be evidently rescued by the caspase inhibitor z-VAD-FMK. Therefore, caspases may also promote NANOG protein degradation in human PS cells as in mouse ES cells (37.Fujita J. Crane A.M. Souza M.K. Dejosez M. Kyba M. Flavell R.A. Thomson J.A. Zwaka T.P. Cell Stem Cell. 2008; 2: 595-601Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), and bFGF may sustain the NANOG level via inhibition of its degradation rather than (or in addition to) stimulation of its transcription. This coincides with our previous observations that NANOG transcription does not decline obviously and rapidly in human ES cells cultured in the absence of bFGF except when TGFβ signaling is inhibited at the same time (9.Xu R.H. Sampsell-Barron T.L. Gu F. Root S. Peck R.M. Pan G. Yu J. Antosiewicz-Bourget J. Tian S. Stewart R. Thomson J.A. Cell Stem Cell. 2008; 3: 196-206Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar).Additionally, we tested whether the anti-anoikis activity of bFGF can be executed by IGF2 because the latter has been shown to act downstream of bFGF through differentiated human ES cells as a niche (24.Bendall S.C. Stewart M.H. Menendez P. George D. Vijayaragavan K. Werbowetski-Ogilvie T. Ramos-Mejia V. Rouleau A. Yang J. Bossé M. Lajoie G. Bhatia M. Nature. 2007; 448: 1015-1021Crossref PubMed Scopus (499) Google Scholar). We found that, although IGF signaling is required to sustain human PS cell self-renewal, IGF2 cannot substitute bFGF for its effects on human PS cells cultured in the defined medium. The discrepancies between our data and those reported by Bendall et al. (24.Bendall S.C. Stewart M.H. Menendez P. George D. Vijayaragavan K. Werbowetski-Ogilvie T. Ramos-Mejia V. Rouleau A. Yang J. Bossé M. Lajoie G. Bhatia M. Nature. 2007; 448: 1015-1021Crossref PubMed Scopus (499) Google Scholar) may result from the different media used in the two studies. The serum replacement in the medium used in their study contained proprietary components, some of which might synergize with IGF2 to support human ES cell culture. However, it is still hard to explain how as high as 98% undifferentiated (OCT4+) cells observed from T1-supported culture can be maintained by endogenous IGF2 produced, if any, by less than the 2% differentiated (OCT4−) cells. A more reasonable explanation is that both FGF and IGF signaling are directly required to sustain human PS cell self-renewal, although they may regulate each other in their target cells.In summary, we have revealed in this study that 1) human pluripotent stem cells including ES and iPS cells are subject to constant anoikis, a critical mechanism that may help maintain their homeostasis; 2) activation of caspases is responsible for the anoikis as well as apoptosis and differentiation of the remaining attached cells; 3) the caspase-ROCK1-myosin signaling is associated to the anoikis; 4) bFGF protects human PS cells from all of these detrimental effects by inhibiting caspases through activation of ERK and AKT and inhibition of BIM (but independent of IGF2), with its anti-anoikis effect playing a dominant role in sustaining human PS cell proliferation; and 5) activated caspases causes NANOG degradation, which then leads to human PS cell differentiation. A model to accommodate all of these findings is schemed in Fig. 7E. It is important to point out, however, that not all apoptotic cells detach and not all detached cells are caused by anoikis (some cells die first and then detach). Because the pan-caspase inhibitor cannot completely replace bFGF to maintain long term propagation of human PS cells, bFGF must also act through other mechanisms in addition to inhibition of caspase activation. To this end, we have at least ascertained that bFGF does not accelerate the cell cycle progression of human PS cells to support their proliferation. IntroductionFibroblast growth factor (FGF) 2The abbreviations used are: FGFfibroblast growth factorbFGFbasic FGFPS cellspluripotent stem cellsES cellsembryonic stem cellsiPS cellsinduced PS cellsIGFinsulin-like growth factorROCKRho-associated kinaseT1mTeSR1and T1/F0T1 without bFGFz-VAD-FMKbenzyloxycarbonyl-valyl-alanyl-aspartyl (O-methyl)-fluoromethyl ketoneTGFtransforming growth factorERKextracellular signal-regulated kinaseMEKmitogen-activated protein kinase/ERK kinaseFACSfluorescence-activated cell sorter7-AAD7-amino-actinomycin DBrdUrd5-bromo2′-deoxy-uridineF-Actinfilamentous actinMLCmyosin light chainDMSOdimethyl sulfoxideBIMBcl-2-interacting mediator of cell death. signaling plays important roles in the regulation of early embryogenesis as well as in embryonic stem (ES) cell self-renewal and differentiation. It supports the self-renewal of human ES cells but is required for differentiation of mouse ES cells into a number of lineages (1.Rossant J. Cell. 2008; 132: 527-531Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 2.McDevitt T.C. Palecek S.P. Curr. Opin. Biotechnol. 2008; 19: 527-533Crossref PubMed Scopus (64) Google Scholar). Basic FGF (bFGF or FGF2), at 4 ng/ml, was first used to supplement the medium used to culture human ES cells on mouse embryonic fibroblast feeder cells (3.Amit M. Carpenter M.K. Inokuma M.S. Chiu C.P. Harris C.P. Waknitz M.A. Itskovitz-Eldor J. Thomson J.A. Dev. Biol. 2000; 227: 271-278Crossref PubMed Scopus (1208) Google Scholar) and then was used to supplement medium conditioned on mouse embryonic fibroblasts for the feeder-free culture of human ES cells on Matrigel (BD Biosciences, San Jose, CA) (4.Xu C. Inokuma M.S. Denham J. Golds K. Kundu P. Gold J.D. Carpenter M.K. Nat. Biotechnol. 2001; 19: 971-974Crossref PubMed Scopus (1573) Google Scholar). We have previously found that high dose (40 ng/ml) bFGF can synergize with Noggin, an antagonist of bone morphogenetic proteins, to maintain human ES cell culture without the need for feeders or feeder-conditioned medium (5.Xu R.H. Peck R.M. Li D.S. Feng X. Ludwig T. Thomson J.A. Nat. Methods. 2005; 2: 185-190Crossref PubMed Scopus (827) Google Scholar). Bone morphogenetic proteins belong to the transforming growth factor β (TGFβ) superfamily and can induce human ES cell differentiation to trophoblast (6.Xu R.H. Chen X. Li D.S. Li R. Addicks G.C. Glennon C. Zwaka T.P. Thomson J.A. Nat. Biotechnol. 2002; 20: 1261-1264Crossref PubMed Scopus (866) Google Scholar) or primitive endoderm (7.Pera M.F. Andrade J. Houssami S. Reubinoff B. Trounson A. Stanley E.G. Ward-van Oostwaard D. Mummery C. J. Cell Sci. 2004; 117: 1269-1280Crossref PubMed Scopus (404) Google Scholar), depending on the culture contexts. Noggin is no longer necessary when the bFGF concentration is increased to 100 ng/ml to compensate for the degradation of bFGF in medium (8.Levenstein M.E. Ludwig T.E. Xu R.H. Llanas R.A. VanDenHeuvel-Kramer K. Manning D. Thomson J.A. Stem Cells. 2006; 24: 568-574Crossref PubMed Scopus (363) Google Scholar). FGF signaling also works concertedly with TGFβ signaling to inhibit bone morphogenetic protein signaling (9.Xu R.H. Sampsell-Barron T.L. Gu F. Root S. Peck R.M. Pan G. Yu J. Antosiewicz-Bourget J. Tian S. Stewart R. Thomson J.A. Cell Stem Cell. 2008; 3: 196-206Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) and synergizes with TGFβ and WNT signaling to support human ES cell culture (1.Rossant J. Cell. 2008; 132: 527-531Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 2.McDevitt T.C. Palecek S.P. Curr. Opin. Biotechnol. 2008; 19: 527-533Crossref PubMed Scopus (64) Google Scholar). The defined medium TeSR1 was formulated as serum-free, animal-free medium that supports feeder-free culture of human ES cells, which contains bFGF (100 ng/ml), TGFβ1, and lithium chloride (an activator of WNT signaling) (10.Ludwig T.E. Levenstein M.E. Jones J.M. Berggren W.T. Mitchen E.R. Frane J.L. Crandall L.J. Daigh C.A. Conard K.R. Piekarczyk M.S. Llanas R.A. Thomson J.A. Nat. Biotechnol. 2006; 24: 185-187Crossref PubMed Scopus (892) Google Scholar). It was later commercialized as mTeSR1 with bovine serum albumin to replace human serum albumin (11.Ludwig T.E. Bergendahl V. Levenstein M.E. Yu J. Probasco M.D. Thomson J.A. Nat. Methods. 2006; 3: 637-646Crossref PubMed Scopus (502) Google Scholar) (Stem Cell Technologies, Inc., Vancouver, Canada). Other defined media have included bFGF, as well, to support human ES cell culture (12.Yao S. Chen S. Clark J. Hao E. Beattie G.M. Hayek A. Ding S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6907-6912Crossref PubMed Scopus (375) Google Scholar, 13.Lu J. Hou R. Booth C.J. Yang S.H. Snyder M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5688-5693Crossref PubMed Scopus (181) Google Scholar, 14.Vallier L. Alexander M. Pedersen R.A. J. Cell Sci. 2005; 118: 4495-4509Crossref PubMed Scopus (744) Google Scholar). bFGF-supplemented media have also been used to derive and culture human induced pluripotent stem (iPS) cells (15.Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14662) Google Scholar, 16.Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar).Extensive studies have been carried out to explore the mechanism whereby FGF signaling acts on human ES cells. Many FGF receptors and ligands are expressed in human ES cells (17.Sperger J.M. Chen X. Draper J.S. Antosiewicz J.E. Chon C.H. Jones S.B. Brooks J.D. Andrews P.W. Brown P.O. Thomson J.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13350-13355Crossref PubMed Scopus (566) Google Scholar, 18.Kang H.B. Kim J.S. Kwon H.J. Nam K.H. Youn H.S. Sok D.E. Lee Y. Stem Cells Dev. 2005; 14: 395-401Crossref PubMed Scopus (84) Google Scholar) with FGFR1 (19.Dvorak P. Hampl A. Folia Histochem. Cytobiol. 2005; 43: 203-208PubMed Google Scholar) and FGF4 (20.Mayshar Y. Rom E. Chumakov I. Kronman A. Yayon A. Benvenisty N. Stem Cells. 2008; 26: 767-774Crossref PubMed Scopus (56) Google Scholar) being the most abundant species. Expression of endogenous bFGF decreases during human ES cell differentiation (21.Eiselleova L. Matulka K. Kriz V. Kunova M. Schmidtova Z. Neradil J. Tichy B. Dvorakova D. Pospisilova S. Hampl A. Dvorak P. Stem Cells. 2009; 27: 1847-1857Crossref PubMed Scopus (167) Google Scholar). Inhibition of FGF receptors with SU5402 decreases phosphorylation/activation of ERK in human ES cells and induces differentiation (22.Greber B. Lehrach H. Adjaye J. BMC Dev. Biol. 2007; 7: 46Crossref PubMed Scopus (61) Google Scholar), whereas exogenous bFGF increases phosphorylation/activation of ERK in the cells (18.Kang H.B. Kim J.S. Kwon H.J. Nam K.H. Youn H.S. Sok D.E. Lee Y. Stem Cells Dev. 2005; 14: 395-401Crossref PubMed Scopus (84) Google Scholar, 19.Dvorak P. Hampl A. Folia Histochem. Cytobiol. 2005; 43: 203-208PubMed Google Scholar). MEK/ERK cascade cooperates with phosphatidylinositol 3-kinase/AKT cascade (also downstream of FGF receptor signaling) to maintain self-renewal of the cells, and inhibition of MEK/ERK activity causes a loss of the self-renewal capacity of human ES cells (23.Li J. Wang G. Wang C. Zhao Y. Zhang H. Tan Z. Song Z. Ding M. Deng H. Differentiation. 2007; 75: 299-307Crossref PubMed Scopus (199) Google Scholar). Moreover, it has been shown that bFGF may also work by stimulating differentiated human ES cells to produce IGF2, which then activates IGF receptors on adjacent undifferentiated ES cells to sustain their self-renewal, the so-called paracrine mechanism (24.Bendall S.C. Stewart M.H. Menendez P. George D. Vijayaragavan K. Werbowetski-Ogilvie T. Ramos-Mejia V. Rouleau A. Yang J. Bossé M. Lajoie G. Bhatia M. Nature. 2007; 448: 1015-1021Crossref PubMed Scopus (499) Google Scholar).We have previously found that withdrawal of bFGF from TeSR1 medium causes a rapid decline of human ES cell proliferation and a slow reduction in the expression of pluripotency genes (9.Xu R.H. Sampsell-Barron T.L. Gu F. Root S. Peck R.M. Pan G. Yu J. Antosiewicz-Bourget J. Tian S. Stewart R. Thomson J.A. Cell Stem Cell. 2008; 3: 196-206Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). However, the detailed mechanism by which bFGF maintains self-renewal of human ES cells and promotes their proliferation remains elusive. We hypothesized that bFGF may also act on human ES cells through another mechanism irrelevant to regulation of the pluripotency genes. With this in mind, we studied cell cycle progression and apoptosis including cell death that is caused by the detachment (anoikis) of human ES and iPS cells. Our results suggest that these human pluripotent stem (PS) cells are subject to constant anoikis in culture, which is inhibited by bFGF via repression of caspase activities, and bFGF withdrawal-induced differentiation of the remaining adherent cells is also mediated by caspase.