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

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 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 a role in processes including and P. E. 2008; PubMed Scopus Google Scholar). Because of the adherent of human PS cells, previous studies have been focused on attached cells with detached cells and By using the defined mTeSR1 medium and collecting the attached and freshly detached cells at we have demonstrated here that human PS cells constant a used by the cells to maintain cell by also differentiated or cells. The caspase-ROCK1-myosin signaling to be associated with the which is the of bFGF via activation of ERK and of and of the caspase Withdrawal of bFGF the and causes loss of the cells and of human PS cell of the caspase activity also bFGF to the remaining attached human PS cells from which also to of the homeostasis and of the cell although on the cell proliferation is the of It has been shown that bFGF apoptosis in other cell 2003; PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). (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 or apoptosis of human ES cells and increases the of the cells by by of cells also the number of 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, the of bFGF on anoikis detachment and apoptosis that human ES cells, which is the role of bFGF in sustaining human PS cell culture as revealed in this the medium used by the serum may that cell the of bFGF and in a of attached cell number in their as also observed in the by Mummery C. M.F. Stem Cell 2007; PubMed Scopus Google Scholar). The absence of the in the defined mTeSR1 medium and of the freshly detached cells have to the of bFGF by 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) and decline of cells We also that this is in mTeSR1 but the medium we that the protein decreases bFGF withdrawal in the of a inhibitor and that the reduction of the protein and cell in human PS cells in the absence of bFGF can be by the caspase inhibitor z-VAD-FMK. may also protein degradation in human PS cells as in mouse ES cells J. M.K. M. M. R.A. Thomson J.A. Zwaka T.P. Cell Stem Cell. 2008; 2: Full Text Full Text PDF PubMed Scopus Google Scholar), and bFGF may sustain the via inhibition of degradation in of with our previous that decline and in human ES cells in the absence of bFGF when TGFβ signaling is inhibited at the (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 we the activity of bFGF can be by IGF2 the has been shown to act downstream of bFGF through differentiated human ES cells as a (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 although IGF signaling is required to sustain human PS cell self-renewal, IGF2 cannot substitute bFGF for effects on human PS cells in the defined The our and reported by (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 from the media used in the two The serum in the medium used in their of which synergize with IGF2 to support human ES cell However, it is to as high as undifferentiated cells observed from culture can be by endogenous IGF2 by the differentiated cells. is that FGF and IGF signaling are required to sustain human PS cell self-renewal, although may other in their we have revealed in this that human pluripotent stem cells including ES and iPS cells are subject to constant a mechanism that may maintain their activation of is for the anoikis as well as apoptosis and differentiation of the remaining attached the caspase-ROCK1-myosin signaling is associated to the bFGF human PS cells from all of these effects by through activation of ERK and AKT and inhibition of independent of with a role in sustaining human PS cell and causes which then to human PS cell differentiation. to all of these findings is in It is important to that all cells and all detached cells are caused by anoikis cells first and then Because the inhibitor cannot replace bFGF to maintain of human PS cells, bFGF also act through other in to inhibition of caspase activation. this we have at that bFGF the cell cycle progression of human PS cells to support their 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

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