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Inhibitors of Src and Focal Adhesion Kinase Promote Endocrine Specification

IMPACT ON THE DERIVATION OF β-CELLS FROM HUMAN PLURIPOTENT STEM CELLS*
  • Ivka Afrikanova
    Footnotes
    Affiliations
    Department of Pediatrics, University of California San Diego, San Diego, California 92121

    Pediatric Diabetes Research Center, University of California San Diego, San Diego, California 92121
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  • Mayra Yebra
    Footnotes
    Affiliations
    Department of Pediatrics, University of California San Diego, San Diego, California 92121

    Pediatric Diabetes Research Center, University of California San Diego, San Diego, California 92121
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  • Megan Simpkinson
    Affiliations
    Department of Pediatrics, University of California San Diego, San Diego, California 92121

    Pediatric Diabetes Research Center, University of California San Diego, San Diego, California 92121
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  • Yang Xu
    Affiliations
    Division of Biological Science, University of California San Diego, San Diego, California 92121
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  • Alberto Hayek
    Affiliations
    Department of Pediatrics, University of California San Diego, San Diego, California 92121

    Pediatric Diabetes Research Center, University of California San Diego, San Diego, California 92121
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  • Anthony Montgomery
    Correspondence
    To whom correspondence should be addressed: The Pediatric Diabetes Research Center, University of California San Diego, 3525 John Hopkins Ct., San Diego, CA 92121. Tel.: 858-822-1995; Fax: 858-558-3495
    Affiliations
    Department of Pediatrics, University of California San Diego, San Diego, California 92121

    Pediatric Diabetes Research Center, University of California San Diego, San Diego, California 92121
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the American Diabetes Association (Innovation Award), by the Juvenile Diabetes Research Foundation (Regular Research Grant), by the California Institute of Regenerative Medicine (Early Translation Grant), and by the Larry L. Hillblom Foundation (network grant).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S9.
    1 Both authors contributed equally to this work.
Open AccessPublished:August 18, 2011DOI:https://doi.org/10.1074/jbc.M111.290825
      Stepwise approaches for the derivation of β-cells from human embryonic stem cells have been described. However, low levels of endocrine specification limit the final yield of insulin-producing β-cells. In this study, we show that the pyrrolo-pyrimidine Src family kinase (SFK) inhibitor PP2 effectively promotes the endocrine specification of human embryonic stem cell derivatives based on its capacity to induce the expression of proendocrine transcription factors (NGN3, NEUROD1, NKX2.2, and PAX4) and to significantly increase the final yield of insulin-positive cells. We further demonstrate that PP2 inhibits the activation of focal adhesion kinase (FAK), and selective inhibition of this kinase is also sufficient to induce early endocrine commitment based on increased expression of NGN3, NEUROD1, and NKX2.2. Additional studies using dominant negative constructs and isolated human fetal pancreata suggest that c-Src is at least partially responsible for inhibiting early endocrine specification. Mechanistically, we propose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGFβR/Smad2/3 pathway. Moreover, we show that inhibition of SFK/FAK signaling suppresses cell growth, increases the expression of the β-cell-associated cyclin-dependent kinase inhibitor p57kip2, and simultaneously suppresses the expression of Id1 and Id2. This study has important implications for the derivation of β-cells for the cell-based therapy of diabetes and sheds new light on the signaling events that regulate early endocrine specification.

      Introduction

      Our understanding of the complex genetic and environmental events involved in islet development has progressed significantly in recent years, and this knowledge has provided the foundation for the development of some sophisticated stage-by-stage approaches for the derivation of β-cells from human embryonic stem cell (hESCs)
      The abbreviations used are: hESC
      human embryonic stem cell
      SFK
      Src family kinase
      FAK
      focal adhesion kinase
      CDKI
      cyclin-dependent kinase inhibitor
      ICC
      islet-like cell cluster
      pAb
      polyclonal antibody
      TGFβR
      TGFβ receptors
      Q-PCR
      quantitative PCR
      Bis-Tris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      (
      • D'Amour K.A.
      • Bang A.G.
      • Eliazer S.
      • Kelly O.G.
      • Agulnick A.D.
      • Smart N.G.
      • Moorman M.A.
      • Kroon E.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Kroon E.
      • Martinson L.A.
      • Kadoya K.
      • Bang A.G.
      • Kelly O.G.
      • Eliazer S.
      • Young H.
      • Richardson M.
      • Smart N.G.
      • Cunningham J.
      • Agulnick A.D.
      • D'Amour K.A.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Zhang D.
      • Jiang W.
      • Liu M.
      • Sui X.
      • Yin X.
      • Chen S.
      • Shi Y.
      • Deng H.
      ,
      • Maehr R.
      • Chen S.
      • Snitow M.
      • Ludwig T.
      • Yagasaki L.
      • Goland R.
      • Leibel R.L.
      • Melton D.A.
      ,
      • Chen S.
      • Borowiak M.
      • Fox J.L.
      • Maehr R.
      • Osafune K.
      • Davidow L.
      • Lam K.
      • Peng L.F.
      • Schreiber S.L.
      • Rubin L.L.
      • Melton D.
      ,
      • Jiang W.
      • Shi Y.
      • Zhao D.
      • Chen S.
      • Yong J.
      • Zhang J.
      • Qing T.
      • Sun X.
      • Zhang P.
      • Ding M.
      • Li D.
      • Deng H.
      ). For the most part, these protocols attempt to recreate in vitro the key developmental stages required for the development of bona fide β-cells including, in order, the derivation of mesendoderm, definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitors, and endocrine progenitors (
      • D'Amour K.A.
      • Bang A.G.
      • Eliazer S.
      • Kelly O.G.
      • Agulnick A.D.
      • Smart N.G.
      • Moorman M.A.
      • Kroon E.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Kroon E.
      • Martinson L.A.
      • Kadoya K.
      • Bang A.G.
      • Kelly O.G.
      • Eliazer S.
      • Young H.
      • Richardson M.
      • Smart N.G.
      • Cunningham J.
      • Agulnick A.D.
      • D'Amour K.A.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Zhang D.
      • Jiang W.
      • Liu M.
      • Sui X.
      • Yin X.
      • Chen S.
      • Shi Y.
      • Deng H.
      ,
      • Maehr R.
      • Chen S.
      • Snitow M.
      • Ludwig T.
      • Yagasaki L.
      • Goland R.
      • Leibel R.L.
      • Melton D.A.
      ,
      • Chen S.
      • Borowiak M.
      • Fox J.L.
      • Maehr R.
      • Osafune K.
      • Davidow L.
      • Lam K.
      • Peng L.F.
      • Schreiber S.L.
      • Rubin L.L.
      • Melton D.
      ,
      • Jiang W.
      • Shi Y.
      • Zhao D.
      • Chen S.
      • Yong J.
      • Zhang J.
      • Qing T.
      • Sun X.
      • Zhang P.
      • Ding M.
      • Li D.
      • Deng H.
      ). Directed differentiation through these stages is primarily achieved by a stepwise exposure to different growth factors and differentiating agents (
      • D'Amour K.A.
      • Bang A.G.
      • Eliazer S.
      • Kelly O.G.
      • Agulnick A.D.
      • Smart N.G.
      • Moorman M.A.
      • Kroon E.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Kroon E.
      • Martinson L.A.
      • Kadoya K.
      • Bang A.G.
      • Kelly O.G.
      • Eliazer S.
      • Young H.
      • Richardson M.
      • Smart N.G.
      • Cunningham J.
      • Agulnick A.D.
      • D'Amour K.A.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Zhang D.
      • Jiang W.
      • Liu M.
      • Sui X.
      • Yin X.
      • Chen S.
      • Shi Y.
      • Deng H.
      ,
      • Maehr R.
      • Chen S.
      • Snitow M.
      • Ludwig T.
      • Yagasaki L.
      • Goland R.
      • Leibel R.L.
      • Melton D.A.
      ,
      • Chen S.
      • Borowiak M.
      • Fox J.L.
      • Maehr R.
      • Osafune K.
      • Davidow L.
      • Lam K.
      • Peng L.F.
      • Schreiber S.L.
      • Rubin L.L.
      • Melton D.
      ,
      • Jiang W.
      • Shi Y.
      • Zhao D.
      • Chen S.
      • Yong J.
      • Zhang J.
      • Qing T.
      • Sun X.
      • Zhang P.
      • Ding M.
      • Li D.
      • Deng H.
      ). However, small compound inhibitors of select signaling pathways have also been used to potentiate specific developmental steps (
      • Champeris Tsaniras S.
      • Jones P.M.
      ). For example, inhibitors of the hedgehog pathway and PI3K have been used to respectively optimize the derivation of pancreatic progenitors and definitive endoderm (
      • D'Amour K.A.
      • Bang A.G.
      • Eliazer S.
      • Kelly O.G.
      • Agulnick A.D.
      • Smart N.G.
      • Moorman M.A.
      • Kroon E.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Zhang D.
      • Jiang W.
      • Liu M.
      • Sui X.
      • Yin X.
      • Chen S.
      • Shi Y.
      • Deng H.
      ,
      • Champeris Tsaniras S.
      • Jones P.M.
      ,
      • McLean A.B.
      • D'Amour K.A.
      • Jones K.L.
      • Krishnamoorthy M.
      • Kulik M.J.
      • Reynolds D.M.
      • Sheppard A.M.
      • Liu H.
      • Xu Y.
      • Baetge E.E.
      • Dalton S.
      ). However, despite such improvements, the subsequent differentiation of pancreatic progenitors into insulin-producing β-cells remains limited because of suboptimal levels of endocrine specification. Unfortunately, attempts to improve levels of endocrine commitment using small compound inhibitors, including inhibitors of the Notch pathway, have been only marginally successful (
      • D'Amour K.A.
      • Bang A.G.
      • Eliazer S.
      • Kelly O.G.
      • Agulnick A.D.
      • Smart N.G.
      • Moorman M.A.
      • Kroon E.
      • Carpenter M.K.
      • Baetge E.E.
      ,
      • Champeris Tsaniras S.
      • Jones P.M.
      ,
      • Phillips B.W.
      • Hentze H.
      • Rust W.L.
      • Chen Q.P.
      • Chipperfield H.
      • Tan E.K.
      • Abraham S.
      • Sadasivam A.
      • Soong P.L.
      • Wang S.T.
      • Lim R.
      • Sun W.
      • Colman A.
      • Dunn N.R.
      ).
      The impact of Notch, hedgehog, and PI3K signaling on β-cell derivation is now well documented (
      • Champeris Tsaniras S.
      • Jones P.M.
      ); however, the contribution of other signaling pathways or intermediates remains unknown or ill defined. Recent studies have shown that individual members of the Src family of protein-tyrosine kinases (SFKs) have a major impact on early ESC differentiation as well as other late stage differentiation events (
      • Annerén C.
      • Cowan C.A.
      • Melton D.A.
      ,
      • Meyn 3rd, M.A.
      • Schreiner S.J.
      • Dumitrescu T.P.
      • Nau G.J.
      • Smithgall T.E.
      ,
      • Tatosyan A.G.
      • Mizenina O.A.
      ,
      • Pala D.
      • Kapoor M.
      • Woods A.
      • Kennedy L.
      • Liu S.
      • Chen S.
      • Bursell L.
      • Lyons K.M.
      • Carter D.E.
      • Beier F.
      • Leask A.
      ,
      • Daoud G.
      • Rassart E.
      • Masse A.
      • Lafond J.
      ,
      • Theus M.H.
      • Wei L.
      • Francis K.
      • Yu S.P.
      ), however, little to nothing is known about how individual SFKs influence β-cell development. SFKs are nonreceptor protein-tyrosine kinases comprising nine members that include Blk, Fgr, Fyn, Hck, Lck, Lyn, Yes, YrK, and prototypical family member c-Src (
      • Tatosyan A.G.
      • Mizenina O.A.
      ). These kinases serve to transduce signals from various cell surface receptors including growth factor receptors, cytokine receptors, integrins, and other cell adhesion molecules (
      • Tatosyan A.G.
      • Mizenina O.A.
      ,
      • Frame M.C.
      ,
      • Bromann P.A.
      • Korkaya H.
      • Courtneidge S.A.
      ,
      • Fizazi K.
      ,
      • Mitra S.K.
      • Schlaepfer D.D.
      ,
      • Avizienyte E.
      • Brunton V.G.
      • Fincham V.J.
      • Frame M.C.
      ). In this intermediary capacity, SFKs have been shown to play an essential role in a wide range of cellular activities including growth and differentiation (
      • Tatosyan A.G.
      • Mizenina O.A.
      ,
      • Frame M.C.
      ,
      • Bromann P.A.
      • Korkaya H.
      • Courtneidge S.A.
      ,
      • Fizazi K.
      ,
      • Mitra S.K.
      • Schlaepfer D.D.
      ,
      • Avizienyte E.
      • Brunton V.G.
      • Fincham V.J.
      • Frame M.C.
      ). Importantly, PP2, a well established SFK inhibitor, has recently been shown to promote the ex vivo differentiation of several cell types including cardiomyocytes (
      • Hakuno D.
      • Takahashi T.
      • Lammerding J.
      • Lee R.T.
      ) and chondrocytes (
      • Pala D.
      • Kapoor M.
      • Woods A.
      • Kennedy L.
      • Liu S.
      • Chen S.
      • Bursell L.
      • Lyons K.M.
      • Carter D.E.
      • Beier F.
      • Leask A.
      ). Interestingly, PP2 appears to induce the differentiation of these cells via a common mechanism that involves the inhibition of focal adhesion kinase (FAK) (
      • Pala D.
      • Kapoor M.
      • Woods A.
      • Kennedy L.
      • Liu S.
      • Chen S.
      • Bursell L.
      • Lyons K.M.
      • Carter D.E.
      • Beier F.
      • Leask A.
      ,
      • Daoud G.
      • Rassart E.
      • Masse A.
      • Lafond J.
      ,
      • Hakuno D.
      • Takahashi T.
      • Lammerding J.
      • Lee R.T.
      ). FAK is a broadly expressed cytoplasmic protein-tyrosine kinase that is activated by integrin ligation and clustering, by growth factor stimulation, and by G-protein-linked receptor activation (
      • Mitra S.K.
      • Schlaepfer D.D.
      ,
      • Lim S.T.
      • Mikolon D.
      • Stupack D.G.
      • Schlaepfer D.D.
      ,
      • Schlaepfer D.D.
      • Mitra S.K.
      ,
      • Vadali K.
      • Cai X.
      • Schaller M.D.
      ). Among other things, FAK recruits and activates various SFKs, including the prototypical c-Src (
      • Mitra S.K.
      • Schlaepfer D.D.
      ,
      • Lim S.T.
      • Mikolon D.
      • Stupack D.G.
      • Schlaepfer D.D.
      ,
      • Schlaepfer D.D.
      • Mitra S.K.
      ,
      • Vadali K.
      • Cai X.
      • Schaller M.D.
      ). The subsequent formation of FAK-SFK complexes results in further FAK phosphorylation, which then triggers the activation of other downstream signaling cascades that then influence growth and differentiation (
      • Mitra S.K.
      • Schlaepfer D.D.
      ,
      • Lim S.T.
      • Mikolon D.
      • Stupack D.G.
      • Schlaepfer D.D.
      ,
      • Schlaepfer D.D.
      • Mitra S.K.
      ,
      • Vadali K.
      • Cai X.
      • Schaller M.D.
      ). The specific mechanism(s) whereby inhibition of SFK/FAK signaling by PP2 induces differentiation remains to be fully defined. However, several studies have shown that inhibition of SFK and FAK signaling alters the association between cells and the underlying extracellular matrix, which in turn can have profound consequences for anchorage-dependent growth and differentiation (
      • Pala D.
      • Kapoor M.
      • Woods A.
      • Kennedy L.
      • Liu S.
      • Chen S.
      • Bursell L.
      • Lyons K.M.
      • Carter D.E.
      • Beier F.
      • Leask A.
      ,
      • Bursell L.
      • Woods A.
      • James C.G.
      • Pala D.
      • Leask A.
      • Beier F.
      ). Interestingly, anchorage-dependent SFK/FAK signaling has also been shown to induce the activation of Smad2/3, possibly as a result of cross-talk between integrins and TGFβ receptors (TGFβRs) (
      • Garamszegi N.
      • Garamszegi S.P.
      • Samavarchi-Tehrani P.
      • Walford E.
      • Schneiderbauer M.M.
      • Wrana J.L.
      • Scully S.P.
      ,
      • Park M.S.
      • Kim Y.H.
      • Lee J.W.
      ). This is significant because TGFβ1-dependent activation of Smad2/3 has also recently been shown to suppress endocrine specification (
      • Nostro M.C.
      • Sarangi F.
      • Ogawa S.
      • Holtzinger A.
      • Corneo B.
      • Li X.
      • Micallef S.J.
      • Park I.H.
      • Basford C.
      • Wheeler M.B.
      • Daley G.Q.
      • Elefanty A.G.
      • Stanley E.G.
      • Keller G.
      ,
      • Rezania A.
      • Riedel M.J.
      • Wideman R.D.
      • Karanu F.
      • Ao Z.
      • Warnock G.L.
      • Kieffer T.J.
      ). Finally, it is noteworthy that PP2 and related SFK antagonists have also been shown to regulate the expression of both cyclin-dependent kinase inhibitors (CDKIs) and inhibitors of differentiation proteins (Ids), which together can have a major can impact on cell fate determination and/or differentiation (
      • Bursell L.
      • Woods A.
      • James C.G.
      • Pala D.
      • Leask A.
      • Beier F.
      ,
      • Walker J.L.
      • Wolff I.M.
      • Zhang L.
      • Menko A.S.
      ,
      • Xing J.
      • Zhang Z.
      • Mao H.
      • Schnellmann R.G.
      • Zhuang S.
      ,
      • Gautschi O.
      • Tepper C.G.
      • Purnell P.R.
      • Izumiya Y.
      • Evans C.P.
      • Green T.P.
      • Desprez P.Y.
      • Lara P.N.
      • Gandara D.R.
      • Mack P.C.
      • Kung H.J.
      ).
      In this study, we show for the first time that the SFK inhibitor PP2 can be used to significantly increase endocrine specification and the subsequent derivation of insulin producing β-cells from hESCs. Moreover, we confirm that this increase in endocrine commitment can be attributed to the inhibition of FAK as well as the prototypical SFK c-Src. Inhibition of SFK and FAK activity is further shown to limit Smad2/3 activation, which in turn promotes endocrine specification (
      • Nostro M.C.
      • Sarangi F.
      • Ogawa S.
      • Holtzinger A.
      • Corneo B.
      • Li X.
      • Micallef S.J.
      • Park I.H.
      • Basford C.
      • Wheeler M.B.
      • Daley G.Q.
      • Elefanty A.G.
      • Stanley E.G.
      • Keller G.
      ,
      • Rezania A.
      • Riedel M.J.
      • Wideman R.D.
      • Karanu F.
      • Ao Z.
      • Warnock G.L.
      • Kieffer T.J.
      ). Finally, we show that inhibition of SFK/FAK activity inhibits progenitor cell proliferation, induces the expression of the human β-cell-associated CDKI p57kip2, and suppresses the expression of both Id1 and Id2.

      DISCUSSION

      We have demonstrated that the SFK inhibitor PP2 promotes endocrine specification and the subsequent derivation of insulin-producing cells. We propose that PP2 induces endocrine specification, at least in part, by inhibiting the activation of FAK. These findings are consistent with prior reports showing that inhibition of FAK by PP2 also promotes the derivation of cardiomyocytes and chondrocytes (
      • Pala D.
      • Kapoor M.
      • Woods A.
      • Kennedy L.
      • Liu S.
      • Chen S.
      • Bursell L.
      • Lyons K.M.
      • Carter D.E.
      • Beier F.
      • Leask A.
      ,
      • Hakuno D.
      • Takahashi T.
      • Lammerding J.
      • Lee R.T.
      ,
      • Bursell L.
      • Woods A.
      • James C.G.
      • Pala D.
      • Leask A.
      • Beier F.
      ). Because SFKs, including c-Src, promote the activation and catalytic activity of FAK, it is not surprising that PP2 can function as a potent inhibitor of FAK activation (
      • Mitra S.K.
      • Schlaepfer D.D.
      ). However, whether inhibition of dual SFK/FAK signaling ultimately serves to induce differentiation is likely to be dependent on the cell type targeted. Thus, PP2 has been shown to actually inhibit the differentiation of certain cell types, including neuroectodermal cells (
      • Hakuno D.
      • Takahashi T.
      • Lammerding J.
      • Lee R.T.
      ) and myofibroblasts (
      • Thannickal V.J.
      • Lee D.Y.
      • White E.S.
      • Cui Z.
      • Larios J.M.
      • Chacon R.
      • Horowitz J.C.
      • Day R.M.
      • Thomas P.E.
      ), and in this study we found no evidence that PP2 induces the emergence of exocrine cells.
      Several lines of evidence suggest that inhibition of SFK/FAK signaling by PP2 potentiates endocrine differentiation by inhibiting the TGFβR/Smad2/3 pathway. Recent studies have shown that pharmacological inhibitors that target the TGFβ type I receptor ALK5 (ALK5 inhibitor II) or ALK5 and its relatives ALK4 and ALK7 (SB431542) promote the endocrine specification of hESC derivatives (
      • Rezania A.
      • Riedel M.J.
      • Wideman R.D.
      • Karanu F.
      • Ao Z.
      • Warnock G.L.
      • Kieffer T.J.
      ) and ultimately increase β-cell yields (
      • Nostro M.C.
      • Sarangi F.
      • Ogawa S.
      • Holtzinger A.
      • Corneo B.
      • Li X.
      • Micallef S.J.
      • Park I.H.
      • Basford C.
      • Wheeler M.B.
      • Daley G.Q.
      • Elefanty A.G.
      • Stanley E.G.
      • Keller G.
      ). Using ALK5 inhibitor II, we have been able to confirm that this is also true using our own differentiation method. Importantly, we can confirm that PP2 and PF228 also inhibit the activation of Smad2. This is consistent with prior reports demonstrating that anchorage-dependent activation of SFK/FAK results in the activation of Smad2/3, possibly because of cross-talk between integrins and TGFβRs (
      • Garamszegi N.
      • Garamszegi S.P.
      • Samavarchi-Tehrani P.
      • Walford E.
      • Schneiderbauer M.M.
      • Wrana J.L.
      • Scully S.P.
      ,
      • Park M.S.
      • Kim Y.H.
      • Lee J.W.
      ). In this regard it has been shown that TGFβ1 ligation results in enhanced FAK activation (
      • Park M.S.
      • Kim Y.H.
      • Lee J.W.
      ). Moreover, it has also been shown that integrin/TGFβR cross-talk can occur even in the absence of TGFβ ligation (
      • Garamszegi N.
      • Garamszegi S.P.
      • Samavarchi-Tehrani P.
      • Walford E.
      • Schneiderbauer M.M.
      • Wrana J.L.
      • Scully S.P.
      ).
      Several studies have shown that SFK inhibitors can promote cell cycle exit and terminal differentiation by inducing the expression of CDKIs including p57kip2 (
      • Bursell L.
      • Woods A.
      • James C.G.
      • Pala D.
      • Leask A.
      • Beier F.
      ,
      • Walker J.L.
      • Wolff I.M.
      • Zhang L.
      • Menko A.S.
      ,
      • Xing J.
      • Zhang Z.
      • Mao H.
      • Schnellmann R.G.
      • Zhuang S.
      ). In this regard, we can confirm that PP2 also induces p57kip2 expression in a subset of NGN3-positive endocrine progenitors. Interestingly, inactivation of the transcription factor HES1 in mice has been shown to induce p57kip2 expression, and this in turn drives pancreatic progenitors out of the cell cycle, forcing them to undergo precocious differentiation (
      • Georgia S.
      • Soliz R.
      • Li M.
      • Zhang P.
      • Bhushan A.
      ). In this mouse model, however, p57kip2 failed to co-localize with endocrine progenitors, and p57kip2 is not expressed by β-cells in the rat (
      • Georgia S.
      • Soliz R.
      • Li M.
      • Zhang P.
      • Bhushan A.
      ,
      • Potikha T.
      • Kassem S.
      • Haber E.P.
      • Ariel I.
      • Glaser B.
      ). However, it should be noted that p57kip2 is expressed by post-mitotic β-cells in human islets (
      • Kassem S.A.
      • Ariel I.
      • Thornton P.S.
      • Hussain K.
      • Smith V.
      • Lindley K.J.
      • Aynsley-Green A.
      • Glaser B.
      ,
      • Bar Y.
      • Russ H.A.
      • Knoller S.
      • Ouziel-Yahalom L.
      • Efrat S.
      ). Based on this observation and our own findings, p57kip2 could have an instructive role in the specification and differentiation of human β-cells, and repressing SFK/FAK signaling should serve to promote this process.
      Based on the induction of NGN3, NKX2.2, NEUROD1, and somatostatin by PF-228, we propose that inhibition of FAK is sufficient to induce endocrine commitment. However, in contrast to PP2, this inhibitor failed to induce the expression of either insulin or glucagon, suggesting that other specification factors associated with the α- and β-cell lineage need to be induced. In this regard, a prior report has shown that ectopic expression of NGN3 in ductal epithelial cells effectively induces markers of early islet cell differentiation (e.g. NEUROD1 and NKX2.2), but subsequent differentiation tended to favor the development of somatostatin-positive cells (
      • Boretti M.I.
      • Gooch K.J.
      ). The authors suggest that this may be due to an intrinsic deficit in the basal expression of other transcription factors such as Pax4, Pax6, or Nkx6.1 (
      • Boretti M.I.
      • Gooch K.J.
      ). This said, it is noteworthy that PP2, which did promote insulin expression, was more effective than PF-228 in inducing the expression of PAX4. Accordingly, FAK inhibition alone may not induce the threshold of PAX4 expression required for β-cell development. One explanation based on our own observations could be that PF228 is less effective in suppressing the expression of Id2. Higher levels of Id2 may then suppress the activity of basic HLH transcription factors required for β-cell development. In this regard, it is noteworthy that Id2 has been shown to inhibit the activity of NeuroD1 and subsequently inhibits PAX6 expression and endocrine differentiation (
      • Hua H.
      • Zhang Y.Q.
      • Dabernat S.
      • Kritzik M.
      • Dietz D.
      • Sterling L.
      • Sarvetnick N.
      ). In addition to inhibiting FAK, PP2 will broadly inhibit all SFK activity, and this may also be important for the optimal induction of PAX4. Moreover, it should be noted that the retraction of cell monolayers into cord-like structures observed in the presence of PP2 was not observed after the addition of PF-228. Thus, it is conceivable that a shift from cell-matrix to cell-cell association is required for the optimal emergence of insulin- and glucagon-positive cells.
      Our work with isolated fetal pancreata indicates that selective inhibition of c-Src is sufficient to induce NGN3, NEUROD1, and insulin expression. Such a role is consistent with prior reports showing that selective inhibition of c-Src inhibits cell growth (
      • Xing J.
      • Zhang Z.
      • Mao H.
      • Schnellmann R.G.
      • Zhuang S.
      ), whereas active Src effectively prevents differentiation (
      • Vardimon L.
      • Fox L.E.
      • Cohen-Kupiec R.
      • Degenstein L.
      • Moscona A.A.
      ). Moreover, c-Src has been shown to activate FAK and to inhibit the expression or stability of both p57kip2 and p27kip1 (
      • Xing J.
      • Zhang Z.
      • Mao H.
      • Schnellmann R.G.
      • Zhuang S.
      ).
      The observation that the SFK inhibitor PP2 can be used to significantly enhance the derivation of β-cells from hESCs has significant translational implications. Islet transplantation into type I diabetics has been shown to provide clear benefits in terms of glucose control, but a critical shortage of donor pancreata means that few can benefit from this approach. Any small compound inhibitors that can be used to significantly enhance the ex vivo derivation of β-cells prior to transplantation should help to ameliorate this problem and thus bring us closer to a universal cell-based therapy for type I diabetes.

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