title: Inhibitors of Src and focal adhesion kinase promote endocrine specification: impact on the derivation of β-cells from human pluripotent stem cells.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ivka
authors.initials: I
authors.lastname: Afrikanova
authors.affiliation: 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.
authors.forename: Mayra
authors.initials: M
authors.lastname: Yebra
authors.affiliation: 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.
authors.forename: Megan
authors.initials: M
authors.lastname: Simpkinson
authors.affiliation: Division of Biological Science, University of California San Diego, San Diego, California 92121.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: 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.
authors.forename: Alberto
authors.initials: A
authors.lastname: Hayek
authors.affiliation: 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. Electronic address: ammontgo@ucsd.edu.
authors.forename: Anthony
authors.initials: A
authors.lastname: Montgomery
mesh_terms: D000943:Antigens, Differentiation / Q000096:biosynthesis; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D064987:Cell- and Tissue-Based Therapy; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000378:metabolism; D003920:Diabetes Mellitus / Q000378:metabolism / Q000628:therapy; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000096:biosynthesis; D051797:Inhibitor of Differentiation Protein 2 / Q000096:biosynthesis; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D014157:Transcription Factors; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C412373:AG 1879; D000943:Antigens, Differentiation; C493964:CDKN1C protein, human; D050761:Cyclin-Dependent Kinase Inhibitor p57; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; C497453:ID1 protein, human; C497450:ID2 protein, human; D051798:Inhibitor of Differentiation Protein 1; D051797:Inhibitor of Differentiation Protein 2; C000715631:NKX2-2 protein, human; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D014157:Transcription Factors; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases