@article{karakis_jabeen_britt_cordiner_mischler_li_miguel_rao_2023, title={Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions}, volume={299}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2023.104650}, abstractNote={Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFβ) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue–laminin-111–switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFβ signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFβ inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFβ inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro. Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFβ) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue–laminin-111–switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFβ signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFβ inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFβ inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro. The placenta is a complex fetal organ with a vast network of villi that ensures efficient exchange of nutrients and waste across the maternal-fetal interface. Epithelial cytotrophoblasts (CTBs) of the early human placenta give rise to all trophoblast cell types in the placenta (1Aplin J.D. Developmental cell biology of human villous trophoblast: current research problems.Int. J. Dev. Biol. 2010; 54: 323-329Crossref PubMed Scopus (95) Google Scholar, 2Knöfler M. Vasicek R. Schreiber M. Key regulatory transcription factors involved in placental trophoblast development - a review.Placenta. 2001; 22: S83-S92Crossref PubMed Scopus (45) Google Scholar, 3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar, 4Loregger T. Pollheimer J. Knöfler M. Regulatory transcription factors controlling function and differentiation of human trophoblast - a review.Placenta. 2003; 24: S104-S110Crossref PubMed Scopus (26) Google Scholar). CTBs undergo cell fusion to form the multinucleate syncytiotrophoblast (STB) that overlays the CTB layer of placental villi (1Aplin J.D. Developmental cell biology of human villous trophoblast: current research problems.Int. J. Dev. Biol. 2010; 54: 323-329Crossref PubMed Scopus (95) Google Scholar, 3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar). The STB is subsequently bathed in maternal blood at ∼10 weeks of gestation, when blood flow to the placenta is established (5Caniggia I. Winter J. Lye S.J. Post M. Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia.Placenta. 2000; 21: S25-S30Crossref PubMed Scopus (313) Google Scholar, 6Tuuli M.G. Longtine M.S. Nelson D.M. Review: oxygen and trophoblast biology - a source of controversy.Placenta. 2011; 32: S109-S118Crossref PubMed Scopus (120) Google Scholar, 7Zhou Y. Genbacev O. Damsky C.H. Fisher S.J. Oxygen regulates human cytotrophoblast differentiation and invasion: implications for endovascular invasion in normal pregnancy and in pre-eclampsia.J. Reprod. Immunol. 1998; 39: 197-213Crossref PubMed Scopus (94) Google Scholar). CTBs of placental villi anchored to the maternal decidua push through the syncytial layer and differentiate to extravillous trophoblasts (EVTs), first forming proliferative column trophoblasts adjacent to the villus tip (1Aplin J.D. Developmental cell biology of human villous trophoblast: current research problems.Int. J. Dev. Biol. 2010; 54: 323-329Crossref PubMed Scopus (95) Google Scholar, 3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar). At the distal end, column trophoblasts undergo an epithelial-to-mesenchymal transition (EMT) to become mature mesenchymal EVTs that invade the maternal decidua and parts of the myometrium (8Vićovac L. Aplin J.D. Epithelial-mesenchymal transition during trophoblast differentiation.Acta Anat. (Basel). 1996; 156: 202-216Crossref PubMed Scopus (209) Google Scholar, 9Thiery J.P. Epithelial-mesenchymal transitions in development and pathologies.Curr. Opin. Cell Biol. 2003; 15: 740-746Crossref PubMed Scopus (1456) Google Scholar, 10Dasilva-Arnold S. James J.L. Al-Khan A. Zamudio S. Illsley N.P. Differentiation of first trimester cytotrophoblast to extravillous trophoblast involves an epithelial-mesenchymal transition.Placenta. 2015; 36: 1412-1418Crossref PubMed Scopus (111) Google Scholar, 11Benirschke K. Burton G.J. Baergen R.N. Pathology of the Human Placenta.6th Ed. Springer, Berlin Heidelberg2012Crossref Scopus (64) Google Scholar). These invasive trophoblasts aid in remodeling maternal spiral arteries and play a critical role in establishing sufficient perfusion of the placenta with maternal blood (12Soares M.J. Iqbal K. Kozai K. Hypoxia and placental development.Birth Defects Res. 2017; 109: 1309-1329Crossref PubMed Scopus (89) Google Scholar, 13Harris L.K. Keogh R.J. Wareing M. Baker P.N. Cartwright J.E. Aplin J.D. et al.Invasive trophoblasts stimulate vascular smooth muscle cell apoptosis by a fas ligand-dependent mechanism.Am. J. Pathol. 2006; 169: 1863-1874Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 14Lash G.E. Pitman H. Morgan H.L. Innes B.A. Agwu C.N. Bulmer J.N. Decidual macrophages: key regulators of vascular remodeling in human pregnancy.J. Leukoc. Biol. 2016; 100: 315-325Crossref PubMed Scopus (121) Google Scholar, 15Pijnenborg R. Vercruysse L. Hanssens M. The uterine spiral arteries in human pregnancy: facts and controversies.Placenta. 2006; 27: 939-958Crossref PubMed Scopus (873) Google Scholar). Many pregnancy complications including preeclampsia, fetal growth restriction, miscarriage, and stillbirth are often a result of impaired arterial remodeling and evidence points to improper EVT differentiation and invasion as a major contributor to these pathologies (3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar, 16Raymond D. Peterson E. A critical review of early-onset and late-onset preeclampsia.Obstet. Gynecol. Surv. 2011; 66: 497-506Crossref PubMed Scopus (376) Google Scholar, 17Prossler J. Chen Q. Chamley L. James J.L. The relationship between TGFβ, low oxygen and the outgrowth of extravillous trophoblasts from anchoring villi during the first trimester of pregnancy.Cytokine. 2014; 68: 9-15Crossref PubMed Scopus (34) Google Scholar, 18Arnholdt H. Meisel F. Fandrey K. Löhrs U. Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies.Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1991; 60: 365-372Crossref PubMed Scopus (148) Google Scholar, 19Silver R.M. Examining the link between placental pathology, growth restriction, and stillbirth.Best Pract. Res. Clin. Obstet. Gynaecol. 2018; 49: 89-102Crossref PubMed Scopus (35) Google Scholar, 20Romero R. Kusanovic J.P. Chaiworapongsa T. Hassan S.S. Placental bed disorders in preterm labor, preterm PROM, spontaneous abortion and abruptio placentae.Best Pract. Res. Clin. Obstet. Gynaecol. 2011; 25: 313-327Crossref PubMed Scopus (144) Google Scholar). For instance, quantitative analyses of images from preeclamptic placenta biopsies demonstrate shallow trophoblast invasion compared with healthy placenta (21Naicker T. Khedun S.M. Moodley J. Pijnenborg R. Quantitative analysis of trophoblast invasion in preeclampsia.Acta Obstet. Gynecol. Scand. 2003; 82: 722-729Crossref PubMed Scopus (153) Google Scholar). Previous studies have also suggested that increased apoptosis and an inability of CTBs to differentiate toward the EVT lineage may underlie shallow trophoblast invasion in preeclampsia (22Zhou Y. Damsky C.H. Fisher S.J. Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype: one cause of defective endovascular invasion in this syndrome?.J. Clin. Invest. 1997; 99: 2152-2164Crossref PubMed Scopus (815) Google Scholar, 23Zhou Y. Gormley M.J. Hunkapiller N.M. Kapidzic M. Stolyarov Y. Feng V. et al.Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia.J. Clin. Invest. 2013; 123: 2862-2872Crossref PubMed Scopus (97) Google Scholar). Yet, despite the importance of EVT differentiation in placental health and pathology, molecular mechanisms underlying EVT differentiation and maturation to mesenchymal invasive EVTs remain poorly understood. Mechanistic studies on early human placental development have been impeded primarily due to lack of suitable model systems. There are substantial restrictions on research with fetal tissue and human embryos and significant differences between placental development in common experimental animals and humans (24Boer G.J. Ethical guidelines for the use of human embryonic or fetal tissue for experimental and clinical neurotransplantation and research.J. Neurol. 1994; 242: 1-13Crossref PubMed Scopus (106) Google Scholar, 25Pera M.F. Human embryo research and the 14-day rule.Development. 2017; 144: 1923-1925Crossref PubMed Scopus (52) Google Scholar, 26Soncin F. Khater M. To C. Pizzo D. Farah O. Wakeland A. et al.Comparative analysis of mouse and human placentae across gestation reveals species-specific regulators of placental development.Development. 2018; 145dev156273Google Scholar, 27Lee K.Y. Demayo F.J. Animal models of implantation.Reproduction. 2004; 128: 679-695Crossref PubMed Scopus (213) Google Scholar, 28Malassine A. Frendo J.-L. Evain-Brion D. A comparison of placental development and endocrine functions between the human and mouse model.Hum. Reprod. Update. 2003; 9: 531-539Crossref PubMed Scopus (350) Google Scholar, 29Kunath T. Yamanaka Y. Detmar J. MacPhee D. Caniggia I. Rossant J. et al.Developmental differences in the expression of FGF receptors between human and mouse embryos.Placenta. 2014; 35: 1079-1088Crossref PubMed Scopus (73) Google Scholar). Additionally, there are significant differences in the transcriptome profiles of immortalized cell lines and primary trophoblasts (30Bilban M. Tauber S. Haslinger P. Pollheimer J. Saleh L. Pehamberger H. et al.Trophoblast invasion: assessment of cellular models using gene expression signatures.Placenta. 2010; 31: 989-996Crossref PubMed Scopus (153) Google Scholar). Further, immortalized cell lines do not model the heterogeneity of cell types observed during trophoblast differentiation in vivo (31Hannan N.J. Paiva P. Dimitriadis E. Salamonsen L.A. Models for study of human embryo implantation: choice of cell lines?.Biol. Reprod. 2010; 82: 235-245Crossref PubMed Scopus (237) Google Scholar). In this context, human trophoblast stem cells (hTSCs) derived from early gestation primary placental samples and blastocysts have emerged as powerful in vitro models of early human placental development (32Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. et al.Derivation of human trophoblast stem cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). hTSCs, which model the CTB in vivo, can be maintained in cell culture and differentiate into STB or EVTs. In recent work, others and we have also shown that hTSCs can be derived from human pluripotent stem cells, including induced pluripotent stem cells (33Yu L. Wei Y. Duan J. Schmitz D.A. Sakurai M. Wang L. et al.Blastocyst-like structures generated from human pluripotent stem cells.Nature. 2021; 591: 620-626Crossref PubMed Scopus (190) Google Scholar, 34Amita M. Adachi K. Alexenko A.P. Sinha S. Schust D.J. Schulz L.C. et al.Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E1212-E1221Crossref PubMed Scopus (184) Google Scholar, 35Castel G. Meistermann D. Bretin B. Firmin J. Blin J. Loubersac S. et al.Induction of human trophoblast stem cells from somatic cells and pluripotent stem cells.Cell Rep. 2020; 33: 108419Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 36Wei Y. Wang T. Ma L. Zhang Y. Zhao Y. Lye K. et al.Efficient derivation of human trophoblast stem cells from primed pluripotent stem cells.Sci. Adv. 2021; 7eabf4416Crossref Scopus (37) Google Scholar, 37Dong C. Beltcheva M. Gontarz P. Zhang B. Popli P. Fischer L.A. et al.Derivation of trophoblast stem cells from naïve human pluripotent stem cells.Elife. 2020; 9e52504Crossref Scopus (143) Google Scholar, 38Mischler A. Karakis V. Mahinthakumar J. Carberry C.K. Miguel A.S. Rager J.E. et al.Two distinct trophectoderm lineage stem cells from human pluripotent stem cells.J. Biol. Chem. 2021; 296: 100386Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 39Tiruthani K. Sarkar P. Rao B. Trophoblast differentiation of human embryonic stem cells.Biotechnol. J. 2013; 8: 421-433Crossref PubMed Scopus (9) Google Scholar, 40Sarkar P. Randall S.M. Collier T.S. Nero A. Russell T.A. Muddiman D.C. et al.Activin/nodal signaling switches the terminal fate of human embryonic stem cell-derived trophoblasts.J. Biol. Chem. 2015; 290: 8834-8848Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 41Cinkornpumin J.K. Kwon S.Y. Guo Y. Hossain I. Sirois J. Russett C.S. et al.Naive human embryonic stem cells can give rise to cells with a trophoblast-like transcriptome and Methylome.Stem Cell Rep. 2020; 15: 198-213Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and villous cytotrophoblasts from term placentas (42Wang L.J. Chen C.P. Lee Y.S. Ng P.S. Chang G.D. Pao Y.H. et al.Functional antagonism between ΔNp63α and GCM1 regulates human trophoblast stemness and differentiation.Nat. Commun. 2022; 13: 1-16PubMed Google Scholar), raising the exciting prospect of investigating pathological trophoblast development using somatic tissues (e.g., placenta) obtained at birth (42Wang L.J. Chen C.P. Lee Y.S. Ng P.S. Chang G.D. Pao Y.H. et al.Functional antagonism between ΔNp63α and GCM1 regulates human trophoblast stemness and differentiation.Nat. Commun. 2022; 13: 1-16PubMed Google Scholar, 43Sheridan M.A. Yang Y. Jain A. Lyons A.S. Yang P. Brahmasani S.R. et al.Early onset preeclampsia in a model for human placental trophoblast.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 4336-4345Crossref PubMed Scopus (42) Google Scholar). Nevertheless, current protocols for hTSC differentiation to EVT and STB limit mechanistic studies. Most current protocols for in vitro differentiation of hTSCs include transforming growth factor-beta (TGFβ) inhibitors during EVT differentiation (32Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. et al.Derivation of human trophoblast stem cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 44Io S. Iemura Y. Takashima Y. Optimized protocol for naive human pluripotent stem cell-derived trophoblast induction.STAR Protoc. 2021; 2: 100921Crossref PubMed Scopus (2) Google Scholar, 45Perez-Garcia V. Lea G. Lopez-Jimenez P. Okkenhaug H. Burton G.J. Moffett A. et al.BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion.Elife. 2021; 10e63254Crossref PubMed Scopus (14) Google Scholar, 46Bai T. Peng C.Y. Aneas I. Sakabe N. Requena D.F. Billstrand C. et al.Establishment of human induced trophoblast stem-like cells from term villous cytotrophoblasts.Stem Cell Res. 2021; 56: 102507Crossref PubMed Scopus (11) Google Scholar, 47Karvas R.M. Khan S.A. Verma S. Yin Y. Kulkarni D. Dong C. et al.Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.Cell Stem Cell. 2022; 29: 810-825.e8Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, this is problematic because TGFβ plays an important role in EVT differentiation and dysregulation of TGFβ signaling can result in placental pathologies (11Benirschke K. Burton G.J. Baergen R.N. Pathology of the Human Placenta.6th Ed. Springer, Berlin Heidelberg2012Crossref Scopus (64) Google Scholar, 17Prossler J. Chen Q. Chamley L. James J.L. The relationship between TGFβ, low oxygen and the outgrowth of extravillous trophoblasts from anchoring villi during the first trimester of pregnancy.Cytokine. 2014; 68: 9-15Crossref PubMed Scopus (34) Google Scholar, 20Romero R. Kusanovic J.P. Chaiworapongsa T. Hassan S.S. Placental bed disorders in preterm labor, preterm PROM, spontaneous abortion and abruptio placentae.Best Pract. Res. Clin. Obstet. Gynaecol. 2011; 25: 313-327Crossref PubMed Scopus (144) Google Scholar, 48Lala P.K. Graham C.H. Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors.Cancer Metastasis Rev. 1990; 9: 369-379Crossref PubMed Scopus (241) Google Scholar, 49Cheng J.C. Chang H.M. Leung P.C.K. Transforming growth factor-β1 inhibits trophoblast cell invasion by inducing snail-mediated down-regulation of vascular endothelial-cadherin protein.J. Biol. Chem. 2013; 288: 33181-33192Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 50Pollheimer J. Knöfler M. Signalling pathways regulating the invasive differentiation of human trophoblasts: a review.Placenta. 2005; 26: S21-S30Crossref PubMed Scopus (169) Google Scholar, 51Xu J. Sivasubramaniyam T. Yinon Y. Tagliaferro A. Ray J. Nevo O. et al.Aberrant TGFβ signaling contributes to altered trophoblast differentiation in preeclampsia.Endocrinology. 2016; 157: 883-899Crossref PubMed Scopus (35) Google Scholar, 52Graham C.H. Lala P.K. Mechanism of control of trophoblast invasion in situ.J. Cell Physiol. 1991; 148: 228-234Crossref PubMed Scopus (321) Google Scholar, 53Haider S. Kunihs V. Fiala C. Pollheimer J. Knöfler M. Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast.Placenta. 2017; 57: 17-25Crossref PubMed Scopus (17) Google Scholar, 54Haider S. Lackner A.I. Dietrich B. Kunihs V. Haslinger P. Meinhardt G. et al.Transforming growth factor-β signaling governs the differentiation program of extravillous trophoblasts in the developing human placenta.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2120667119Crossref PubMed Scopus (13) Google Scholar). Inhibition of TGFβ during EVT differentiation precludes investigation into the role of TGFβ signaling during normal or pathological trophoblast differentiation. Similarly, STB differentiation in vitro predominantly relies on the use of forskolin to induce cell fusion. Forskolin raises cyclic AMP concentrations causing upregulation of GCM1, which controls expression of fusion genes, syncytin-1 and syncytin-2 (55Chang C.-W. Chang G.-D. Chen H. A novel cyclic AMP/Epac1/CaMKI signaling cascade promotes GCM1 desumoylation and placental cell fusion.Mol. Cell. Biol. 2011; 31: 3820-3831Crossref PubMed Scopus (39) Google Scholar). The use of forskolin impedes studies on extracellular cues regulating STB differentiation. Finally, it is important to note that heterogeneity of cell types is a central feature of EVT differentiation in vivo. Initially, epithelial CTBs form proliferative proximal cell columns that express column markers MYC, VE-cadherin, Notch1, as well as CTB markers EGFR and ITGA6 (3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar, 8Vićovac L. Aplin J.D. Epithelial-mesenchymal transition during trophoblast differentiation.Acta Anat. (Basel). 1996; 156: 202-216Crossref PubMed Scopus (209) Google Scholar, 11Benirschke K. Burton G.J. Baergen R.N. Pathology of the Human Placenta.6th Ed. Springer, Berlin Heidelberg2012Crossref Scopus (64) Google Scholar, 32Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. et al.Derivation of human trophoblast stem cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 50Pollheimer J. Knöfler M. Signalling pathways regulating the invasive differentiation of human trophoblasts: a review.Placenta. 2005; 26: S21-S30Crossref PubMed Scopus (169) Google Scholar, 56Rosati E. Sabatini R. Rampino G. Tabilio A. Di Ianni M. Fettucciari K. et al.Constitutively activated notch signaling is involved in survival and apoptosis resistance of B-CLL cells.Blood. 2009; 113: 856-865Crossref PubMed Scopus (250) Google Scholar, 57Haider S. Meinhardt G. Saleh L. Kunihs V. Gamperl M. Kaindl U. et al.Self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta.Stem Cell Rep. 2018; 11: 537-551Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 58Haider S. Meinhardt G. Velicky P. Otti G.R. Whitley G. Fiala C. et al.Notch signaling plays a critical role in motility and differentiation of human first-trimester cytotrophoblasts.Endocrinology. 2014; 155: 263-274Crossref PubMed Scopus (46) Google Scholar, 59Haider S. Meinhardt G. Saleh L. Fiala C. Pollheimer J. Knöfler M. Notch1 controls development of the extravillous trophoblast lineage in the human placenta.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E7710-E7719Crossref PubMed Scopus (88) Google Scholar, 60Meinhardt G. Haider S. Haslinger P. Proestling K. Fiala C. Pollheimer J. et al.Wnt-dependent T-cell factor-4 controls human etravillous trophoblast motility.Endocrinology. 2014; 155: 1908-1920Crossref PubMed Scopus (46) Google Scholar, 61Hunkapiller N.M. Gasperowicz M. Kapidzic M. Plaks V. Maltepe E. Kitajewski J. et al.A role for notch signaling in trophoblast endovascular invasion and in the pathogenesis of pre-eclampsia.Development. 2011; 138: 2987-2998Crossref PubMed Scopus (130) Google Scholar, 62Plessl K. Haider S. Fiala C. Pollheimer J. Knöfler M. Expression pattern and function of notch2 in different subtypes of first trimester cytotrophoblast.Placenta. 2015; 36: 365-371Crossref PubMed Scopus (17) Google Scholar, 63Meinhardt G. Kaltenberger S. Fiala C. Knöfler M. Pollheimer J. ERBB2 gene amplification increases during the transition of proximal EGFR+ to distal HLA-G+ first trimester cell column trophoblasts.Placenta. 2015; 36: 803-808Crossref PubMed Scopus (16) Google Scholar, 64Pawar S.C. Demetriou M.C. Nagle R.B. Bowden G.T. Cress A.E. Integrin α6 cleavage: a novel modification to modulate cell migration.Exp. Cell Res. 2007; 313: 1080-1089Crossref PubMed Scopus (49) Google Scholar). Differentiating EVTs at the distal end of the epithelial column remain proliferative; however, they begin to express ITGA5 and low levels of the mature EVT marker HLA-G and lose CTB marker expression (11Benirschke K. Burton G.J. Baergen R.N. Pathology of the Human Placenta.6th Ed. Springer, Berlin Heidelberg2012Crossref Scopus (64) Google Scholar, 57Haider S. Meinhardt G. Saleh L. Kunihs V. Gamperl M. Kaindl U. et al.Self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta.Stem Cell Rep. 2018; 11: 537-551Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 62Plessl K. Haider S. Fiala C. Pollheimer J. Knöfler M. Expression pattern and function of notch2 in different subtypes of first trimester cytotrophoblast.Placenta. 2015; 36: 365-371Crossref PubMed Scopus (17) Google Scholar, 65Mccormick J. Whitley G.S.J. Le Bouteiller P. Cartwright J.E. Soluble HLA-G regulates motility and invasion of the trophoblast-derived cell line SGHPL-4.Hum. Reprod. 2009; 24: 1339-1345Crossref PubMed Scopus (35) Google Scholar). Subsequently, when these distal EVTs undergo EMT, they gain expression of ITGA1, express higher levels of HLA-G, and lose expression of markers that characterize the column trophoblasts (3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar, 66Fukushima K. Miyamoto S. Tsukimori K. Kobayashi H. Seki H. Takeda S. et al.Tumor necrosis factor and vascular endothelial growth factor induce endothelial integrin repertories, regulating endovascular differentiation and apoptosis in a human extravillous trophoblast cell Line1.Biol. Reprod. 2005; 73: 172-179Crossref PubMed Scopus (52) Google Scholar, 67Natenzon A. McFadden P. DaSilva-Arnold S.C. Zamudio S. Illsley N.P. Diminished trophoblast differentiation in early onset preeclampsia.Placenta. 2022; 120: 25-31Crossref PubMed Scopus (4) Google Scholar). Current protocols for EVT differentiation that include a passage step (32Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. et al.Derivation of human trophoblast stem cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 47Karvas R.M. Khan S.A. Verma S. Yin Y. Kulkarni D. Dong C. et al.Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.Cell Stem Cell. 2022; 29: 810-825.e8Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) do not capture EVT heterogeneity and the sequential nature of CTB differentiation as mature mesenchymal EVTs are formed. Here we present chemically defined culture conditions for differentiation of placenta- and hiPSC-derived hTSCs to EVTs and STB. Notably, our conditions do not involve a passage step and exclude forskolin and TGFβ inhibition during STB and EVT differentiation, respectively. Under these culture conditions, we identified laminin-111-mediated upregulation of hypoxia-inducible factor-alpha (HIFα) as the critical input that switches differentiation hTSCs from STB to the EVT lineage. We also investigated the effect of inhibiting TGFβ signaling on EVT and STB differentiation. Placenta-derived CT29 and CT30 hTSCs and hiPSC-derived SC102A-1 hTSCs were cultured in trophoblast stem cell medium (TSCM) as described previously (32Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. et al.Derivation of human trophoblast stem cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 38Mischler A. Karakis V. Mahinthakumar J. Carberry C.K. Miguel A.S. Rager J.E. et al.Two distinct trophectoderm lineage stem cells from human pluripotent stem cells.J. Biol. Chem. 2021; 296: 100386Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Differentiation was induced by passaging hTSCs into a defined trophoblast differentiation medium (DTDM) supplemented with epidermal growth factor (EGF) and the ROCK inhibitor, Y-27632, at passage for 2 days, and culturing them for an additional 4 days in DTDM (Fig. 1A). Upon passage, we initially observed an increase in cell number, but b}, number={5}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Karakis, Victoria and Jabeen, Mahe and Britt, John W. and Cordiner, Abigail and Mischler, Adam and Li, Feng and Miguel, Adriana San and Rao, Balaji M.}, year={2023}, month={May} }
 @article{mcarthur_cruz-teran_thatavarty_reeves_rao_2022, title={Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase}, ISSN={["2470-1343"]}, DOI={10.1021/acsomega.2c02319}, abstractNote={The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting nonoverlapping epitopes on the target analyte were each fused to nonactive fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provides the foundation for the development of split luciferase assays for detection and quantification of various targets.}, journal={ACS OMEGA}, author={McArthur, Nikki and Cruz-Teran, Carlos and Thatavarty, Apoorva and Reeves, Gregory T. and Rao, Balaji M.}, year={2022}, month={Jul} }
 @article{mcarthur_cruz-teran_thatavarty_reeves_rao_2022, title={Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase}, volume={7}, ISSN={["2470-1343"]}, DOI={10.1021/acsomega.2c0231924551ACS}, number={28}, journal={ACS OMEGA}, author={McArthur, Nikki and Cruz-Teran, Carlos and Thatavarty, Apoorva and Reeves, Gregory T. and Rao, Balaji M.}, year={2022}, month={Jul}, pages={24551–24560} }
 @article{meanor_keung_rao_2022, title={Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity}, volume={23}, ISSN={["1422-0067"]}, DOI={10.3390/ijms23031691}, abstractNote={Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.}, number={3}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Meanor, Jenna N. and Keung, Albert J. and Rao, Balaji M.}, year={2022}, month={Feb} }
 @article{waldman_rao_keung_2021, title={Mapping the residue specificities of epigenome enzymes by yeast surface display}, volume={28}, ISSN={["2451-9448"]}, DOI={10.1016/j.chembiol.2021.05.022}, abstractNote={Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here, we describe a versatile and scalable approach which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first use this system to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, a large panel of commercially available anti-acetylation antibodies are screened for their specificities, identifying many suitable and unsuitable reagents. Furthermore, this approach enables efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4 and uncovers residue interdependencies affecting p300 activity. These results show that using yeast surface display to study histone modifications is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.}, number={12}, journal={CELL CHEMICAL BIOLOGY}, author={Waldman, Alison C. and Rao, Balaji M. and Keung, Albert J.}, year={2021}, month={Dec}, pages={1772-+} }
 @article{bacon_blain_bowen_burroughs_mcarthur_menegatti_rao_2021, title={Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions}, volume={10}, ISSN={["2161-5063"]}, DOI={10.1021/acssynbio.0c00472}, abstractNote={Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.}, number={3}, journal={ACS SYNTHETIC BIOLOGY}, author={Bacon, Kaitlyn and Blain, Abigail and Bowen, John and Burroughs, Matthew and McArthur, Nikki and Menegatti, Stefano and Rao, Balaji M.}, year={2021}, month={Mar}, pages={505–514} }
 @article{bowen_schneible_bacon_labar_menegatti_rao_2021, title={Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands}, volume={22}, ISSN={["1422-0067"]}, url={https://www.mdpi.com/1422-0067/22/4/1634}, DOI={10.3390/ijms22041634}, abstractNote={We present the construction and screening of yeast display libraries of cyclic peptides wherein site-selective enzymatic cyclization of linear peptides is achieved using bacterial transglu-taminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve cyclization in the endoplasmic reticulum prior to yeast surface display. The cyclization yield was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-cyclized peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified cyclic peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.67 µM for YAP and 0.84 µM for WW as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of yeast surface display for screening transglutaminase-cyclized peptide libraries, and efficient identification of cyclic peptide ligands.}, number={4}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Bowen, John and Schneible, John and Bacon, Kaitlyn and Labar, Collin and Menegatti, Stefano and Rao, Balaji M.}, year={2021}, month={Feb} }
 @article{mischler_karakis_mahinthakumar_carberry_san miguel_rager_fry_rao_2021, title={Two distinct trophectoderm lineage stem cells from human pluripotent stem cells}, volume={296}, ISSN={["1083-351X"]}, url={http://dx.doi.org/10.1016/j.jbc.2021.100386}, DOI={10.1016/j.jbc.2021.100386}, abstractNote={The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells—they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development. The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells—they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development. Specification of the trophectoderm and the inner cell mass is the first differentiation event during human embryonic development. The trophectoderm mediates blastocyst implantation in the uterus and is the precursor to all trophoblast cells in the placenta. Upon embryo implantation, the trophectoderm forms the cytotrophoblast (CTB), a putative stem cell that can differentiate to form the two major cell types in the placenta, the extravillous trophoblast (EVT) and the syncytiotrophoblast (STB) (1Bischof P. Irminger-Finger I. The human cytotrophoblastic cell, a mononuclear chameleon.Int. J. Biochem. Cel. Biol. 2005; 37: 1-16Crossref PubMed Scopus (125) Google Scholar, 2Benirschke Kurt. Baergen R.N. Burton G. Graham J. Pathology of the Human Placenta [electronic Resource]. Springer, Heidelberg2012Crossref Scopus (48) Google Scholar). The EVTs are involved in remodeling of uterine arteries, which is critical to ensure adequate perfusion of the placenta with maternal blood, whereas the multinucleated STB mediates the nutrient and gas exchange at the maternal–fetal interface (3Yabe S. Alexenko A.P. Amita M. Yang Y. Schust D.J. Sadovsky Y. Ezashi T. Roberts R.M. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E2598-E2607Crossref PubMed Scopus (68) Google Scholar, 4Moser G. Orendi K. Gauster M. Siwetz M. Helige C. Huppertz B. The art of identification of extravillous trophoblast.Placenta. 2011; 32: 197-199Crossref PubMed Scopus (29) Google Scholar). Abnormalities in trophoblast development are associated with pregnancy-related pathologies such as miscarriage, preeclampsia, and placenta accreta. Yet, despite its relevance to maternal and fetal health, constraints on research with human embryos and early fetal tissue impede mechanistic insight into early trophoblast development. Trophoblast stem (TS) cells derived from first-trimester human placental samples and blastocyst-stage embryos have emerged as an attractive in vitro model system for early human trophoblast (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). However, restricted accessibility of embryos and placental samples from early gestation and low genetic diversity of existing cell lines limit the use of this model. In contrast, human pluripotent stem cells (hPSCs) are a more accessible source for generating in vitro models of human trophoblast. Of more importance, unlike early gestation primary samples where the projected pregnancy outcome is uncertain, human induced pluripotent stem cells (hiPSCs) can potentially provide models of validated normal and pathological trophoblast development (6Sheridan M.A. Yang Y. Jain A. Lyons A.S. Yang P. Brahmasani S.R. Dai A. Tian Y. Ellersieck M.R. Tuteja G. Schust D.J. Schulz L.C. Ezashi T. Roberts R.M. Early onset preeclampsia in a model for human placental trophoblast.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 4336-4345Crossref PubMed Scopus (20) Google Scholar). However, whether bona fide trophoblast can be obtained from hPSCs has been a subject of intense debate (7Roberts R.M. Loh K.M. Amita M. Bernardo A.S. Adachi K. Alexenko A.P. Schust D.J. Schulz L.C. Telugu B.P.V.L. Ezashi T. Pedersen R.A. Differentiation of trophoblast cells from human embryonic stem cells: To be or not to be?.Reproduction (Cambridge, England). 2014; 147: D1-D12Crossref PubMed Scopus (45) Google Scholar). A rigorous head-to-head comparison between trophoblast derived from hPSCs and their in vivo counterparts has proven difficult owing to multiple reasons. Previous studies have used varying experimental protocols (8Roberts R.M. Ezashi T. Sheridan M.A. Yang Y. Specification of trophoblast from embryonic stem cells exposed to BMP4†.Biol. Reprod. 2018; 99: 212-224Crossref PubMed Scopus (22) Google Scholar); both primary placental samples and cultures of terminally differentiated trophoblast obtained from hPSCs exhibit heterogeneity and contain many cell types, and until recently self-renewing TS-like cells had not been derived from hPSCs (9Dong C. Beltcheva M. Gontarz P. Zhang B. Popli P. Fischer L.A. Khan S.A. Park K.-M. Yoon E.-J. Xing X. Kommagani R. Wang T. Solnica-Krezel L. Theunissen T.W. Derivation of trophoblast stem cells from naïve human pluripotent stem cells.eLife. 2020; 9: e52504Crossref PubMed Scopus (57) Google Scholar, 10Cinkornpumin J.K. Kwon S.Y. Guo Y. Hossain I. Sirois J. Russett C.S. Tseng H.W. Okae H. Arima T. Duchaine T.F. Liu W. Pastor W.A. Naive human embryonic stem cells can give rise to cells with a trophoblast-like transcriptome and Methylome.Stem Cell Rep. 2020; 15: 198-213Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Li Z. Kurosawa O. Iwata H. Development of trophoblast cystic structures from human induced pluripotent stem cells in limited-area cell culture.Biochem. Biophysical Res. Commun. 2018; 505: 671-676Crossref PubMed Scopus (5) Google Scholar, 12Gao X. Nowak-Imialek M. Chen X. Chen D. Herrmann D. Ruan D. Chen A.C.H. Eckersley-Maslin M.A. Ahmad S. Lee Y.L. Kobayashi T. Ryan D. Zhong J. Zhu J. Wu J. et al.Establishment of porcine and human expanded potential stem cells.Nat. Cell Biol. 2019; 21: 687-699Crossref PubMed Scopus (120) Google Scholar). In this study, we report the derivation and maintenance of two distinct trophectoderm lineage stem cell types from hPSCs, specifically human embryonic stem cells (hESCs) and hiPSCs, in chemically defined culture conditions. The first is a CDX2- stem cell that is comparable with TS cells derived from early-gestation placental samples and similar to the villous CTB. The second is a CDX2+ cell type with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Critically, the isolation of self-renewing stem cell populations allowed a direct comparison of placenta-derived TS cells with TS cells from hPSCs; genome-wide transcriptomic analysis and functional differentiation assays demonstrate very high similarity between placenta- and hPSC-derived CDX2- TS cells. The routine derivation of TS cells from hPSCs will provide powerful tools for mechanistic studies on normal and pathological early trophoblast development. Media formulations in previous studies on trophoblast differentiation of hESCs included components such as knockout serum replacement (KSR) or bovine serum albumin (BSA) that act as carriers for lipids. Albumin-associated lipids have been implicated in activation of G-protein–coupled receptor–mediated signaling (13Yu F.-X. Zhao B. Panupinthu N. Jewell J.L. Lian I. Wang L.H. Zhao J. Yuan H. Tumaneng K. Li H. Fu X.-D. Mills G.B. Guan K.-L. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.Cell. 2012; 150: 780-791Abstract Full Text Full Text PDF PubMed Scopus (974) Google Scholar, 14Mendelson K. Evans T. Hla T. Sphingosine 1-phosphate signalling.Development (Cambridge, England). 2014; 141: 5-9Crossref PubMed Scopus (165) Google Scholar). For instance, the phospholipid sphingosine-1 phosphate (S1P) present in KSR can activate YAP signaling. YAP plays a critical role in specification of the trophectoderm in mouse (15Yagi R. Kohn M.J. Karavanova I. Kaneko K.J. Vullhorst D. DePamphilis M.L. Buonanno A. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.Development (Cambridge, England). 2007; 134: 3827-3836Crossref PubMed Scopus (353) Google Scholar, 16Knott J.G. Paul S. Transcriptional regulators of the trophoblast lineage in mammals with hemochorial placentation.Reproduction (Cambridge, England). 2014; 148: R121-R136Crossref PubMed Scopus (40) Google Scholar, 17Nishioka N. Yamamoto S. Kiyonari H. Sato H. Sawada A. Ota M. Nakao K. Sasaki H. Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos.Mech. Dev. 2008; 125: 270-283Crossref PubMed Scopus (331) Google Scholar), as well as human trophoblast development (18Saha B. Ganguly A. Home P. Bhattacharya B. Ray S. Ghosh A. Rumi M.A.K. Marsh C. French V. Gunewardena S. Paul S. TEAD4 ensures postimplantation development by promoting trophoblast self-renewal: An implication in early human pregnancy loss.Proc. Natl. Acad. Sci. 2020; 117: 202002449Crossref Scopus (20) Google Scholar, 19Meinhardt G. Haider S. Kunihs V. Saleh L. Pollheimer J. Fiala C. Hetey S. Feher Z. Szilagyi A. Than N.G. Knöfler M. Pivotal role of the transcriptional co-activator YAP in trophoblast stemness of the developing human placenta.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13562-13570Crossref PubMed Scopus (25) Google Scholar). We investigated the use of S1P in the context of trophoblast differentiation of hESCs under chemically defined culture conditions, by modifying our previous protocol that utilized KSR (20Sarkar P. Randall S.M. Collier T.S. Nero A. Russell T.A. Muddiman D.C. Rao B.M. Activin/nodal signaling Switches the terminal fate of human embryonic stem cell-derived trophoblasts.J. Biol. Chem. 2015; 290: 8834-8848Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 21Sarkar P. Mischler A. Randall S.M. Collier T.S. Dorman K.F. Boggess K.A. Muddiman D.C. Rao B.M. Identification of epigenetic factor proteins expressed in human embryonic stem cell-derived trophoblasts and in human placental trophoblasts.J. Proteome Res. 2016; 15: 2433-2444Crossref PubMed Scopus (6) Google Scholar). H1 and H9 hESCs cultured in E8 medium were differentiated for 6 days in E7 medium (E8 without transforming growth factor-beta1 [TGFβ1]) supplemented with S1P, by treatment with BMP4 and the activin/nodal inhibitor SB431542 (Fig. 1A). Under these conditions, we observed upregulation of the trophectoderm marker CDX2 and the CTB marker ELF5 (Fig. S1, A and B). Upregulation of TBX4 was observed after 6 days. However, overall there were no significant changes in markers associated with neural or mesodermal differentiation after 6 days suggesting that differentiation to these lineages did not occur (Fig. S1, A and B). Immunofluorescence analysis at day 6 confirmed expression of the pan-trophoblast marker KRT7, and CTB markers P63 and GATA3; expression of CDX2 was not observed (Figs. 1B and S1C). The putative CTB cells obtained at day 6 were investigated for their ability to differentiate to EVTs and STB, using protocols similar to those previously employed (20Sarkar P. Randall S.M. Collier T.S. Nero A. Russell T.A. Muddiman D.C. Rao B.M. Activin/nodal signaling Switches the terminal fate of human embryonic stem cell-derived trophoblasts.J. Biol. Chem. 2015; 290: 8834-8848Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). We observed formation of mesenchymal cells from epithelial cells over a 6-day period when passaged into E8 medium supplemented with epidermal growth factor (EGF) and SB431542. Immunofluorescence analysis showed expression of KRT7 and the EVT markers VE-Cadherin and HLA-G (Figs. 1C, S1D). Alternatively, passaging CTB-like cells in E6 medium (E8 without TGFβ1 and fibroblast growth factor-2 [FGF2]) supplemented with activin and EGF resulted in the formation of KRT7+ multinucleate cells expressing the STB markers hCG and syncytin over an 8-day period (Figs. 1D, S1E). Removal of S1P from the medium during hESC differentiation to CTB-like cells abolished the formation of EVTs that express HLA-G and VE-Cadherin (Figs. 1E, S2A) under identical differentiation conditions (Fig. 1A). Differentiation to STB also did not occur in the absence of S1P, as evidenced by lack of expression of syncytin and KRT7 (Figs. 1F, S2B). Also, downregulation of the trophectoderm marker CDX2 and upregulation of transcripts of neural and mesoderm markers was observed in cells after 6 days of differentiation, upon removal of S1P (Fig. S2C). Taken together these results show that CTB-like cells, similar to those in previous studies utilizing more complex culture conditions (20Sarkar P. Randall S.M. Collier T.S. Nero A. Russell T.A. Muddiman D.C. Rao B.M. Activin/nodal signaling Switches the terminal fate of human embryonic stem cell-derived trophoblasts.J. Biol. Chem. 2015; 290: 8834-8848Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), can be obtained by differentiation of hESCs in a chemically defined medium containing S1P. Furthermore, addition of exogenous S1P is necessary for hESC differentiation to trophoblast in our chemically defined culture medium. Rho GTPase signaling, downstream of G-protein–coupled receptors activated by S1P, has been implicated in nuclear localization of YAP (22Ohgushi M. Minaguchi M. Sasai Y. Rho-signaling-directed YAP/TAZ activity Underlies the long-term Survival and Expansion of human embryonic stem cells.Cell stem cell. 2015; 17: 448-461Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 23Mo J.-S. Yu F.-X. Gong R. Brown J.H. Guan K.-L. Regulation of the Hippo-YAP pathway by protease-activated receptors (PARs).Genes Dev. 2012; 26: 2138-2143Crossref PubMed Scopus (195) Google Scholar). Both Rho/RhoA associated kinase (ROCK) and nuclear YAP play a critical role in trophectoderm specification in the mouse (24Nishioka N. Inoue K. Adachi K. Kiyonari H. Ota M. Ralston A. Yabuta N. Hirahara S. Stephenson R.O. Ogonuki N. Makita R. Kurihara H. Morin-Kensicki E.M. Nojima H. Rossant J. et al.The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.Dev. Cel. 2009; 16: 398-410Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 25Kono K. Tamashiro D.A.A. Alarcon V.B. Inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling in the mouse blastocyst.Dev. Biol. 2014; 394: 142-155Crossref PubMed Scopus (79) Google Scholar). Therefore, we investigated the role of Rho/ROCK signaling and YAP in trophoblast differentiation of hESCs. The Rho/ROCK inhibitor Y-27632 was included during differentiation of hESCs to CTB-like cells and subsequent differentiation to EVT and STB to investigate the role of Rho/ROCK signaling. Under these conditions, HLA-G expression was observed in cells obtained from H9 hESCs; however, VE-Cadherin expression was weak and observed in only a few cells (Fig. S3A). On the other hand, expression of EVT markers was not observed in cells derived from H1 hESCs. In addition, presence of ROCK inhibition abolished STB formation, as shown by the lack of expression of syncytin and KRT7 (Fig. S3B). To investigate the role of YAP signaling in CTB formation from hESCs, we used an hESC cell line (H9) that expresses an inducible shRNA against YAP (H9-YAP-ishRNA) or a scrambled shRNA control (26Hsiao C. Lampe M. Nillasithanukroh S. Han W. Lian X. Palecek S.P. Human pluripotent stem cell culture density modulates YAP signaling.Biotechnol. J. 2016; 11: 662-675Crossref PubMed Scopus (26) Google Scholar). YAP knockdown abolished differentiation to EVT and STB, as evidenced by lack of expression of the relevant markers. It is notable that high cell death was observed (Fig. S3, A and B). Gene expression analysis revealed a significant reduction in ELF5 upon YAP knockdown, relative to the scrambled shRNA control (Fig. S3C). Significant downregulation of the mesodermal genes TBX4 and LMO2 was observed, whereas T was upregulated, in H9-YAP-ishRNA, relative to the scrambled control. Taken together, these results show that Rho/ROCK signaling and YAP are necessary for differentiation of hESCs to functional CTB that can give rise to both EVTs and STB, in our chemically defined culture medium. S1P acts through both receptor-mediated and receptor-independent pathways (14Mendelson K. Evans T. Hla T. Sphingosine 1-phosphate signalling.Development (Cambridge, England). 2014; 141: 5-9Crossref PubMed Scopus (165) Google Scholar, 27Maceyka M. Harikumar K.B. Milstien S. Spiegel S. Sphingosine-1-phosphate signaling and its role in disease.Trends Cell Biol. 2012; 22: 50-60Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar). To investigate the specific mechanism of S1P action during hESC differentiation to trophoblast, we replaced S1P with D-erythro-dihydrospingosine-1-phosphate (dhS1P) in our protocol. dhS1P acts as an agonist for the S1P receptors (S1PRs) but does not mediate an intracellular effect (28Van Brocklyn J.R. Lee M.-J. Menzeleev R. Olivera A. Edsall L. Cuvillier O. Thomas D.M. Coopman P.J.P. Thangada S. Liu C.H. Hla T. Spiegel S. Dual actions of sphingosine-1-phosphate: Extracellular through the G i -coupled receptor Edg-1 and intracellular to regulate proliferation and Survival.J. Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (444) Google Scholar). Replacing S1P with dhS1P yielded similar results—CTB-like cells showed expression of CDX2, GATA3, P63, and TEAD4 (Figs. 2A and S4A). Upon further differentiation as previously described (Fig. 1A), STB expressing KRT7 and hCG, and EVT expressing HLA-G and VE-Cadherin were obtained (Fig. 2, B and C; Fig. S4, B and C). These results suggest that S1PR signaling mediates the effect of exogenous S1P during hESC differentiation to trophoblast in our chemically defined medium. S1P acts extracellularly through S1PR1-5 (14Mendelson K. Evans T. Hla T. Sphingosine 1-phosphate signalling.Development (Cambridge, England). 2014; 141: 5-9Crossref PubMed Scopus (165) Google Scholar, 27Maceyka M. Harikumar K.B. Milstien S. Spiegel S. Sphingosine-1-phosphate signaling and its role in disease.Trends Cell Biol. 2012; 22: 50-60Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar); however, TBs have been shown to only express S1PR1-3 (29Johnstone E.D. Chan G. Sibley C.P. Davidge S.T. Lowen B. Guilbert L.J. Sphingosine-1-phosphate inhibition of placental trophoblast differentiation through a G(i)-coupled receptor response.J. lipid Res. 2005; 46: 1833-1839Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). We further used selective chemical agonists for S1PR1-3—CYM5442 hydrochloride, CYM5520, and CYM5541, respectively—to replace S1P in differentiation protocols previously discussed. Expression of CDX2, GATA3, P63, and TEAD4 was observed in CTB-like cells for all three agonists (Figs. 2A and S4A). Similarly, use of each agonist resulted in expression of the EVT markers HLA-G and VE-Cadherin and formation of multinucleate STB expressing KRT7 and hCG (Fig. 2, B and C; Fig. S4, B and C). However, we observed some variability between the agonists (Fig. S5). For instance, use of the S1PR2 agonist resulted in strong cytoplasmic expression of P63 and high heterogeneity in staining at day 6 relative to the other agonists. Formation of large multinucleated STB was more pronounced when the S1PR2 or S1PR3 agonists were used, as compared with the S1PR1 agonist. On the other hand, the S1PR1 and S1PR3 agonists enhanced the formation of mesenchymal EVTs, relative to the S1PR2 agonist. Taken together, our results further confirmed that S1PR signaling mediates effects of exogenous S1P during trophoblast differentiation of hESCs in our culture system. Since our qualitative observations showed that use of the S1PR3 agonist resulted in expression of CTB markers, and both multinucleate STB and mesenchymal EVTs could be obtained when the S1PR3 agonist was used, we chose the S1PR3 agonist for subsequent studies. We investigated whether CTB-like cells obtained by treatment of hESCs with BMP4 and SB431542 in E7 medium supplemented with the S1PR3 agonist CYM5541 for 6 days could be passaged and maintained under conditions used for culture of blastocyst- and placenta-derived primary TS cells (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Upon plating in trophoblast stem cell medium (TSCM) developed by Okae et al. (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), hESC-derived CTB-like cells underwent differentiation and epithelial colonies could not be retained after a single passage. CDX2 expression is upregulated significantly in as little as 2 days after initiation of hESC differentiation but decreases by day 6 (Fig. S1, A and B). In addition, previous studies have reported differentiation of hESCs to CDX2+/p63+ cells upon treatment with BMP for 4 days (30Horii M. Li Y. Wakeland A.K. Pizzo D.P. Nelson K.K. Sabatini K. Laurent L.C. Liu Y. Parast M.M. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease.Proc. Natl. Acad. Sci. United States America. 2016; 113: E3882-E3891Crossref PubMed Scopus (66) Google Scholar). Therefore, we explored the use of a shorter differentiation step for obtaining CTB-like cells (Fig. 3A). After 3 days of differentiation, H9 and H1 hESCs expressed nuclear CDX2, P63, and TEAD4 uniformly (Fig. 3B). However, by day 6 most differentiated H1 and H9 hESCs lose expression of CDX2 (Fig. 3C). Quantitative image analysis showed that nearly all cells are CDX2+ at day 3, in contrast to CTB-like cells at day 6. Of note, use of a 6-day protocol resulted in a significantly reduced fraction of CDX2+ cells in the case of H1 hESCs in comparison with the 3-day protocol; on the other hand, a significant fraction of H9 cells retained CDX2+ at day 6 (Fig. 3D). Transcriptome analysis using RNA sequencing identified 291 genes with significantly higher expression levels and 330 genes with significantly lower expression levels in day 3 differentiated hESCs versus undifferentiated hESCs (Tables S1 and S2).Expression of other trophectoderm-associated markers such as HAND1, GATA3, and TFAP2A, in addition to CDX2, was upregulated in differentiated hESCs at day 3, whereas expression of pluripotency-associated NANOG was downregulated. Gene set enrichment analysis of differentially expressed genes identified 567 and 202 gene ontology (GO) categories (of 9996 queried categories) associated with higher and lower gene expression in day 3 differentiated cells versus undifferentiated hESCs, respectively (Tables S3 and S4). Consistent with differentiation to epithelial trophoblast, genes associated with the GO terms for epithelium development, epithelial cell proliferation, and epithelial cell differentiation were upregulated in day 3 differentiated hESCs. CDX2+ cells at day 3 were passaged into a chemically defined medium containing four major components (denoted TM4), the S1PR3 agonist CYM5541, the GSK3β inhibitor CHIR99021, the TGFβ inhibitor A83-01, and FGF10. CHIR99021 and A83-01 are components of TSCM used for culture of primary TS cells; FGF10 was included because FGFR2b signaling is active in blastocyst- and placenta-derived TS cells and the early placenta (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Cells in TM4 could be maintained as epithelial colonies for 30+ passages over the course of 5 months. In TM4 medium, cells derived from H9 and H1 hESCs retained expression of the trophoblast markers CDX2, TFAP2C, YAP, TEAD4, and GATA3 (Figs. 3E and S6) (15Yagi R. Kohn M.J. Karavanova I. Kaneko K.J. Vullhorst D. DePamphilis M.L. Buonanno A. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.Development (Cambridge, England). 2007; 134: 3827-3836Crossref PubMed Scopus (353) Google Scholar, 17Nishioka N. Yamamoto S. Kiyonari H. Sato H. Sawada A. Ota M. Nakao K. Sasaki H. Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos.Mech. Dev. 2008; 125: 270-283Crossref PubMed Scopus (331) Google Scholar, 31Choi I. Carey T.S. Wilson C.A. Knott J.G. Transcription factor AP-2γ is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis.Development (Cambridge, England). 2012; 139: 4623-4632Crossref PubMed Scopus (40) Google Scholar, 32Home P. Saha B. Ray S. Dutta D. Gunewardena S. Yoo B. Pal A. Vivian J.L. Larson M. Petroff M. Gallagher P.G. Schulz V.P. White K.L. Golos T.G. Behr B. et al.Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 7362-7367Crossref PubMed Scopus (113) Google Scholar, 33Ralston A. Cox B.J. Nishioka N. Sasaki H. Chea E. Rugg-Gunn P. Guo G. Robson P. Draper J.S. Rossant J. Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2.Development (Cambridge, England). 2010; 137: 395-403Crossref PubMed Scopus (299) Google Scholar, 34Niwa H. Toyooka Y. Shimosato D. Strumpf D. Takahashi K. Yagi R. Rossant J. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation.Cell. 2005; 123: 917-929Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). In addition, cells expressed the pan-trophoblast marker KRT7 and low levels of P63. Of note, CDX2 expression has been strongly associated with the trophectoderm and is lost once placental villi are formed (30Horii M. Li Y. Wakeland A.K. Pizzo D.P. Nelson K.K. Sabatini K. Laurent L.C. Liu Y. Parast M.M. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease.Proc. Natl. Acad. Sci. United States America. 2016; 113: E3882-E3891Crossref PubMed Scopus (66) Google Scholar, 35Hemberger M. Udayashankar R. Tesar P. Moore H. Burton G.J. ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.Hum. Mol. Genet. 2010; 19: 2456-2467Crossref PubMed Scopus (126) Google Scholar, 36Blakeley P. Fogarty N.M.E. del Valle I. Wamaitha S.E. Hu T.X. Elder K. Snell P. Christie L. Robson P. Niakan K.K. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.Development. 2015; 142: 3151-3165Crossref PubMed Scopus (262) Google Scholar, 37Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: Key molecular mechanisms and model systems.Cell Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (144) Google Scholar). To indicate that these cells are derived from hPSCs, and to distinguish these cells from TS cells that do not express CDX2, these cells are denoted as hPSC-TSCDX2 cells. We further evaluated the differentiation potential of hPSC-TSCDX2 cells using same protocols as those used by Okae et al. for differentiation of primary TS cells to EVTs and STB (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Cells were able to form multinucleate STB that expressed hCG and KRT7 (Fig. 3F). However, upon EVT treatment, cells did not form mesenchymal elongated cells but acquired a flattened morphology. Upon passage, cells showed no HLA-G and minimal VE-Cadherin expression (Fig. 3G). Furthermore, cells maintained an epithelial flattened morp}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={Elsevier BV}, author={Mischler, Adam and Karakis, Victoria and Mahinthakumar, Jessica and Carberry, Celeste K. and San Miguel, Adriana and Rager, Julia E. and Fry, Rebecca C. and Rao, Balaji M.}, year={2021} }
 @article{mukherjee_singh_udayan_biswas_reddy_manmadhan_george_kumar_das_rao_et al._2020, title={A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells}, volume={9}, ISSN={["2050-084X"]}, DOI={10.7554/eLife.50571}, abstractNote={Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.}, journal={ELIFE}, author={Mukherjee, Ananya and Singh, Randhir and Udayan, Sreeram and Biswas, Sayan and Reddy, Pothula Purushotham and Manmadhan, Saumya and George, Geen and Kumar, Shilpa and Das, Ranabir and Rao, Balaji M. and et al.}, year={2020}, month={Feb} }
 @article{bowen_schloop_reeves_menegatti_rao_2020, title={Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display}, volume={31}, ISSN={["1520-4812"]}, DOI={10.1021/acs.bioconjchem.0c00413}, abstractNote={Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.}, number={10}, journal={BIOCONJUGATE CHEMISTRY}, author={Bowen, John and Schloop, Allison E. and Reeves, Gregory T. and Menegatti, Stefano and Rao, Balaji M.}, year={2020}, month={Oct}, pages={2325–2338} }
 @article{kesarwani_lama_chandra_reddy_jijumon_bodakuntla_rao_janke_das_sirajuddin_2020, title={Genetically encoded live-cell sensor for tyrosinated microtubules}, volume={219}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.201912107}, abstractNote={Intracellular nanobodies as fluorogenic sensors have been developed for many cellular components. Here, we report tyrosination sensor, the first genetically encoded sensor against tyrosinated microtubules. The sensor described here has potential applications to study microtubules and their PTMs in living cells.}, number={10}, journal={JOURNAL OF CELL BIOLOGY}, author={Kesarwani, Shubham and Lama, Prakash and Chandra, Anchal and Reddy, P. Purushotam and Jijumon, A. S. and Bodakuntla, Satish and Rao, Balaji M. and Janke, Carsten and Das, Ranabir and Sirajuddin, Minhajuddin}, year={2020}, month={Oct} }
 @article{bacon_blain_burroughs_mcarthrur_rao_menegatti_2020, title={Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display}, volume={22}, ISSN={["2156-8944"]}, DOI={10.1021/acscombsci.0c00076}, abstractNote={Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups with-in a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ~ 1.2 - 3.7 M). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative es-timates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human in-terleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identi-fied a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (KD ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.}, number={10}, journal={ACS COMBINATORIAL SCIENCE}, author={Bacon, Kaitlyn and Blain, Abigail and Burroughs, Matthew and McArthrur, Nikki and Rao, Balaji M. and Menegatti, Stefano}, year={2020}, month={Oct}, pages={519–532} }
 @misc{bacon_lavoie_rao_daniele_menegatti_2020, title={Past, Present, and Future of Affinity-based Cell Separation Technologies}, volume={112}, ISSN={["1878-7568"]}, DOI={10.1016/j.actbio.2020.05.004}, abstractNote={Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. STATEMENT OF SIGNIFICANCE: Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs.}, journal={ACTA BIOMATERIALIA}, author={Bacon, Kaitlyn and Lavoie, Ashton and Rao, Balaji M. and Daniele, Michael and Menegatti, Stefano}, year={2020}, month={Aug}, pages={29–51} }
 @article{cruz-teran_bacon_rao_2020, title={Simultaneous Soluble Secretion and Surface Display of Proteins in Saccharomyces cerevisiae Using Inefficient Ribosomal Skipping}, volume={2070}, ISBN={["978-1-4939-9852-4"]}, ISSN={["1940-6029"]}, DOI={10.1007/978-1-4939-9853-1_18}, abstractNote={Combinatorial library screening platforms, such as yeast surface display, typically identify several candidate proteins that need further characterization and validation using soluble recombinant protein. However, recombinant production of these candidate proteins involves tedious and time-consuming subcloning steps. This, in turn, limits the number of candidate proteins that can be characterized. To address this bottleneck, we have developed a platform that exploits inefficient ribosomal skipping by the F2A peptide for simultaneous soluble secretion and cell surface display of protein in the yeast Saccharomyces cerevisiae. Here we provide detailed protocols utilizing this F2A-based yeast display system. We discuss specific recommendations for the purification of the secreted protein. Additionally, we provide suggestions for testing the functionality and binding specificity of the soluble secreted proteins using flow cytometry analysis.}, journal={GENOTYPE PHENOTYPE COUPLING: METHODS AND PROTOCOLS}, author={Cruz-Teran, Carlos A. and Bacon, Kaitlyn and Rao, Balaji M.}, year={2020}, pages={321–334} }
 @article{bacon_bowen_reese_rao_menegatti_2020, title={Use of Target-Displaying Magnetized Yeast in Screening mRNA-Display Peptide Libraries to Identify Ligands}, volume={22}, ISSN={["2156-8944"]}, DOI={10.1021/acscombsci.0c00171}, abstractNote={This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of select WW-YAP-binding peptides: KD values of 37 and 4 μM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 μM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.}, number={12}, journal={ACS COMBINATORIAL SCIENCE}, author={Bacon, Kaitlyn and Bowen, John and Reese, Hannah and Rao, Balaji M. and Menegatti, Stefano}, year={2020}, month={Dec}, pages={738–744} }
 @article{mischler_karakis_san miguel_rao_2019, title={DERIVATION OF HUMAN TROPHOBLAST STEM CELLS FROM HUMAN PLURIPOTENT STEM CELLS}, volume={83}, ISSN={["1532-3102"]}, DOI={10.1016/j.placenta.2019.06.193}, abstractNote={Conventional soil maps contain valuable knowledge on soil–environment relationships. Such knowledge can be extracted for use when updating conventional soil maps with improved environmental data. Existing methods take all polygons of the same map unit on a map as a whole to extract the soil–environment relationship. Such approach ignores the difference in the environmental conditions represented by individual soil polygons of the same map unit. This paper proposes a method of mining soil–environment relationships from individual soil polygons to update conventional soil maps. The proposed method consists of three major steps. Firstly, the soil–environment relationships represented by each individual polygon on a conventional soil map are extracted in the form of frequency distribution curves for the involved environmental covariates. Secondly, for each environmental covariate, these frequency distribution curves from individual polygons of the same soil map unit are synthesized to form the overall soil–environment relationship for that soil map unit across the mapped area. And lastly, the extracted soil–environment relationships are applied to updating the conventional soil map with new, improved environmental data by adopting a soil land inference model (SoLIM) framework. This study applied the proposed method to updating a conventional soil map of the Raffelson watershed in La Crosse County, Wisconsin, United States. The result from the proposed method was compared with that from the previous method of taking all polygons within the same soil map unit on a map as a whole. Evaluation results with independent soil samples showed that the proposed method exhibited better performance and produced higher accuracy.}, journal={PLACENTA}, author={Mischler, Adam and Karakis, Victoria and San Miguel, Adriana and Rao, Balaji}, year={2019}, month={Aug}, pages={E59–E59} }
 @article{tiruthani_mischler_ahmed_mahinthakumar_haugh_rao_2019, title={Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation}, volume={12}, ISSN={["1937-9145"]}, DOI={10.1126/scisignal.aap7584}, abstractNote={Engineered biosensors accurately report the kinetics of EGFR activation. Building better biosensors The accuracy of a biosensor used to measure signaling events in live cells depends on both the specificity of the biosensor for its intended target and the absence of interference with the signaling pathway. Phosphotyrosine-binding SH2 domains have been used as biosensors for receptor tyrosine kinase activation. Tiruthani et al. showed that a paired SH2 domain biosensor for monitoring phosphorylation of the epidermal growth factor receptor (EGFR) at Tyr992 was not specific for EGFR and was not recruited to the membrane in a manner that accurately reflected the kinetics of EGFR signaling. Hence, the authors used two different mutagenesis and screening approaches to engineer new biosensors, mSH2 and SPY992, that exhibited greater specificity for EGFR Tyr992 and more accurately reported EGFR signaling kinetics. These approaches were extended to develop SPY1148, a biosensor for phosphorylation of EGFR at Tyr1148, and could be extended to generate phospho-specific biosensors for various targets. Live-cell fluorescence microscopy is broadly applied to study the dynamics of receptor-mediated cell signaling, but the availability of intracellular biosensors is limited. A biosensor based on the tandem SH2 domains from phospholipase C–γ1 (PLCγ1), tSH2-WT, has been used to measure phosphorylation of the epidermal growth factor receptor (EGFR). Here, we found that tSH2-WT lacked specificity for phosphorylated EGFR, consistent with the known promiscuity of SH2 domains. Further, EGF-stimulated membrane recruitment of tSH2-WT differed qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model suggested, and experiments confirmed, that the high avidity of tSH2-WT resulted in saturation of its target and interference with EGFR endocytosis. To overcome the apparent target specificity and saturation issues, we implemented two protein engineering strategies. In the first approach, we screened a combinatorial library generated by random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1 and isolated a mutant form (mSH2) with enhanced specificity for phosphorylated Tyr992 (pTyr992) of EGFR. A biosensor based on mSH2 closely reported the kinetics of EGFR phosphorylation but retained cross-reactivity similar to tSH2-WT. In the second approach, we isolated a pTyr992-binding protein (SPY992) from a combinatorial library generated by mutagenesis of the Sso7d protein scaffold. Compared to tSH2-WT and mSH2, SPY992 exhibited superior performance as a specific, moderate-affinity biosensor. We extended this approach to isolate a biosensor for EGFR pTyr1148 (SPY1148). This approach of integrating theoretical considerations with protein engineering strategies can be generalized to design and evaluate suitable biosensors for various phospho-specific targets.}, number={584}, journal={SCIENCE SIGNALING}, author={Tiruthani, Karthik and Mischler, Adam and Ahmed, Shoeb and Mahinthakumar, Jessica and Haugh, Jason M. and Rao, Balaji M.}, year={2019}, month={Jun} }
 @article{bacon_burroughs_blain_menegatti_rao_2019, title={Screening Yeast Display Libraries against Magnetized Yeast Cell Targets Enables Efficient Isolation of Membrane Protein Binders}, volume={21}, ISSN={["2156-8944"]}, DOI={10.1021/acscombsci.9b00147}, abstractNote={When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder–target pair with modest binding affinity (KD ≈ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ≈ 272–1934 nM) and the extracellular domain of the c-Kit receptor (KD ≈ 93 to KD > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion—in contrast to recombinant soluble expression—makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.}, number={12}, journal={ACS COMBINATORIAL SCIENCE}, author={Bacon, Kaitlyn and Burroughs, Matthew and Blain, Abigail and Menegatti, Stefano and Rao, Balaji M.}, year={2019}, month={Dec}, pages={817–832} }
 @article{cruz-teran_bacon_mcarthur_rao_2018, title={An Engineered Sso7d Variant Enables Efficient Magnetization of Yeast Cells}, volume={20}, ISSN={["2156-8944"]}, DOI={10.1021/acscombsci.8b00084}, abstractNote={Magnetization using cheap and minimally toxic materials, such as iron oxide nanoparticles can enable easy separation of cells from culture medium and is relevant to several industrial applications. Here, we show that cell surface expression of a mutant protein that binds iron oxide can enable efficient magnetization of yeast cells. We screened a combinatorial library of mutants derived from the Sso7d protein scaffold to isolate proteins that exhibit preferential binding to iron oxide. One of the isolated mutants, SsoFe2, was chosen for further characterization. Yeast cells expressing SsoFe2 as fusions to a cell wall protein-but not other Sso7d mutants with similar overall protein charge or amino acid composition-preferentially bind iron oxide when present in a solution with high protein concentration and in the presence of 1000-fold excess of competitor yeast cells. Moreover, coexpression of cell surface SsoFe2 enables efficient magnetic capture and separation of yeast cells expressing an enzyme (glucose oxidase) on the cell surface from yeast culture medium, and solutions with high protein concentration or containing other metal oxides. Therefore, SsoFe2-enabled magnetization can enable a range of industrial and biotechnology applications, where easy separation of cells or organelles from complex media is desirable.}, number={10}, journal={ACS COMBINATORIAL SCIENCE}, author={Cruz-Teran, Carlos A. and Bacon, Kaitlyn and McArthur, Nikki and Rao, Balaji M.}, year={2018}, month={Oct}, pages={579–584} }
 @article{patil_miles_ko_datta_rao_kiserow_genzer_2018, title={Kinetic Study of Degrafting Poly(methyl methacrylate) Brushes from Flat Substrates by Tetrabutylammonium Fluoride}, volume={51}, ISSN={["1520-5835"]}, DOI={10.1021/acs.macromol.8b01832}, abstractNote={Polymer degrafting is a process in which surface-attached polymer brushes are removed from the substrate by breaking a chemical bond in proximity to the substrate. This paper provides insight into the kinetics of degrafting poly(methyl methacrylate) (PMMA) brushes using tetrabutylammonium fluoride (TBAF) and demonstrates how the process can be modeled using a series of degrafting reactions. The trichlorosilane-based polymerization initiator utilized here to synthesize PMMA grafts by surface-initiated atom transfer radical polymerization anchors to the silica substrate by up to three potential attachment points. During the degrafting sequence this anchoring reduces to two and one chemical bond and finally results in complete liberation of the PMMA macromolecule from the substrate. We investigate the effect of TBAF concentration, the initial grafting density of PMMA grafts on the substrate, and TBAF exposure time on degrafting of PMMA by monitoring the instantaneous areal grafting density of PMMA on the sub...}, number={24}, journal={MACROMOLECULES}, author={Patil, Rohan and Miles, Jason and Ko, Yeongun and Datta, Preeta and Rao, Balaji M. and Kiserow, Douglas and Genzer, Jan}, year={2018}, month={Dec}, pages={10237–10245} }
 @article{mischler_rao_2017, title={A defined and serum-free culture system for trophoblast differentiation of human embryonic stem cells}, volume={57}, ISSN={0143-4004}, url={http://dx.doi.org/10.1016/J.PLACENTA.2017.07.059}, DOI={10.1016/J.PLACENTA.2017.07.059}, abstractNote={TGF-β superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts.Expression and localisation of the canonical TGF-β targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium.Smad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs.The lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-β could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells.}, journal={Placenta}, publisher={Elsevier BV}, author={Mischler, Adam and Rao, Balaji}, year={2017}, month={Sep}, pages={237–238} }
 @article{miles_schlenker_ko_patil_rao_genzer_2017, title={Design and Fabrication of Wettability Gradients with Tunable Profiles through Degrafting Organosilane Layers from Silica Surfaces by Tetrabutylammonium Fluoride}, volume={33}, ISSN={["0743-7463"]}, DOI={10.1021/acs.langmuir.7b02961}, abstractNote={Surface-bound wettability gradients allow for a high-throughput approach to evaluate surface interactions for many biological and chemical processes. Here we describe the fabrication of surface wettability gradients on flat surfaces by a simple, two-step procedure that permits precise tuning of the gradient profile. This process involves the deposition of homogeneous silane SAMs followed by the formation of a surface coverage gradient through the selective removal of silanes from the substrate. Removal of silanes from the surface is achieved by using tetrabutylammonium fluoride which selectively cleaves the Si-O bonds at the headgroup of the silane. The kinetics of degrafting has been modeled by using a series of first order rate equations, based on the number of attachment points broken to remove a silane from the surface. Degrafting of monofunctional silanes exhibits a single exponential decay in surface coverage; however, there is a delay in degrafting of trifunctional silanes due to the presence of multiple attachment points. The effects of degrafting temperature and time are examined in detail and demonstrate the ability to reliably and precisely control the gradient profile on the surface. We observe a relatively homogeneous coverage of silane (i.e., without the presence of islands or holes) throughout the degrafting process, providing a much more uniform surface when compared to additive approaches of gradient formation. Linear gradients were formed on the substrates to demonstrate the reproducibility and tuneability of this subtractive approach.}, number={51}, journal={LANGMUIR}, author={Miles, Jason and Schlenker, Spencer and Ko, Yeongun and Patil, Rohan and Rao, Balaji M. and Genzer, Jan}, year={2017}, month={Dec}, pages={14556–14564} }
 @article{albrecht_baker_blundell_chavez_carbone_chamley_hannibal_illsley_kurre_laurent_et al._2017, title={IFPA meeting 2016 workshop report I: Genomic communication, bioinformatics, trophoblast biology and transport systems}, volume={60}, ISSN={0143-4004}, url={http://dx.doi.org/10.1016/J.PLACENTA.2017.01.103}, DOI={10.1016/J.PLACENTA.2017.01.103}, abstractNote={Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops covered innovative technologies applied to new and traditional areas of placental research: 1) genomic communication; 2) bioinformatics; 3) trophoblast biology and pathology; 4) placental transport systems.}, journal={Placenta}, publisher={Elsevier BV}, author={Albrecht, Christiane and Baker, Julie C. and Blundell, Cassidy and Chavez, Shawn L. and Carbone, Lucia and Chamley, Larry and Hannibal, Roberta L. and Illsley, Nick and Kurre, Peter and Laurent, Louise C. and et al.}, year={2017}, month={Dec}, pages={S5–S9} }
 @article{gocha_rao_dasgupta_2017, title={Identification and characterization of a novel Sso7d scaffold-based binder against Notch1}, volume={7}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-017-12246-1}, abstractNote={Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11-13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.}, journal={SCIENTIFIC REPORTS}, author={Gocha, Tenzin and Rao, Balaji M. and DasGupta, Ramanuj}, year={2017}, month={Sep} }
 @article{cruz-teran_tiruthani_mischler_rao_2017, title={Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae}, volume={6}, ISSN={["2161-5063"]}, DOI={10.1021/acssynbio.7b00144}, abstractNote={The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface. We show that binding proteins derived from the Sso7d scaffold and the homodimeric enzyme glucose oxidase can be simultaneously secreted solubly and expressed as yeast cell surface fusions using the F2A-based system. Furthermore, a combinatorial library of Sso7d mutants can be screened to isolate binders with higher affinity for a model target (lysozyme), and the pool of higher affinity binders can be characterized in soluble form. Significantly, we show that both N- and C-terminal fusions to Aga2 can be simultaneously secreted solubly and displayed on the cell surface; this is particularly advantageous because protein functionality can be affected by the specific position of Aga2 in the protein fusion. We expect that the F2A-based yeast surface display and secretion system will be a useful tool for protein engineering and enable efficient characterization of individual clones isolated from combinatorial libraries.}, number={11}, journal={ACS SYNTHETIC BIOLOGY}, author={Cruz-Teran, Carlos A. and Tiruthani, Karthik and Mischler, Adam and Rao, Balaji M.}, year={2017}, month={Nov}, pages={2096–2107} }
 @article{sarkar_mischler_rao_2016, title={BMP signaling plays a biphasic role during trophoblast differentiation of human embryonic stem cells}, volume={45}, ISSN={0143-4004}, url={http://dx.doi.org/10.1016/J.PLACENTA.2016.06.135}, DOI={10.1016/J.PLACENTA.2016.06.135}, journal={Placenta}, publisher={Elsevier BV}, author={Sarkar, Prasenjit and Mischler, Adam and Rao, Balaji}, year={2016}, month={Sep}, pages={99–100} }
 @article{carlin_cruz-teran_kumar_gomes_rao_2016, title={Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme}, volume={5}, ISSN={["2161-5063"]}, DOI={10.1021/acssynbio.6b00034}, abstractNote={We show that a combinatorial library constructed by random pairwise assembly of low affinity binders can efficiently generate binders with increased affinity. Such a library based on the Sso7d scaffold, from a pool of low affinity binders subjected to random mutagenesis, contained putative high affinity clones for a model target (lysozyme) at higher frequency than a library of monovalent mutants generated by random mutagenesis alone. Increased binding affinity was due to intramolecular avidity generated by linking binders targeting nonoverlapping epitopes; individual binders of KD ∼ 1.3 μM and 250 nM produced a bivalent binder with apparent KD ∼ 2 nM. Furthermore, the bivalent protein retained thermal stability (TM = 84.5 °C) and high recombinant expression yields in E. coli. Finally, when binders comprising the bivalent protein are fused to two of the three fragments of tripartite split-green fluorescent protein (GFP), target-dependent reconstitution of fluorescence occurs, thereby enabling a "mix-and-read" assay for target quantification.}, number={12}, journal={ACS SYNTHETIC BIOLOGY}, author={Carlin, Kevin B. and Cruz-Teran, Carlos A. and Kumar, Jay Prakash and Gomes, Catherina and Rao, Balaji M.}, year={2016}, month={Dec}, pages={1348–1354} }
 @article{sarkar_mischler_randall_collier_dorman_boggess_muddiman_rao_2016, title={Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts}, volume={15}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.5b01118}, abstractNote={Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development.}, number={8}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Sarkar, Prasenjit and Mischler, Adam and Randall, Shan M. and Collier, Timothy S. and Dorman, Karen F. and Boggess, Kim A. and Muddiman, David C. and Rao, Balaji M.}, year={2016}, month={Aug}, pages={2433–2444} }
 @article{cruz-teran_carlin_efimenko_genzer_rao_2016, title={Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins}, volume={32}, ISSN={["0743-7463"]}, DOI={10.1021/acs.langmuir.6b01446}, abstractNote={While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts.}, number={34}, journal={LANGMUIR}, author={Cruz-Teran, Carlos A. and Carlin, Kevin B. and Efimenko, Kirill and Genzer, Jan and Rao, Balaji M.}, year={2016}, month={Aug}, pages={8660–8667} }
 @article{sarkar_randall_collier_nero_russell_muddiman_rao_2015, title={Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts}, volume={290}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m114.620641}, abstractNote={Background: Specification of terminal fate in trophoblasts derived from human embryonic stem cells is not understood. Results: Inhibition of activin/nodal signaling triggers extravillous fate, but loss of inhibition causes syncytial fate. Conclusion: Activin/nodal signaling switches the terminal fate of trophoblasts. Significance: We provide a model system that allows for targeted derivation of extravillous trophoblasts and syncytiotrophoblasts. Human embryonic stem cells (hESCs) have been routinely treated with bone morphogenetic protein and/or inhibitors of activin/nodal signaling to obtain cells that express trophoblast markers. Trophoblasts can terminally differentiate to either extravillous trophoblasts or syncytiotrophoblasts. The signaling pathways that govern the terminal fate of these trophoblasts are not understood. We show that activin/nodal signaling switches the terminal fate of these hESC-derived trophoblasts. Inhibition of activin/nodal signaling leads to formation of extravillous trophoblast, whereas loss of activin/nodal inhibition leads to the formation of syncytiotrophoblasts. Also, the ability of hESCs to form bona fide trophoblasts has been intensely debated. We have examined hESC-derived trophoblasts in the light of stringent criteria that were proposed recently, such as hypomethylation of the ELF5-2b promoter region and down-regulation of HLA class I antigens. We report that trophoblasts that possess these properties can indeed be obtained from hESCs.}, number={14}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Sarkar, Prasenjit and Randall, Shan M. and Collier, Timothy S. and Nero, Anthony and Russell, Teal A. and Muddiman, David C. and Rao, Balaji M.}, year={2015}, month={Apr}, pages={8834–8848} }
 @article{hussain_lockney_wang_gera_rao_2013, title={Avidity-mediated virus separation using a hyperthermophilic affinity ligand}, volume={29}, ISSN={["8756-7938"]}, DOI={10.1002/btpr.1655}, abstractNote={Immunoaffinity separation of large multivalent species such as viruses is limited by the stringent elution conditions necessary to overcome their strong and highly avid interaction with immobilized affinity ligands on the capture surface. Here we present an alternate strategy that harnesses the avidity effect to overcome this limitation. Red clover necrotic mosaic virus (RCNMV), a plant virus relevant to drug delivery applications, was chosen as a model target for this study. An RCNMV binding protein (RBP) with modest binding affinity (KD ∼100 nM) was generated through mutagenesis of the Sso7d protein from Sulfolobus solfataricus and used as the affinity ligand. In our separation scheme, RCNMV is captured by a highly avid interaction with RBP immobilized on a nickel surface through a hexahistidine (6xHis) tag. Subsequently, disruption of the multivalent interaction and release of RCNMV is achieved by elution of RBP from the nickel surface. Finally, RCNMV is separated from RBP by exploiting the large difference in their molecular weights (∼8 MDa vs. ∼10 kDa). Our strategy not only eliminates the need for harsh elution conditions, but also bypasses chemical conjugation of the affinity ligand to the capture surface. Stable non‐antibody affinity ligands to a wide spectrum of targets can be generated through mutagenesis of Sso7d and other hyperthermophilic proteins. Therefore, our approach may be broadly relevant to cases where capture of large multivalent species from complex mixtures and subsequent release without the use of harsh elution conditions is necessary. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013}, number={1}, journal={BIOTECHNOLOGY PROGRESS}, author={Hussain, Mahmud and Lockney, Dustin and Wang, Ruqi and Gera, Nimish and Rao, Balaji M.}, year={2013}, pages={237–246} }
 @article{goli_gera_liu_rao_rojas_genzer_2013, title={Generation and Properties of Antibacterial Coatings Based on Electrostatic Attachment of Silver Nanoparticles to Protein-Coated Polypropylene Fibers}, volume={5}, ISSN={["1944-8252"]}, DOI={10.1021/am4011644}, abstractNote={We present a simple method for attaching silver nanoparticles to polypropylene (PP) fibers in a two-step process to impart antibacterial properties. Specifically, PP fibers are pretreated by the adsorption from an aqueous solution of heat-denatured lysozyme (LYS) followed by LYS cross-linking using glutaraldehyde and sodium borohydride. At neutral pH, the surface of the adsorbed LYS layer is enriched with numerous positive charges. Silver nanoparticles (AgNPs) capped with trisodium citrate are subsequently deposited onto the protein-coated PP. Nanoparticle binding is mediated by electrostatic interactions between the positively charged LYS layer and the negatively charged AgNPs. The density of AgNPs deposited on PP depends on the amount of protein adsorbed on the surface. UV-vis spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy are employed to follow all preparation steps and to characterize the resulting functional surfaces. The antibacterial activity of the modified surfaces is tested against gram negative bacteria Escherichia coli (E. coli). Overall, our results show that PP surfaces coated with AgNPs exhibit excellent antibacterial activity with 100% removal efficiency.}, number={11}, journal={ACS APPLIED MATERIALS & INTERFACES}, author={Goli, Kiran K. and Gera, Nimish and Liu, Xiaomeng and Rao, Balaji M. and Rojas, Orlando J. and Genzer, Jan}, year={2013}, month={Jun}, pages={5298–5306} }
 @article{canbolat_gera_tang_monian_rao_pourdeyhimi_khan_2013, title={Preservation of Cell Viability and Protein Conformation on Immobilization within Nanofibers via Electrospinning Functionalized Yeast}, volume={5}, ISSN={["1944-8252"]}, DOI={10.1021/am4022768}, abstractNote={We investigate the immobilization of a model system of functionalized yeast that surface-display enhanced green fluorescent protein (eGFP) within chemically crosslinked polyvinyl alcohol (PVA) nanofibers. Yeast is incorporated into water insoluble nanofibrous materials by direct electrospinning with PVA followed by vapor phase chemical crosslinking of the polymer. Incorporation of yeast into the fibers is confirmed by elemental analysis and the viability is indicated by live/dead staining. Following electrospinning and crosslinking, we confirm that the yeast maintains its viability as well as the ability to express eGFP in the correct conformation. This method of processing functionalized yeast may thus be a powerful tool in the direct immobilization of properly folded, active enzymes within electrospun nanofibers with potential applications in biocatalysis.}, number={19}, journal={ACS APPLIED MATERIALS & INTERFACES}, author={Canbolat, M. Fatih and Gera, Nimish and Tang, Christina and Monian, Brinda and Rao, Balaji M. and Pourdeyhimi, Behnam and Khan, Saad A.}, year={2013}, month={Oct}, pages={9349–9354} }
 @misc{gera_hussain_rao_2013, title={Protein selection using yeast surface display}, volume={60}, ISSN={["1046-2023"]}, DOI={10.1016/j.ymeth.2012.03.014}, abstractNote={Binding proteins are typically isolated from combinatorial libraries of scaffold proteins using one of the many library screening tools available, such as phage display, yeast surface display or mRNA display. A key principle underlying these screening technologies is the establishment of a link between each unique mutant protein and its corresponding genetic code. The mutant proteins binding a desired target species are separated and subsequently identified using the genetic code. In this review, we largely focus on the use of yeast surface display for the isolation of binding proteins from combinatorial libraries. In yeast surface display, the yeast cell links the mutant protein to its coding DNA. Each yeast cell expresses the mutant proteins as fusions to a yeast cell wall protein; the yeast cell also carries plasmid DNA that codes for the mutant protein. Over the years, the yeast surface display platform has emerged as a powerful tool for protein engineering, and has been used in a variety of applications including affinity maturation, epitope mapping and biophysical characterization of proteins. Here we present a broad overview of the yeast surface display system and its applications, and compare it with other contemporary screening platforms. Further, we present detailed protocols for the use of yeast surface display to isolate de novo binding proteins from combinatorial libraries, and subsequent biophysical characterization of binders. These protocols can also be easily modified for affinity maturation of the isolated de novo binders.}, number={1}, journal={METHODS}, author={Gera, Nimish and Hussain, Mahmud and Rao, Balaji M.}, year={2013}, month={Mar}, pages={15–26} }
 @article{hussain_gera_hill_rao_2013, title={Scaffold Diversification Enhances Effectiveness of a Super library of Hyperthermophilic Proteins}, volume={2}, ISSN={["2161-5063"]}, DOI={10.1021/sb300029m}, abstractNote={The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a "superlibrary" or "library-of-libraries" of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.}, number={1}, journal={ACS SYNTHETIC BIOLOGY}, author={Hussain, Mahmud and Gera, Nimish and Hill, Andrew B. and Rao, Balaji M.}, year={2013}, month={Jan}, pages={6–13} }
 @misc{tiruthani_sarkar_rao_2013, title={Trophoblast differentiation of human embryonic stem cells}, volume={8}, ISSN={["1860-7314"]}, DOI={10.1002/biot.201200203}, abstractNote={Molecular mechanisms regulating human trophoblast differentiation remain poorly understood due to difficulties in obtaining primary tissues from very early developmental stages in humans. Therefore, the use of human embryonic stem cells (hESCs) as a source for generating trophoblast tissues is of significant interest. Trophoblast‐like cells have been obtained through treatment of hESCs with bone morphogenetic protein (BMP) or inhibitors of activin/nodal/transforming growth factor‐β signaling, or through protocols involving formation of embryoid bodies (EBs); however, there is controversy over whether hESC‐derived cells are indeed analogous to true trophoblasts found in vivo. In this review, we provide an overview of previously described efforts to obtain trophoblasts from hESCs. We also discuss the merits and limitations of hESCs as a source of trophoblast derivatives.}, number={4}, journal={BIOTECHNOLOGY JOURNAL}, author={Tiruthani, Karthik and Sarkar, Prasenjit and Rao, Balaji}, year={2013}, month={Apr}, pages={421-+} }
 @article{menegatti_hussain_naik_carbonell_rao_2013, title={mRNA display selection and solid-phase synthesis of Fc-binding cyclic peptide affinity ligands}, volume={110}, DOI={10.1002/bit.24760}, abstractNote={Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.}, number={3}, journal={Biotechnology and Bioengineering}, author={Menegatti, S. and Hussain, M. and Naik, A. D. and Carbonell, R. G. and Rao, B. M.}, year={2013}, pages={857–870} }
 @article{gera_hill_white_carbonell_rao_2012, title={Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold}, volume={7}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0048928}, abstractNote={We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG1–4), and recognize hIgG1–4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.}, number={11}, journal={PLOS ONE}, author={Gera, Nimish and Hill, Andrew B. and White, Dalon P. and Carbonell, Ruben G. and Rao, Balaji M.}, year={2012}, month={Nov} }
 @article{sarkar_randall_muddiman_rao_2012, title={Targeted Proteomics of the Secretory Pathway Reveals the Secretome of Mouse Embryonic Fibroblasts and Human Embryonic Stem Cells}, volume={11}, ISSN={["1535-9484"]}, DOI={10.1074/mcp.m112.020503}, abstractNote={Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of hESC self-renewal and differentiation. Current MS-based approaches for identifying secreted proteins rely predominantly on MS analysis of cell culture supernatants. Here we show that targeted proteomics of secretory pathway organelles is a powerful alternate approach for interrogating the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse embryonic fibroblasts (MEFs) and hESCs that are enriched in secretory pathway organelles while ensuring retention of the secretory cargo. MS analysis of these fractions from hESCs cultured in MEF conditioned medium (MEF-CM) or MEFs exposed to hESC medium revealed 99 and 129 proteins putatively secreted by hESCs and MEFs, respectively. Of these, 53 and 62 proteins have been previously identified in cell culture supernatants of MEFs and hESCs, respectively, thus establishing the validity of our approach. Furthermore, 76 and 37 putatively secreted proteins identified in this study in MEFs and hESCs, respectively, have not been reported in previous MS analyses. The identification of low abundance secreted proteins via MS analysis of cell culture supernatants typically necessitates the use of altered culture conditions such as serum-free medium. However, an altered medium formulation might directly influence the cellular secretome. Indeed, we observed significant differences between the abundances of several secreted proteins in subcellular fractions isolated from hESCs cultured in MEF-CM and those exposed to unconditioned hESC medium for 24 h. In contrast, targeted proteomics of secretory pathway organelles does not require the use of customized media. We expect that our approach will be particularly valuable in two contexts highly relevant to hESC biology: obtaining a temporal snapshot of proteins secreted in response to a differentiation trigger, and identifying proteins secreted by cells that are isolated from a heterogeneous population.}, number={12}, journal={MOLECULAR & CELLULAR PROTEOMICS}, author={Sarkar, Prasenjit and Randall, Shan M. and Muddiman, David C. and Rao, Balaji M.}, year={2012}, month={Dec}, pages={1829–1839} }
 @article{sarkar_collier_randall_muddiman_rao_2012, title={The subcellular proteome of undifferentiated human embryonic stem cells}, volume={12}, ISSN={["1615-9853"]}, DOI={10.1002/pmic.201100507}, abstractNote={We have characterized the subcellular proteome of human embryonic stem cells (hESCs) through MS analysis of the membrane, cytosolic, and nuclear fractions, isolated from the same sample of undifferentiated hESCs. Strikingly, 74% of all proteins identified were detected in a single subcellular fraction; we also carried out immunofluorescence studies to validate the subcellular localization suggested by proteomic analysis, for a subset of proteins. Our approach resulted in deeper proteome coverage – peptides mapping to 893, 2475, and 1185 proteins were identified in the nuclear, cytosolic, and membrane fractions, respectively. Additionally, we used spectral counting to estimate the relative abundance of all cytosolic proteins. A large number of proteins relevant to hESC biology, including growth factor receptors, cell junction proteins, transcription factors, chromatin remodeling proteins, and histone modifying enzymes were identified. Our analysis shows that components of a large number of interacting signaling pathways are expressed in hESCs. Finally, we show that proteomic analysis of the endoplasmic reticulum (ER) and Golgi compartments is a powerful alternative approach to identify secreted proteins since these are synthesized in the ER and transit through the Golgi. Taken together, our results show that systematic subcellular proteomic analysis is a valuable tool for studying hESC biology.}, number={3}, journal={PROTEOMICS}, author={Sarkar, Prasenjit and Collier, Timothy S. and Randall, Shan M. and Muddiman, David C. and Rao, Balaji M.}, year={2012}, month={Feb}, pages={421–430} }
 @article{collier_randall_sarkar_rao_dean_muddiman_2011, title={Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression}, volume={25}, ISSN={["1097-0231"]}, DOI={10.1002/rcm.5151}, abstractNote={Protein quantification is one of the principal goals of mass spectrometry (MS)-based proteomics, and many strategies exist to achieve it. Several approaches involve the incorporation of a stable-isotope label using either chemical derivatization, enzymatically catalyzed incorporation of (18)O, or metabolic labeling in a cell or tissue culture. These techniques can be cost or time prohibitive or not amenable to the biological system of interest. Label-free techniques including those utilizing integrated ion abundance and spectral counting offer an alternative to stable-isotope-based methodologies. Herein, we present the comparison of stable-isotope labeling of amino acids in cell culture (SILAC) with spectral counting for the quantification of human embryonic stem cells as they differentiate toward the trophectoderm at three time points. Our spectral counting experimental strategy resulted in the identification of 2641 protein groups across three time points with an average sequence coverage of 30.3%, of which 1837 could be quantified with more than five spectral counts. SILAC quantification was able to identify 1369 protein groups with an average coverage of 24.7%, of which 1027 could be quantified across all time points. Within this context we further explore the capacity of each strategy for proteome coverage, variation in quantification, and the relative sensitivity of each technique to the detection of change in relative protein expression.}, number={17}, journal={RAPID COMMUNICATIONS IN MASS SPECTROMETRY}, author={Collier, Timothy S. and Randall, Shan M. and Sarkar, Prasenjit and Rao, Balaji M. and Dean, Ralph A. and Muddiman, David C.}, year={2011}, month={Sep}, pages={2524–2532} }
 @article{gera_hussain_wright_rao_2011, title={Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold}, volume={409}, ISSN={["0022-2836"]}, DOI={10.1016/j.jmb.2011.04.020}, abstractNote={We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (∼ 7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 °C. We generated a library of 108 Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Binding proteins for a diverse set of model targets could be isolated from this library; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Despite extensive mutagenesis, Sso7d mutants have high thermal stability; five of six mutants analyzed have melting temperatures > 89 °C. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine.}, number={4}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Gera, Nimish and Hussain, Mahmud and Wright, Robert C. and Rao, Balaji M.}, year={2011}, month={Jun}, pages={601–616} }
 @article{sarkar_rao_2011, title={Role of Signaling Pathways and Epigenetic Factors in Lineage Determination During Human Embryonic Stem Cell Differentiation}, volume={1}, journal={Embryonic Stem Cells - Differentiation and Pluripotent Alternatives}, author={Sarkar, P. and Rao, B.M}, year={2011}, pages={1} }
 @article{collier_sarkar_franck_rao_dean_muddiman_2010, title={Direct Comparison of Stable Isotope Labeling by Amino Acids in Cell Culture and Spectral Counting for Quantitative Proteomics}, volume={82}, ISSN={["1520-6882"]}, DOI={10.1021/ac101978b}, abstractNote={Numerous experimental strategies exist for relative protein quantification, one of the primary objectives of mass spectrometry based proteomics analysis. These strategies mostly involve the incorporation of a stable isotope label via either metabolic incorporation in cell or tissue culture (¹⁵N/¹⁴N metabolic labeling, stable isotope labeling by amino acids in cell culture (SILAC)), chemical derivatization (ICAT, iTRAQ, TMT), or enzymatically catalyzed incorporation (¹⁸O labeling). Also, these techniques can be cost or time prohibitive or not amenable to the biological system of interest (i.e., metabolic labeling of clinical samples, most animals, or fungi). This is the case with the quantification of fungal proteomes, which often require auxotroph mutants to fully metabolically label. Alternatively, label-free strategies for protein quantification such as using integrated ion abundance and spectral counting have been demonstrated for quantification affording over 2 orders of magnitude of dynamic range which is comparable to metabolic labeling strategies. Direct comparisons of these quantitative techniques are largely lacking in the literature but are highly warranted in order to evaluate the capabilities, limitations, and analytical variability of available quantitative strategies. Here, we present the direct comparison of SILAC to label-free quantification by spectral counting of an identical set of data from the bottom-up proteomic analysis of human embryonic stem cells, which are readily able to be quantified using both strategies, finding that both strategies result in a similar number of protein identifications. We also discuss necessary constraints for accurate quantification using spectral counting and assess the potential of this label-free strategy as a viable alternative for quantitative proteomics.}, number={20}, journal={ANALYTICAL CHEMISTRY}, author={Collier, Timothy S. and Sarkar, Prasenjit and Franck, William L. and Rao, Balaji M. and Dean, Ralph A. and Muddiman, David C.}, year={2010}, month={Oct}, pages={8696–8702} }
 @article{collier_sarkar_rao_muddiman_2010, title={Quantitative top-down proteomics of SILAC labeled human embryonic stem cells}, volume={21}, DOI={10.1016/j.jasms.2010.01.031}, abstractNote={Human embryonic stem cells (hESCs) are self-renewing pluripotent cells with relevance to treatment of numerous medical conditions. However, a global understanding of the role of the hESC proteome in maintaining pluripotency or triggering differentiation is still largely lacking. The emergence of top-down proteomics has facilitated the identification and characterization of intact protein forms that are not readily apparent in bottom-up studies. Combined with metabolic labeling techniques such as stable isotope labeling by amino acids in cell culture (SILAC), quantitative comparison of intact protein expression under differing experimental conditions is possible. Herein, quantitative top-down proteomics of hESCs is demonstrated using the SILAC method and nano-flow reverse phase chromatography directly coupled to a linear-ion-trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FT-ICR-MS). In this study, which to the best of our knowledge represents the first top-down analysis of hESCs, we have confidently identified 11 proteins by accurate intact mass, MS/MS, and amino acid counting facilitated by SILAC labeling. Although quantification is challenging due to the incorporation of multiple labeled amino acids (i.e., lysine and arginine) and arginine to proline conversion, we are able to quantitatively account for these phenomena using a mathematical model.}, number={6}, journal={Journal of the American Society for Mass Spectrometry}, author={Collier, T. S. and Sarkar, P. and Rao, B. and Muddiman, David}, year={2010}, pages={879–889} }
 @article{sarkar_rao_2009, title={Molecular aspects of cardiac differentiation in embryonic stem cells}, volume={37}, DOI={10.1615/critrevbiomedeng.v37.i4-5.10}, abstractNote={Embryonic stem cells (ESCs) are culture-adapted pluripotent cells derived from the inner cell mass of the blastocyst-stage embryo. ESCs provide unique opportunities to study the molecular basis of the process of differentiation that gives rise to all the somatic cell types of the body, including cells of the cardiac lineages. Many protocols have been established to obtain cells of the cardiac lineage from ESCs in vitro. More recently, a clearer picture of the regulatory networks governing cardiac differentiation has begun to emerge. Herein, we summarize the current methods to obtain cells of the cardiac lineages from ESCs and present a review of the signaling pathways, transcription factors, and target genes involved in cardiac differentiation.}, number={4-5}, journal={Critical Reviews in Biomedical Engineering}, author={Sarkar, P. and Rao, B. M.}, year={2009}, pages={283–320} }
 @article{varelas_sakuma_samavarchi-tehrani_peerani_rao_dembowy_yaffe_zandstra_wrana_2008, title={TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal}, volume={10}, ISSN={["1476-4679"]}, DOI={10.1038/ncb1748}, abstractNote={Transforming growth factor-beta (TGFbeta) family members regulate many developmental and pathological events through Smad transcriptional modulators. How nuclear accumulation of Smad is coupled to the transcriptional machinery is poorly understood. Here we demonstrate that in response to TGFbeta stimulation the transcriptional regulator TAZ binds heteromeric Smad2/3-4 complexes and is recruited to TGFbeta response elements. In human embryonic stem cells TAZ is required to maintain self-renewal markers and loss of TAZ leads to inhibition of TGFbeta signalling and differentiation into a neuroectoderm lineage. In the absence of TAZ, Smad2/3-4 complexes fail to accumulate in the nucleus and activate transcription. Furthermore, TAZ, which itself engages in shuttling, dominantly controls Smad nucleocytoplasmic localization and can be retained in the nucleus by transcriptional co-factors such as ARC105, a component of the Mediator complex. TAZ thus defines a hierarchical system regulating Smad nuclear accumulation and coupling to the transcriptional machinery.}, number={7}, journal={NATURE CELL BIOLOGY}, author={Varelas, Xaralabos and Sakuma, Rui and Samavarchi-Tehrani, Payman and Peerani, Raheem and Rao, Balaji M. and Dembowy, Joanna and Yaffe, Michael B. and Zandstra, Peter W. and Wrana, Jeffrey L.}, year={2008}, month={Jul}, pages={837–848} }
 @article{peerani_rao_bauwens_yin_wood_nagy_kumacheva_zandstra_2007, title={Niche-mediated control of human embryonic stem cell self-renewal and differentiation}, volume={26}, ISSN={["1460-2075"]}, DOI={10.1038/sj.emboj.7601896}, abstractNote={Complexity in the spatial organization of human embryonic stem cell (hESC) cultures creates heterogeneous microenvironments (niches) that influence hESC fate. This study demonstrates that the rate and trajectory of hESC differentiation can be controlled by engineering hESC niche properties. Niche size and composition regulate the balance between differentiation-inducing and -inhibiting factors. Mechanistically, a niche size-dependent spatial gradient of Smad1 signaling is generated as a result of antagonistic interactions between hESCs and hESC-derived extra-embryonic endoderm (ExE). These interactions are mediated by the localized secretion of bone morphogenetic protein-2 (BMP2) by ExE and its antagonist, growth differentiation factor-3 (GDF3) by hESCs. Micropatterning of hESCs treated with small interfering (si) RNA against GDF3, BMP2 and Smad1, as well treatments with a Rho-associated kinase (ROCK) inhibitor demonstrate that independent control of Smad1 activation can rescue the colony size-dependent differentiation of hESCs. Our results illustrate, for the first time, a role for Smad1 in the integration of spatial information and in the niche-size-dependent control of hESC self-renewal and differentiation.}, number={22}, journal={EMBO JOURNAL}, author={Peerani, Raheem and Rao, Balaji M. and Bauwens, Celine and Yin, Ting and Wood, Geoffrey A. and Nagy, Andras and Kumacheva, Eugenia and Zandstra, Peter W.}, year={2007}, month={Nov}, pages={4744–4755} }
 @article{cochran_kim_lippow_rao_wittrup_2006, title={Improved mutants from directed evolution are biased to orthologous substitutions}, volume={19}, ISSN={["1741-0134"]}, DOI={10.1093/protein/gzl006}, abstractNote={We have engineered human epidermal growth factor (EGF) by directed evolution through yeast surface display for significantly enhanced affinity for the EGF receptor (EGFR). Statistical analysis of improved EGF mutants isolated from randomly mutated yeast-displayed libraries indicates that mutations are biased towards substitutions at positions exhibiting significant phylogenetic variation. In particular, mutations in high-affinity EGF mutants are statistically biased towards residues found in orthologous EGF species. This same trend was also observed with other proteins engineered through directed evolution in our laboratory (EGFR, interleukin-2) and in a meta-analysis of reported results for engineered subtilisin. By contrast, reported loss-of-function mutations in EGF were biased towards highly conserved positions. Based on these findings, orthologous mutations were introduced into a yeast-displayed EGF library by a process we term shotgun ortholog scanning mutagenesis (SOSM). EGF mutants with a high frequency of the introduced ortholog mutations were isolated through screening the library for enhanced binding affinity to soluble EGFR ectodomain. These mutants possess a 30-fold increase in binding affinity over wild-type EGF to EGFR-transfected fibroblasts and are among the highest affinity EGF proteins to be engineered to date. Collectively, our findings highlight a general approach for harnessing information present in phylogenetic variability to create useful genetic diversity for directed evolution. Our SOSM method exploits the benefits of library diversity obtained through complementary methods of error-prone PCR and DNA shuffling, while circumventing the need for acquisition of multiple genes for family or synthetic shuffling.}, number={6}, journal={PROTEIN ENGINEERING DESIGN & SELECTION}, author={Cochran, Jennifer R. and Kim, Yong-Sung and Lippow, Shaun M. and Rao, Balaji and Wittrup, K. Dane}, year={2006}, month={Jun}, pages={245–253} }
 @misc{rao_zandstra_2005, title={Culture development for human embryonic stem cell propagation: molecular aspects and challenges}, volume={16}, ISSN={["1879-0429"]}, DOI={10.1016/j.copbio.2005.08.001}, abstractNote={Basic fibroblast growth factor and members of the transforming growth factor-β superfamily are important regulators of human embryonic stem cell (hESC) self-renewal. Extensive cross-talk between the intracellular signaling pathways activated by these factors contributes to maintenance of the undifferentiated hESC state. Understanding the molecular regulation of hESC self-renewal will facilitate the design of improved systems for hESC propagation and provide a foundation for strategies to direct the differentiation of hESCs to clinically relevant cell types.}, number={5}, journal={CURRENT OPINION IN BIOTECHNOLOGY}, author={Rao, BM and Zandstra, PW}, year={2005}, month={Oct}, pages={568–576} }
 @article{rao_lauffenburger_wittrup_2005, title={Engineering protein therapeutics within a systems-level computational context}, volume={23}, number={2}, journal={Nature Biotechnology}, author={Rao, B.M. and Lauffenburger, D. A. and Wittrup, K. D.}, year={2005}, pages={191–194} }
 @article{rao_driver_lauffenburger_wittrup_2005, title={High-Affinity CD25-Binding IL-2 Mutants Potently Stimulate Persistent T Cell Growth†}, volume={44}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi050436x}, DOI={10.1021/bi050436x}, abstractNote={We have used directed evolution to construct IL-2 mutants that bind the IL-2 alpha receptor subunit (IL-2Ralpha, CD25) with affinities comparable to that of the IL-15-IL-15 alpha receptor subunit (IL-15Ralpha) interaction. T cells proliferate for up to 6 days following a 30 minute incubation with these IL-2 mutants, which may lead to potential applications for cancer and viral immunotherapy. Several alternative mechanisms have been proposed to explain the contrasting effects of IL-2 and IL-15 on T cell proliferation and death. These IL-2 mutants exhibit T cell growth response-receptor occupancy curves indistinguishable from that for IL-15, suggesting that much of the difference between wild-type IL-2 and IL-15 effects arises simply from their 1000-fold differing affinities for their private alpha receptor subunits.}, number={31}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Rao, Balaji M. and Driver, Ian and Lauffenburger, Douglas A. and Wittrup, K. Dane}, year={2005}, month={Aug}, pages={10696–10701} }
 @article{rao_lauffenburger_wittrup_2005, title={Integrating cell-level kinetic modeling into the design of engineered protein therapeutics}, volume={23}, ISSN={["1546-1696"]}, DOI={10.1038/nbt1064}, abstractNote={Functional genomics and proteomics are identifying many potential drug targets for novel therapeutic proteins, and both rational and combinatorial protein engineering methods are available for creating drug candidates. A central challenge is the definition of the most appropriate design criteria, which will benefit critically from computational kinetic models that incorporate integration from the molecular level to the whole systems level. Interpretation of these processes will require mathematical models that are refined in combination with relevant data derived from quantitative assays, to correctly set biophysical objectives for protein design.}, number={2}, journal={NATURE BIOTECHNOLOGY}, author={Rao, BM and Lauffenburger, DA and Wittrup, KD}, year={2005}, month={Feb}, pages={191–194} }
 @article{rao_driver_lauffenburger_wittrup_2004, title={Interleukin 2 (IL-2) variants engineered for increased IL-2 receptor alpha-subunit affinity exhibit increased potency arising from a cell surface ligand reservoir effect}, volume={66}, number={4}, journal={Molecular Pharmacology}, author={Rao, B. M. and Driver, I. and Lauffenburger, D. A. and Wittrup, K. D.}, year={2004}, pages={864–869} }
 @article{rao_girvin_ciardelli_lauffenburger_wittrup_2003, title={Interleukin-2 mutants with enhanced alpha-receptor subunit binding affinity}, volume={16}, ISSN={["0269-2139"]}, DOI={10.1093/protein/gzg111}, abstractNote={Stimulation of T-cells by IL-2 has been exploited for treatment of metastatic renal carcinoma and melanoma. However, a narrow therapeutic window delimited by negligible stimulation of T-cells at low picomolar concentrations and undesirable stimulation of NK cells at nanomolar concentrations hampers IL-2-based therapies. We hypothesized that increasing the affinity of IL-2 for IL-2Ralpha may create a class of IL-2 mutants with increased biological potency as compared with wild-type IL-2. Towards this end, we have screened libraries of mutated IL-2 displayed on the surface of yeast and isolated mutants with a 15-30-fold improved affinity for the IL-2Ralpha subunit. These mutants do not exhibit appreciably altered bioactivity at 0.5-5 pM in steady-state bioassays, concentrations well below the IL-2Ralpha equilibrium binding constant for both the mutant and wild-type IL-2. A mutant was serendipitously identified that exhibited somewhat improved potency, perhaps via altered endocytic trafficking mechanisms described previously.}, number={12}, journal={PROTEIN ENGINEERING}, author={Rao, BM and Girvin, AT and Ciardelli, T and Lauffenburger, DA and Wittrup, KD}, year={2003}, month={Dec}, pages={1081–1087} }