@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. 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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{huang_ashwell_fry_lloyd_flowers_spears_2015, title={Effect of dietary copper amount and source on copper metabolism and oxidative stress of weanling pigs in short-term feeding}, volume={93}, ISSN={["1525-3163"]}, DOI={10.2527/jas.2014-8082}, abstractNote={Forty-eight weanling barrows were used to determine the effects of amount and source of dietary Cu on Cu metabolism, oxidative stress in the duodenum, and VFA ratios in the cecum of weanling pigs in short-term feeding. At 21 d of age, newly weaned pigs were stratified by BW (7.03 ± 1.20 kg) and equally assigned to 1 of the following dietary treatments: 1) control (5 mg supplemental Cu/kg diet from CuSO4), 2) 225 mg supplemental Cu/kg diet from CuSO4, or 3) 225 mg supplemental Cu/kg diet from tribasic Cu chloride (TBCC). Pigs were housed 2 pigs per pen and were fed a complex diet until harvest on d 11 and 12. During harvest, bile and liver were obtained for mineral analysis, and liver samples were obtained for analysis of mRNA expression of Cu regulatory proteins. Digesta of duodenum, proximal jejunum, and ileum were collected for soluble Cu analysis. Mucosal scrapings of duodenum, proximal jejunum, and ileum were obtained for analysis of mucosal Cu concentration and mRNA expression of Cu regulatory proteins. Duodenal mucosal scrapings were also collected for analysis of malondialdehyde (MDA). Pigs fed high Cu had markedly greater (P < 0.0001) Cu concentrations in the duodenal, proximal jejunal, and ileal mucosa than controls. Copper in the duodenal mucosa was greater (P = 0.003) in CuSO4 than TBCC pigs. Duodenal MDA concentrations were greater (P = 0.003) in CuSO4 vs. control pigs and tended (P = 0.06) to be greater than in TBCC pigs. Duodenal antioxidant 1 (Atox1) mRNA was downregulated (P < 0.01) in pigs fed high Cu compared to controls and was not affected by Cu source. Compared with control pigs, those fed CuSO4 and TBCC had greater (P < 0.001) liver and bile Cu concentrations. Liver Cu was also greater (P = 0.0007) in TBCC than CuSO4-fed pigs. Hepatic Cu transporting β-polypeptide ATPase (Atp7b) was upregulated (P = 0.02) in the Cu-supplemented pigs compared with controls and did not differ among Cu sources. The acetate:propionate ratio in cecal contents was much greater in pigs supplemented with 225 mg Cu/kg diet than in controls. When fed at 225 mg Cu/kg diet, TBCC may cause less oxidative stress in the duodenum than CuSO4. Feeding weanling pigs increased Cu resulted in modulation of duodenal and liver at the transcription level.}, number={6}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Huang, Y. L. and Ashwell, M. S. and Fry, R. S. and Lloyd, K. E. and Flowers, W. L. and Spears, J. W.}, year={2015}, month={Jun}, pages={2948–2955} }
 @article{fry_spears_lloyd_o'nan_ashwell_2013, title={Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and use in Angus and Simmental cows and fetuses}, volume={91}, ISSN={["1525-3163"]}, DOI={10.2527/jas.2011-3888}, abstractNote={Copper (Cu) deficiency is a widespread problem in cattle across the United States and breed differences in Cu metabolism may contribute to this issue. Intracellular Cu is tightly regulated by transport and chaperone proteins, and to date, these mechanisms have not been elucidated to address breed differences in Cu metabolism, nor have these proteins been characterized in bovine fetal liver. Mature, pregnant Angus (n = 8) and Simmental (n = 8) cows (∼4 mo into gestation) were used in a 2 × 2 factorial arrangement of treatments. All cows were bred to Angus sires resulting in an Angus vs. Simmental × Angus comparison for fetuses. Cows were randomly assigned to corn silage-based diets that were either adequate (+Cu) or deficient (-Cu; 6.6 mg Cu/kg DM) in Cu. Diets were individually fed for 112 d. At the end of the study, cows were harvested to collect duodenal mucosa scrapes, liver samples, and fetal liver samples for mineral analysis and also for mRNA and protein analysis of Cu transport and chaperone proteins. Placentomes were also obtained for mineral analysis. Plasma Cu and liver Cu were affected by Cu, breed, and Cu × breed. Both of these Cu indices were less (P ≤ 0.05) in-Cu Simmentals (-CuS) than in-Cu Angus (- uA), but were similar among +Cu Simmental (+CuS) and +Cu Angus cows (+CuA). Duodenal Cu was less (P = 0.01) in-Cu vs. +Cu cows. Placentome Cu was less (P = 0.003) in-Cu vs. +Cu cows, and was also less (P = 0.03) in Simmentals vs. Angus. Fetal liver Cu was less (P = 0.002) in-Cu vs. +Cu fetuses, and was also less (P = 0.05) in Simmental × Angus vs. Angus. Abundance of Cu transporter1 (CTR1) protein and transcripts for Cu transporters and chaperones were not affected by Cu or breed in liver and were not affected by Cu in the intestine. Duodenal Ctr1 was less (P = 0.04) and CTR1 tended (P = 0.10) to be less in Simmentals vs. Angus. Expression of Atp7a tended (P = 0.08) to be less in Simmentals than in Angus. In fetal liver, expression of antioxidant 1 (Atox1), cytochrome c oxidase assembly protein 17 (Cox17), and Cu metabolism MURR1 domain 1 (Commd1) were up-regulated (P ≤ 0.05) in-Cu vs. +Cu fetuses. In conclusion, less expression of duodenal Ctr1 and a tendency for less CTR1 (P = 0.10) and Atp7a (P = 0.08) suggest that Simmentals have a lesser ability to absorb and utilize dietary Cu, and may explain why Simmentals are more prone to Cu deficiency than Angus. Up-regulation of fetal liver Atox1, Cox17, and Commd1 in-Cu fetuses may reflect the great Cu demand by the fetus.}, number={2}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Fry, R. S. and Spears, J. W. and Lloyd, K. E. and O'Nan, A. T. and Ashwell, M. S.}, year={2013}, month={Feb}, pages={861–871} }
 @article{fry_ashwell_lloyd_o'nan_flowers_stewart_spears_2012, title={Amount and source of dietary copper affects small intestine morphology, duodenal lipid peroxidation, hepatic oxidative stress, and mRNA expression of hepatic copper regulatory proteins in weanling pigs}, volume={90}, ISSN={["1525-3163"]}, DOI={10.2527/jas.2011-4403}, abstractNote={Thirty weanling, crossbred barrows (SUS SCROFA) were used to determine the effects of amount and source of dietary Cu on small intestinal morphology and lipid peroxidation, Cu metabolism, and mRNA expression of proteins involved in hepatic Cu homeostasis. At 21 d of age, pigs were stratified by BW (6.33 ± 0.23 kg) and allocated to 1 of the following dietary treatments: i) control (no supplemental Cu; 6.7 mg Cu/kg), ii) 225 mg supplemental Cu/kg diet from Cu sulfate (CuSO(4)), or iii) 225 mg supplemental Cu/kg diet from tribasic Cu chloride (TBCC). Pigs were housed 2 pigs per pen and were fed a 3-phase diet regimen until d 35 or 36 of the study. During harvest, bile and liver were obtained for mineral analysis, and liver samples were also obtained for analysis of liver glutathione (GSH) and mRNA expression of Cu regulatory proteins. Segments of duodenum, proximal jejunum, and ileum were obtained for mucosal morphology, and duodenal mucosal scrapings were collected from all pigs for analysis of malondialdehyde (MDA). Duodenal villus height was reduced in CuSO(4) pigs compared with control (P = 0.001) and TBCC (P = 0.03) pigs. Villus height in the proximal jejunum of CuSO(4) pigs was reduced (P = 0.03) compared with control pigs, but ileal villus height was not affected (P = 0.82) by treatment. Duodenal MDA concentrations were greater (P = 0.03) in CuSO(4) pigs and tended to be greater (P = 0.10) in pigs supplemented with TBCC compared with control pigs. Liver Cu was greater (P = 0.01) in CuSO(4) vs. control pigs, and tended (P = 0.07) to be greater in TBCC pigs than control pigs. Bile Cu concentrations were greater (P < 0.001) in CuSO(4) and TBCC pigs vs. controls and were also greater (P = 0.04) in TBCC vs. CuSO(4) pigs. Total liver GSH concentrations were less (P = 0.02) in pigs fed diets supplemented with CuSO(4) vs. pigs fed control diets but total liver GSH did not differ (P = 0.11) between control and TBCC pigs. Hepatic mRNA of cytochrome c oxidase assembly protein 17 was less (P = 0.01) in CuSO(4) and tended to be less (P = 0.08) in TBCC pigs vs. control pigs. Expression of antioxidant 1 mRNA was greater (P = 0.04) in TBCC pigs and tended to be greater (P = 0.06) in CuSO(4) pigs compared with control pigs. Results of this study indicated that, when fed at 225 mg Cu/kg diet, TBCC may cause less oxidative stress in the duodenum than CuSO(4). Feeding weanling pigs increased Cu resulted in modulation of certain Cu transporters and chaperones at the transcription level.}, number={9}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Fry, R. S. and Ashwell, M. S. and Lloyd, K. E. and O'Nan, A. T. and Flowers, W. L. and Stewart, K. R. and Spears, J. W.}, year={2012}, month={Sep}, pages={3112–3119} }
 @article{spears_whisnant_huntington_lloyd_fry_krafka_lamptey_hyda_2012, title={Chromium propionate enhances insulin sensitivity in growing cattle}, volume={95}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2011-4845}, abstractNote={Thirty-six Angus and Angus×Simmental heifers, averaging 291 kg, were used to determine the effects of dietary Cr, in the form of Cr propionate (Cr Prop), on glucose metabolism and serum insulin concentrations following glucose administration. Heifers were stratified by body weight (BW) within a breed and randomly assigned to treatments. Treatments consisted of 0, 3, 6, or 9 mg of supplemental Cr/d from Cr Prop. Based on dry matter (DM) intakes, the daily doses of Cr were equivalent to 0.47, 0.94, and 1.42 mg of supplemental Cr/kg of DM. Heifers were individually fed a corn silage-based diet at a level of 2% of BW. Each heifer was also fed 0.45 kg of a ground corn supplement daily that served as a carrier for supplemental Cr. Glucose tolerance tests were performed on d 44 of the study. Glucose was infused via jugular catheters at a level of 0.45 g/kg of BW(0.75) over a course of 1 to 2 min. Blood samples were collected at -10, 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, and 180 min relative to glucose dosing for glucose and insulin determination. Area under the glucose response curve was lower (1,603 vs. 1,964 mg/dL per minute) in heifers supplemented with Cr from 0 to 45 min following glucose challenge. Serum insulin concentrations were lower in Cr-supplemented heifers than in controls following glucose infusion. The molar ratio of insulin to glucose was also lower in Cr-supplemented heifers relative to controls. Serum insulin and serum insulin to glucose ratios did not differ among heifers supplemented with 3, 6, or 9 mg of Cr/d. Results indicate that Cr Prop supplementation increased tissue sensitivity to insulin in growing heifers. Based on insulin sensitivity, Cr requirements (as Cr Prop) of growing heifers can be met by supplementing with 3 mg of Cr/d or 0.47 mg of Cr/kg of DM.}, number={4}, journal={JOURNAL OF DAIRY SCIENCE}, author={Spears, J. W. and Whisnant, C. S. and Huntington, G. B. and Lloyd, K. E. and Fry, R. S. and Krafka, K. and Lamptey, A. and Hyda, J.}, year={2012}, month={Apr}, pages={2037–2045} }
 @article{fry_brown_lloyd_hansen_legleiter_robarge_spears_2011, title={Effect of dietary boron on physiological responses in growing steers inoculated with bovine herpesvirus type-1}, volume={90}, ISSN={["1532-2661"]}, DOI={10.1016/j.rvsc.2010.04.016}, abstractNote={Thirty-six Angus and Angus×Simmental steers were fed one of three dietary treatments; (1) control (no supplemental B), (2) 5 mg supplemental B/kg, and (3) 15 mg supplemental B/kg for 47 days to determine the effects of dietary boron (B) on disease resistance following an inoculation with bovine herpesvirus type-1 (BHV-1). On day 34 of the study steers were inoculated intranasally with BHV-1. Rectal temperatures began to elevate at day 2, and plasma tumor necrosis factor-α concentrations increased (P<0.05) by day 2 following BHV-1 inoculation. Plasma acute phase proteins were increased (P<0.01) while plasma interferon-γ was decreased (P<0.05) by day 4 post-inoculation. Supplementation of B increased (P<0.001) plasma B concentrations in a dose-responsive manner. However, dietary B did not affect the duration and severity of clinical signs of BHV-1 and had minimal effects on plasma acute phase proteins and cytokines.}, number={1}, journal={RESEARCH IN VETERINARY SCIENCE}, author={Fry, R. S. and Brown, T. T., Jr. and Lloyd, K. E. and Hansen, S. L. and Legleiter, L. R. and Robarge, W. P. and Spears, J. W.}, year={2011}, month={Feb}, pages={78–83} }
 @article{ashwell_fry_spears_o'nan_maltecca_2011, title={Investigation of breed and sex effects on cytochrome P450 gene expression in cattle liver}, volume={90}, ISSN={["0034-5288"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79951771053&partnerID=MN8TOARS}, DOI={10.1016/j.rvsc.2010.05.029}, abstractNote={Many cytochrome P450 enzymes are involved in xenobiotic metabolism and elimination. In humans, genetic variation in some of these enzymes contributes to inter-individual drug responses, sometimes having significant clinical effects. Transcript levels of eight P450 genes were evaluated in liver to investigate potential differences in breed and sex in cattle. In Angus calves, heifers appeared to have higher gene expression than steers for two of the eight genes. In Angus and Simmental pregnant cows, Angus appeared to have higher gene expression for three of the eight genes. Transcript evaluation is just the first step toward determining if differences exist between breeds and sexes in enzyme catalytic activity. However, others (Giantin et al., 2008) have shown correlations between transcript levels and catalytic activity in other cattle breeds. Therefore breed and/or sex of an animal may need to be considered before administering a dose of a xenobiotic due to the potential for harmful drug residues in foodstuffs as well as improper treatment of disease conditions.}, number={2}, journal={RESEARCH IN VETERINARY SCIENCE}, author={Ashwell, M. S. and Fry, R. S. and Spears, J. W. and O'Nan, A. T. and Maltecca, C.}, year={2011}, month={Apr}, pages={235–237} }
 @article{spears_lloyd_fry_2011, title={Tolerance of cattle to increased dietary sulfur and effect of dietary cation-anion balance}, volume={89}, ISSN={["1525-3163"]}, DOI={10.2527/jas.2010-3265}, abstractNote={The objective of this study was to determine if dietary cation-anion balance (DCAB) affects the concentration of S that can be tolerated by growing and finishing cattle without adversely affecting performance. Angus cross and Bradford steers (n=114; average initial BW=252.8 kg) were blocked by BW and breed, and randomly assigned within a block to treatment. The design was a 3 × 2 factorial arrangement of treatments with S (from NH(4)SO(4)) supplemented at 0, 0.15, or 0.30% of DM, and NaHCO(3) added at 0 or 1.0% of DM. Each treatment consisted of 3 pens containing 5 steers and 1 pen containing 4 steers. Steers were used in an 84-d growing study followed by a finishing study. A corn silage-based diet was fed during the growing study and a corn-based diet was fed during the finishing study. Steers were not randomized between experiments. The analyzed concentrations of S in the growing diets were 0.12, 0.30, and 0.46%, whereas the analyzed concentrations of S in the finishing diets were 0.13, 0.31, and 0.46% for treatments supplemented with 0, 0.15, and 0.30% S, respectively. Increasing DCAB by approximately 15 mEq/100 g of DM, by the addition of NaHCO(3,) did not affect (P > 0.36) performance during the growing or finishing studies. During the growing study DMI was not affected (P=0.29) by dietary S. Steers fed diets containing 0.30% S had greater ADG (P=0.02) and G:F (P=0.01) than those receiving 0.46% S, but similar (P > 0.36) performance to steers fed 0.12% S. During the finishing study, steers fed diets containing 0.46% S had less ADG than steers fed 0.13 (P=0.004) or 0.31% S (P=0.07), whereas ADG did not differ (P=0.18) among steers fed 0.13 and 0.31% S. Steers fed diets containing 0.31 (P=0.01) or 0.46% S (P=0.001) had less DMI than controls, but G:F was not affected (P=0.52) by S during the finishing study. Carcass characteristics did not differ (P > 0.18) among steers fed diets containing 0.13 and 0.31% S. Steers receiving diets containing 0.46% S had decreased HCW (P=0.001), quality (P=0.02), and yield grades (P=0.04) than steers receiving 0.13% S. Plasma Cu concentrations on d 101 of the finishing phase and liver Cu concentrations at slaughter were greater (P ≤ 0.05) in control steers compared with those fed diets containing 0.31 or 0.46% S. This study indicates that steers fed growing diets can tolerate up to 0.46% S with minimum effects on performance. Finishing steers tolerated diets containing 0.31% S without adverse affects on ADG or G:F. However, 0.46% S greatly decreased ADG and DMI, and increasing DCAB did not prevent these depressions.}, number={8}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Spears, J. W. and Lloyd, K. E. and Fry, R. S.}, year={2011}, month={Aug}, pages={2502–2509} }
 @article{nose_wood_kim_prohaska_fry_spears_thiele_2010, title={Ctr1 Is an Apical Copper Transporter in Mammalian Intestinal Epithelial Cells in Vivo That Is Controlled at the Level of Protein Stability}, volume={285}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m110.143826}, abstractNote={Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu+ importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.}, number={42}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Nose, Yasuhiro and Wood, L. Kent and Kim, Byung-Eun and Prohaska, Joseph R. and Fry, Robert S. and Spears, Jerry W. and Thiele, Dennis J.}, year={2010}, month={Oct}, pages={32385–32392} }
 @article{fry_lloyd_jacobi_siciliano_robarge_spears_2010, title={Effect of dietary boron on immune function in growing beef steers*}, volume={94}, ISSN={["0931-2439"]}, DOI={10.1111/j.1439-0396.2008.00906.x}, abstractNote={Summary}, number={3}, journal={JOURNAL OF ANIMAL PHYSIOLOGY AND ANIMAL NUTRITION}, author={Fry, R. S. and Lloyd, K. E. and Jacobi, S. K. and Siciliano, P. D. and Robarge, W. P. and Spears, J. W.}, year={2010}, month={Jun}, pages={273–279} }
 @article{lloyd_fellner_mcleod_fry_krafka_lamptey_spears_2010, title={Effects of supplementing dairy cows with chromium propionate on milk and tissue chromium concentrations}, volume={93}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2010-3198}, abstractNote={Eight primiparous and 8 multiparous Holstein cows were used to determine the effects of Cr supplementation, in the form of Cr propionate (Cr Prop), on milk and tissue Cr concentrations. Cows were randomly assigned by parity to one of 2 diets: 1) control diet or 2) 2 mg of supplemental Cr/kg of DM. The level of Cr Prop supplemented exceeded by 4-fold the concentration of 0.5 mg of Cr/kg permitted by the FDA. Experimental diets were fed from approximately 30 d prepartum until at least 91 d postpartum, resulting in a minimum of 121 d of exposure to supplemental Cr. The control prepartum and postpartum diets analyzed 0.48 and 0.38 mg of Cr/kg of DM, respectively. Milk samples were obtained from the a.m. milking on d 0 (colostrum), 7, 14, 21, 28, 42, 56, 77, and 90 and on the final day of the study for Cr analysis. Cows were harvested after lactating for a minimum of 91 d and samples of liver, kidney, semitendinosus muscle, and fat were obtained for Cr analysis. Chromium was measured using electrothermal atomic absorption spectrophotometry. Milk Cr concentration averaged 1.7 ng/mL and was affected by day of lactation but not by Cr or a Cr × day interaction. Supplementation of 2 mg of Cr/kg of DM increased kidney Cr by approximately 3-fold and liver Cr concentrations by approximately 2-fold. Chromium concentrations in muscle and fat were not affected by Cr supplementation. In summary, supplementation of Cr Prop at a level of 2 mg of Cr/kg of DM did not affect Cr concentration in milk, muscle, or fat, the major bovine products consumed by humans.}, number={10}, journal={JOURNAL OF DAIRY SCIENCE}, author={Lloyd, K. E. and Fellner, V. and McLeod, S. J. and Fry, R. S. and Krafka, K. and Lamptey, A. and Spears, J. W.}, year={2010}, month={Oct}, pages={4774–4780} }
 @article{hansen_ashwell_moeser_fry_knutson_spears_2010, title={High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves}, volume={93}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2009-2341}, abstractNote={A 56-d experiment was designed to examine the effect of high dietary Fe on metal transporters involved in Fe and Mn metabolism. Fourteen weaned Holstein calves were stratified by weight and randomly assigned to 1 of 2 treatments: 1) no supplemental Fe (normal Fe) or 2) 750mg of supplemental Fe/kg of dry matter (high Fe). Jugular blood was collected on d 0, 35, and 56. At the end of the trial, 6 calves per treatment were humanely killed and duodenal scrapings, liver, and heart were collected for analysis. Additionally, proximal duodenum was mounted on Ussing chambers to assess intestinal barrier integrity. Calves receiving high dietary Fe displayed decreased transepithelial resistance and increased apical-to-basolateral flux of radiolabeled mannitol, suggesting that high Fe created increased intestinal permeability. Feeding calves a diet high in Fe decreased average daily gain, dry matter intake, and feed efficiency. Hemoglobin and serum Fe concentrations did not differ due to dietary treatment. High dietary Fe increased concentrations of Fe in the liver, but did not affect heart or duodenal Fe concentrations. Duodenal Mn concentrations were lowered by feeding a high Fe diet, but liver and heart Mn concentrations were not affected. As determined by real-time reverse transcription PCR, relative hepatic expression of the gene that encodes the Fe regulatory hormone hepcidin was 5-fold greater in calves fed high dietary Fe. Hepcidin is released in response to increased Fe status and binds to the Fe export protein ferroportin causing ferroportin to be degraded, thereby reducing dietary Fe absorption. Confirmation of this result was achieved through Western blotting of duodenal protein, which revealed that ferroportin was decreased in calves fed high dietary Fe. Duodenal protein expression of divalent metal transporter 1 (DMT1), a Fe import protein that can also transport Mn, tended to be reduced by high dietary Fe. Transcript levels of several genes involved in Fe metabolism in liver and duodenum were unchanged by treatment. In summary, feeding calves a diet high in Fe induced a signal cascade (hepcidin) designed to reduce absorption of Fe (via reduced protein expression of ferroportin and DMT1) in a manner similar to that reported in rodents. Additionally, reduced levels of DMT1 protein appeared to decrease duodenal Mn, suggesting that Mn may also be a substrate for DMT1 in cattle.}, number={2}, journal={JOURNAL OF DAIRY SCIENCE}, author={Hansen, S. L. and Ashwell, M. S. and Moeser, A. J. and Fry, R. S. and Knutson, M. D. and Spears, J. W.}, year={2010}, month={Feb}, pages={656–665} }
 @article{hansen_ashwell_legleiter_fry_lloyd_spears_2009, title={The addition of high manganese to a copper-deficient diet further depresses copper status and growth of cattle}, volume={101}, ISSN={["1475-2662"]}, DOI={10.1017/S0007114508057589}, abstractNote={A study was conducted evaluating the effect of long-term Cu deficiency, with or without high Mn, on growth, gene expression and Cu status of beef cattle. Twenty-one Angus calves were born to cows receiving one of the following treatments: (1) 10 mg supplemental Cu/kg DM (+Cu); (2) no supplemental Cu and 2 mg Mo/kg DM ( − Cu); (3) − Cu diet plus 500 mg supplemental Mn/kg DM ( − Cu+Mn). Calves were weaned at approximately 183 d of age and individually fed throughout the growing and finishing phases. Plasma Cu was lower (P < 0·01) in − Cu calves compared with +Cu calves while high dietary Mn further depressed (P < 0·01) plasma Cu in − Cu+Mn calvesv.− Cu calves. Liver Cu concentrations in +Cu calves were greater (P < 0·01) than in − Cu calves, with no differences between − Cu and − Cu+Mn calves. The daily body-weight gain of +Cu calves was greater (P < 0·01) than − Cu calves during the period from birth to weaning, but did not differ during the growing phase. − Cu+Mn calves gained less (P < 0·05) than − Cu calves during the growing phase. DM intake was lower (P < 0·01) in − Cu+Mn calvesv.− Cu calves, and did not differ among +Cu and − Cu calves. The relative gene expression of cytochrome c oxidase in the liver was lower (P < 0·05) in − Cu calves compared with +Cu or − Cu+Mn calves. In conclusion, feeding a Cu − deficient diet in combination with high Mn negatively affected the growth and Cu status of beef cattle.}, number={7}, journal={BRITISH JOURNAL OF NUTRITION}, author={Hansen, Stephanie L. and Ashwell, Melissa S. and Legleiter, Leon R. and Fry, Robert S. and Lloyd, Karen E. and Spears, Jerry W.}, year={2009}, month={Apr}, pages={1068–1078} }
 @article{legleiter_liu_lloyd_hansen_fry_spears_2007, title={Exposure to low dietary copper or low copper coupled with high dietary manganese for one year does not alter brain prion protein characteristics in the mature cow}, volume={85}, ISSN={["0021-8812"]}, DOI={10.2527/jas.2007-0215}, abstractNote={It is now widely accepted that abnormal prion proteins are the likely causative agent in bovine spongiform encephalopathy. Cellular prion proteins (PrP(c)) bind Cu, which appears to be required to maintain functional characteristics of the protein. The replacement of Cu on PrP(c) with Mn has resulted in loss of function and increased protease resistance. Twelve mature cows were used to determine the effects of Cu deficiency, alone and coupled with high dietary Mn, on brain Cu and Mn concentrations and on PrP(c) functional characteristics. Copper-adequate cows were randomly assigned to treatments: 1) control (adequate in Cu and Mn), 2) Cu-deficient (-Cu), and 3) Cu-deficient plus high dietary Mn (-Cu+Mn). Cows assigned to treatments -Cu and -Cu+Mn received no supplemental Cu and were supplemented with Mo to further induce Cu deficiency. After 360 d, Cu-deficient cows (-Cu and -Cu+Mn) tended to have lesser concentrations of Cu (P = 0.09) in the obex region of the brain stem. Brain Mn tended (P = 0.09) to be greater in -Cu+Mn cattle compared with -Cu cattle. Western blots revealed that PrP(c) relative optical densities, proteinase K degradability, elution profiles, molecular weights, and glycoform distributions were not different among treatments. The concentration of PrP(c), as determined by ELISA, was similar across treatment groups. Brain tissue (obex) Mn superoxide dismutase activity was greatest (P = 0.04) in cattle receiving -Cu+Mn, whereas immunopurified PrP(c) had similar superoxide dismutase-like activities among treatments. Immunopurified PrP(c) had similar Cu concentrations across treatments, whereas Mn was undetectable. We concluded that Cu deficiency, coupled with excessive Mn intake, in the bovine may decrease brain Cu and increase brain Mn. Copper deficiency, alone or coupled with high dietary Mn, did not cause detectable alterations in PrP(c) functional characteristics.}, number={11}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Legleiter, L. R. and Liu, H. C. and Lloyd, K. E. and Hansen, S. L. and Fry, R. S. and Spears, J. W.}, year={2007}, month={Nov}, pages={2895–2903} }