@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. 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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{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.}, 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{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.}, 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{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_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} }