{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,2]],"date-time":"2025-11-02T05:35:29Z","timestamp":1762061729500,"version":"build-2065373602"},"reference-count":35,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2022,8,8]],"date-time":"2022-08-08T00:00:00Z","timestamp":1659916800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Research, Development, and Innovation Fund of Hungary","award":["K124045","K128369","FIEK_16-1-2016\u20130005","HunProtExc 2018-1.2.1-NKP-2018\u201300005","K\u00d6-39\/2021","RRF-2.3.1-21-2022-00015"],"award-info":[{"award-number":["K124045","K128369","FIEK_16-1-2016\u20130005","HunProtExc 2018-1.2.1-NKP-2018\u201300005","K\u00d6-39\/2021","RRF-2.3.1-21-2022-00015"]}]},{"name":"Cooperation between ELKH TTK and PTE SzKK in Areas of Internationally Significant Biomedical Research","award":["K124045","K128369","FIEK_16-1-2016\u20130005","HunProtExc 2018-1.2.1-NKP-2018\u201300005","K\u00d6-39\/2021","RRF-2.3.1-21-2022-00015"],"award-info":[{"award-number":["K124045","K128369","FIEK_16-1-2016\u20130005","HunProtExc 2018-1.2.1-NKP-2018\u201300005","K\u00d6-39\/2021","RRF-2.3.1-21-2022-00015"]}]},{"name":"National Research, Development and Innovation Office","award":["K124045","K128369","FIEK_16-1-2016\u20130005","HunProtExc 2018-1.2.1-NKP-2018\u201300005","K\u00d6-39\/2021","RRF-2.3.1-21-2022-00015"],"award-info":[{"award-number":["K124045","K128369","FIEK_16-1-2016\u20130005","HunProtExc 2018-1.2.1-NKP-2018\u201300005","K\u00d6-39\/2021","RRF-2.3.1-21-2022-00015"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Tyrosine kinase substrate with four SH3 domains (Tks4) scaffold protein plays roles in cell migration and podosome formation and regulates systemic mechanisms such as adult bone homeostasis and adipogenesis. Mutations in the Tks4 gene (SH3PXD2b) cause a rare developmental disorder called Frank-Ter Haar syndrome (FTHS), which leads to heart abnormalities, bone tissue defects, and reduced adiposity. We aimed to produce a human stem cell-based in vitro FTHS model system to study the effects of the loss of the Tks4 protein in different cell lineages and the accompanying effects on the cell signalome. To this end, we used CRISPR\/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)\/CRISPR associated (Cas9)) to knock out the SH3PXD2b gene in the HUES9 human embryonic stem cell line (hESC), and we obtained stable homo- and heterozygous knock out clones for use in studying the potential regulatory roles of Tks4 protein in embryonic stem cell biology. Based on pluripotency marker measurements and spontaneous differentiation capacity assays, we concluded that the newly generated Tks4-KO HUES9 cells retained their embryonic stem cell characteristics. We propose that the Tks4-KO HUES9 cells could serve as a tool for further cell differentiation studies to investigate the involvement of Tks4 in the complex disorder FTHS. Moreover, we successfully differentiated all of the clones into mesenchymal stem cells (MSCs). The derived MSC cultures showed mesenchymal morphology and expressed MSC markers, although the expression levels of mesodermal and osteogenic marker genes were reduced, and several EMT (epithelial mesenchymal transition)-related features were altered in the Tks4-KO MSCs. Our results suggest that the loss of Tks4 leads to FTHS by altering cell lineage differentiation and cell maturation processes, rather than by regulating embryonic stem cell potential.<\/jats:p>","DOI":"10.3390\/ijms23158803","type":"journal-article","created":{"date-parts":[[2022,8,8]],"date-time":"2022-08-08T21:09:18Z","timestamp":1659992958000},"page":"8803","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0367-4900","authenticated-orcid":false,"given":"Loretta","family":"L\u00e1szl\u00f3","sequence":"first","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"},{"name":"National Laboratory for Drug Research and Development, 1117 Budapest, Hungary"},{"name":"Doctoral School of Biology, Institute of Biology, ELTE E\u00f6tv\u00f6s Lor\u00e1nd University, 1117 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"H\u00e9di","family":"Maczelka","sequence":"additional","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"},{"name":"Basic and Translational Medicine Doctoral School, Semmelweis University, 1085 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tam\u00e1s","family":"Tak\u00e1cs","sequence":"additional","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"},{"name":"National Laboratory for Drug Research and Development, 1117 Budapest, Hungary"},{"name":"Doctoral School of Biology, Institute of Biology, ELTE E\u00f6tv\u00f6s Lor\u00e1nd University, 1117 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anita","family":"Kurilla","sequence":"additional","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"},{"name":"Doctoral School of Biology, Institute of Biology, ELTE E\u00f6tv\u00f6s Lor\u00e1nd University, 1117 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c1lmos","family":"Tilajka","sequence":"additional","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"},{"name":"National Laboratory for Drug Research and Development, 1117 Budapest, Hungary"},{"name":"Doctoral School of Biology, Institute of Biology, ELTE E\u00f6tv\u00f6s Lor\u00e1nd University, 1117 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"L\u00e1szl\u00f3","family":"Buday","sequence":"additional","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"},{"name":"Doctoral School of Biology, Institute of Biology, ELTE E\u00f6tv\u00f6s Lor\u00e1nd University, 1117 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7249-6816","authenticated-orcid":false,"given":"Virag","family":"Vas","sequence":"additional","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"},{"name":"Doctoral School of Biology, Institute of Biology, ELTE E\u00f6tv\u00f6s Lor\u00e1nd University, 1117 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0380-8139","authenticated-orcid":false,"given":"\u00c1gota","family":"Ap\u00e1ti","sequence":"additional","affiliation":[{"name":"Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,8,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4186","DOI":"10.1021\/acs.biochem.8b00084","article-title":"EGF Regulates the Interaction of Tks4 with Src through Its SH2 and SH3 Domains","volume":"57","author":"Szeder","year":"2018","journal-title":"Biochemistry"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"31321","DOI":"10.1074\/jbc.M111.324897","article-title":"Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration","volume":"287","author":"Geiszt","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4348","DOI":"10.1111\/j.1742-4658.2010.07864.x","article-title":"Functional classification of scaffold proteins and related molecules","volume":"277","author":"Buday","year":"2010","journal-title":"FEBS J."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Vas, V., H\u00e1hner, T., Kudlik, G., Ernszt, D., Kvell, K., Kuti, D., Kov\u00e1cs, K.J., T\u00f3v\u00e1ri, J., Trexler, M., and Mer\u0151, B.L. (2019). Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing. Cells, 8.","DOI":"10.3390\/cells8080831"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5781","DOI":"10.1038\/s41598-019-42250-6","article-title":"Significance of the Tks4 scaffold protein in bone tissue homeostasis","volume":"9","author":"Vas","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kudlik, G., Tak\u00e1cs, T., Radnai, L., Kurilla, A., Szeder, B., Koprivanacz, K., Mer\u0151, B.L., Buday, L., and Vas, V. (2020). Advances in Understanding TKS4 and TKS5: Molecular Scaffolds Regulating Cellular Processes from Podosome and Invadopodium Formation to Differentiation and Tissue Homeostasis. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21218117"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Massadeh, S., Alhabshan, F., Alsudairi, H.N., Alkwai, S., Alswailm, M., Kabbani, M.S., Chaikhouni, F., and Alaamery, M. (2022). The Role of the Disrupted Podosome Adaptor Protein (SH3PXD2B) in Frank\u2013Ter Haar Syndrome. Genes, 13.","DOI":"10.3390\/genes13020236"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1097\/MCD.0000000000000389","article-title":"Novel SH3PXD2B variant identified by whole-exome sequencing in a Turkish newborn with frank-ter haar Syndrome","volume":"31","author":"Sager","year":"2022","journal-title":"Clin. Dysmorphol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"103857","DOI":"10.1016\/j.ejmg.2020.103857","article-title":"A severe case of Frank-ter Haar syndrome and literature review: Further delineation of the phenotypical spectrum","volume":"63","author":"Durand","year":"2020","journal-title":"Eur. J. Med. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"514","DOI":"10.1016\/j.jaapos.2017.07.216","article-title":"Ophthalmic findings in Frank-ter Haar syndrome: Report of a sibling pair","volume":"21","author":"Chang","year":"2017","journal-title":"J. AAPOS"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1007\/s40368-013-0092-x","article-title":"Common dental features and craniofacial development of three siblings with Ter Haar syndrome","volume":"15","author":"Parker","year":"2014","journal-title":"Eur. Arch. Paediatr. Dent."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1016\/j.ajhg.2010.01.009","article-title":"Disruption of the Podosome Adaptor Protein TKS4 (SH3PXD2B) Causes the Skeletal Dysplasia, Eye, and Cardiac Abnormalities of Frank-Ter Haar Syndrome","volume":"86","author":"Iqbal","year":"2010","journal-title":"Am. J. Hum. Genet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1302","DOI":"10.1091\/mbc.e08-09-0949","article-title":"The novel adaptor protein Tks4 (SH3PXD2B) is required for functional podosome formation","volume":"20","author":"Buschman","year":"2009","journal-title":"Mol. Biol. Cell"},{"key":"ref_14","first-page":"D77","article-title":"Expression Atlas update: From tissues to single cells","volume":"48","author":"Papatheodorou","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.neuron.2014.05.013","article-title":"NeuroResource CORTECON: A Temporal Transcriptome Analysis of In Vitro Human Cerebral Cortex Development from Human Embryonic Stem Cells","volume":"83","author":"Boles","year":"2014","journal-title":"Neuron"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1136\/gutjnl-2017-314817","article-title":"DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development","volume":"68","author":"Kraiczy","year":"2019","journal-title":"Gut"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.omtm.2020.10.019","article-title":"CRISPR\/Cas9-mediated generation and analysis of N terminus polymorphic models of \u03b22AR in isogenic hPSC-derived cardiomyocytes","volume":"20","author":"Kondrashov","year":"2021","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Klementieva, N., Goliusova, D., Krupinova, J., Yanvarev, V., Panova, A., Mokrysheva, N., and Kiselev, S.L. (2021). A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR\/Cas9 Genome Editing. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222112054"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Szeder, B., T\u00e1rnoki-Z\u00e1ch, J., Lakatos, D., Vas, V., Kudlik, G., Mer\u0151, B., Koprivanacz, K., B\u00e1nyai, L., H\u00e1mori, L., and R\u00f3na, G. (2019). Absence of the Tks4 Scaffold Protein Induces Epithelial-Mesenchymal Transition-Like Changes in Human Colon Cancer Cells. Cells, 8.","DOI":"10.3390\/cells8111343"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1016\/j.bbrc.2011.09.089","article-title":"Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth","volume":"414","author":"Varga","year":"2011","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3811","DOI":"10.1242\/dev.126.17.3811","article-title":"Goosecoid acts cell autonomously in mesenchyme-derived tissues during craniofacial development","volume":"126","author":"Wakamiya","year":"1999","journal-title":"Development"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"115819","DOI":"10.1016\/j.bone.2020.115819","article-title":"GATA4 regulates mesenchymal stem cells via direct transcriptional regulation of the WNT signalosome","volume":"144","author":"Khalid","year":"2021","journal-title":"Bone"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"11254","DOI":"10.1158\/0008-5472.CAN-07-2253","article-title":"Epithelial-Mesenchymal Transition Events during Human Embryonic Stem Cell Differentiation","volume":"67","author":"Eastham","year":"2007","journal-title":"Cancer Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.gene.2017.07.011","article-title":"Identification of two novel SH3PXD2B gene mutations in Frank-Ter Haar syndrome by exome sequencing: Case report and review of the literature","volume":"628","author":"Zrhidri","year":"2017","journal-title":"Gene"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1097\/IJG.0000000000001420","article-title":"A Rare Case Report of Frank ter Haar Syndrome in a Sibling Pair Presenting with Congenital Glaucoma","volume":"29","author":"Ratukondla","year":"2020","journal-title":"J. Glaucoma"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1007\/s00335-009-9210-9","article-title":"The podosomal-adaptor protein SH3PXD2B is essential for normal postnatal development","volume":"20","author":"Mao","year":"2009","journal-title":"Mamm. Genome"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"34280","DOI":"10.1038\/srep34280","article-title":"The scaffold protein Tks4 is required for the differentiation of mesenchymal stromal cells (MSCs) into adipogenic and osteogenic lineages","volume":"6","author":"Kudlik","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1007\/s00018-016-2358-z","article-title":"DNA repair mechanisms in embryonic stem cells","volume":"74","author":"Fu","year":"2016","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_29","first-page":"1000165","article-title":"Genomic Instability and Cancer","volume":"5","author":"Yao","year":"2014","journal-title":"J. Carcinog. Mutagen."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"12865","DOI":"10.1073\/pnas.202215399","article-title":"Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility","volume":"99","author":"Gabant","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"102134","DOI":"10.1016\/j.scr.2020.102134","article-title":"Establishing a human embryonic stem cell clone with a heterozygous mutation in the DGCR8 gene","volume":"50","author":"Borsy","year":"2021","journal-title":"Stem Cell Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1002\/cytob.21168","article-title":"Expression pattern of the human ABC transporters in pluripotent embryonic stem cells and in their derivatives","volume":"86","author":"Erdei","year":"2014","journal-title":"Cytom. Part B Clin. Cytom."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"L\u00e1nyi, \u00c1., Bar\u00e1th, M., P\u00e9terfi, Z., B\u00f6gel, G., Orient, A., Simon, T., Petrovszki, E., Kis-T\u00f3th, K., Sirokm\u00e1ny, G., and Rajnav\u00f6lgyi, \u00c9. (2011). The Homolog of the Five SH3-Domain Protein (HOFI\/SH3PXD2B) Regulates Lamellipodia Formation and Cell Spreading. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0023653"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"8343","DOI":"10.1038\/s41598-019-44593-6","article-title":"Human osteogenic differentiation in Space: Proteomic and epigenetic clues to better understand osteoporosis","volume":"9","author":"Gambacurta","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2748","DOI":"10.1002\/stem.2078","article-title":"Human Induced Pluripotent Stem Cell-Derived Microvesicles Transmit RNAs and Proteins to Recipient Mature Heart Cells Modulating Cell Fate and Behavior","volume":"33","author":"Kmiotek","year":"2015","journal-title":"Stem Cells"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8803\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:05:38Z","timestamp":1760141138000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8803"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,8,8]]},"references-count":35,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2022,8]]}},"alternative-id":["ijms23158803"],"URL":"https:\/\/doi.org\/10.3390\/ijms23158803","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2022,8,8]]}}}