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
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 (<i>SH3PXD2b</i>) cause a rare developmental disorder...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/15/8803 |
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| author | Loretta László Hédi Maczelka Tamás Takács Anita Kurilla Álmos Tilajka László Buday Virag Vas Ágota Apáti |
| author_facet | Loretta László Hédi Maczelka Tamás Takács Anita Kurilla Álmos Tilajka László Buday Virag Vas Ágota Apáti |
| author_sort | Loretta László |
| collection | DOAJ |
| description | 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 (<i>SH3PXD2b</i>) 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 <i>SH3PXD2b</i> 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. |
| first_indexed | 2024-03-09T05:18:53Z |
| format | Article |
| id | doaj.art-23a6830da489494b871292c266b40b23 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T05:18:53Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-23a6830da489494b871292c266b40b232023-12-03T12:42:03ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-08-012315880310.3390/ijms23158803A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model SystemLoretta László0Hédi Maczelka1Tamás Takács2Anita Kurilla3Álmos Tilajka4László Buday5Virag Vas6Ágota Apáti7Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryInstitute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryInstitute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryInstitute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryInstitute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryInstitute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryInstitute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryInstitute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, HungaryTyrosine 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 (<i>SH3PXD2b</i>) 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 <i>SH3PXD2b</i> 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.https://www.mdpi.com/1422-0067/23/15/8803FTHSFrank-Ter Haar syndromeCRISPR-Cas9Tks4human embryonic stem cellscell commitment |
| spellingShingle | Loretta László Hédi Maczelka Tamás Takács Anita Kurilla Álmos Tilajka László Buday Virag Vas Ágota Apáti 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 International Journal of Molecular Sciences FTHS Frank-Ter Haar syndrome CRISPR-Cas9 Tks4 human embryonic stem cells cell commitment |
| 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | novel cell based model for a rare disease the tks4 ko human embryonic stem cell line as a frank ter haar syndrome model system |
| topic | FTHS Frank-Ter Haar syndrome CRISPR-Cas9 Tks4 human embryonic stem cells cell commitment |
| url | https://www.mdpi.com/1422-0067/23/15/8803 |
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