Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia
IntroductionBesides skeletal symptoms, dental abnormalities are a typical feature of the rare inherited disorder hypophosphatasia (HPP), which is caused by loss of function mutations in the ALPL gene (alkaline phosphatase, biomineralization associated) coding for tissue-nonspecific alkaline phosphat...
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Frontiers Media S.A.
2025-06-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| author | Jana Schiffmaier Jana Schiffmaier Sofia Rehling Sofia Rehling Katharina Marnet Katharina Marnet Angela Borst Drenka Trivanović Drenka Trivanović Drenka Trivanović Denitsa Docheva Franz Jakob Stephanie Graser Marietta Herrmann Daniel Liedtke |
| author_facet | Jana Schiffmaier Jana Schiffmaier Sofia Rehling Sofia Rehling Katharina Marnet Katharina Marnet Angela Borst Drenka Trivanović Drenka Trivanović Drenka Trivanović Denitsa Docheva Franz Jakob Stephanie Graser Marietta Herrmann Daniel Liedtke |
| author_sort | Jana Schiffmaier |
| collection | DOAJ |
| description | IntroductionBesides skeletal symptoms, dental abnormalities are a typical feature of the rare inherited disorder hypophosphatasia (HPP), which is caused by loss of function mutations in the ALPL gene (alkaline phosphatase, biomineralization associated) coding for tissue-nonspecific alkaline phosphatase (TNAP). Dental symptoms include premature loss of deciduous teeth, disturbance in dentin and cementum mineralization, and an increased risk for periodontitis. However, the underlying molecular mechanisms are not fully understood and experimental cell lines for in vitro analyses of these processes are missing.MethodsWe aimed to develop a physiologically relevant cellular model of dental origin with genetic ALPL variants to investigate the molecular consequences of TNAP deficiencies in vitro. For this purpose, we used immortalized periodontal ligament stem cells (PDL-hTERT cells) to establish five independent clonal cell lines via CRISPR/Cas9, harboring different ALPL genetic variants.ResultsDetailed investigation of their genetic properties revealed that four different genotypes were successfully established at two different positions within the ALPL gene locus. The detected variants either result in mis-splicing of ALPL mRNAs or in frameshift mutations. All determined variants implied severe consequences on TNAP function, as indicated by in silico modeling and comparison to reported human pathogenic variants. Subsequent detailed cell culture experiments demonstrated TNAP residual gene expression and altered TNAP activity in the newly established ALPLtg PDL-hTERT lines. Further assessment of cell line features showed significantly reduced cell growth, partly lower levels of intracellular ATP as well as mitochondrial function proteins. TNAP activity was furthermore investigated during in vitro osteogenic differentiation and strong suppression during this process in nearly all newly established lines was observed.DiscussionWe report the generation of a new set of immortalized ALPLtg PDL-hTERT cells for investigation of TNAP cellular function in PDL cells, which can be used in subsequent studies for deciphering molecular processes in dental cells affected by reduction of TNAP function. |
| format | Article |
| id | doaj-art-dbd9596e3d714197b98febe15a1a018f |
| institution | DOAJ |
| issn | 2296-634X |
| language | English |
| publishDate | 2025-06-01 |
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| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj-art-dbd9596e3d714197b98febe15a1a018f2025-08-20T03:07:17ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2025-06-011310.3389/fcell.2025.15725711572571Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasiaJana Schiffmaier0Jana Schiffmaier1Sofia Rehling2Sofia Rehling3Katharina Marnet4Katharina Marnet5Angela Borst6Drenka Trivanović7Drenka Trivanović8Drenka Trivanović9Denitsa Docheva10Franz Jakob11Stephanie Graser12Marietta Herrmann13Daniel Liedtke14IZKF Group Tissue Regeneration in Musculoskeletal Diseases, University Hospital Würzburg, Würzburg, GermanyBernhard-Heine-Center for Locomotion Research, University of Würzburg, Würzburg, GermanyIZKF Group Tissue Regeneration in Musculoskeletal Diseases, University Hospital Würzburg, Würzburg, GermanyBernhard-Heine-Center for Locomotion Research, University of Würzburg, Würzburg, GermanyIZKF Group Tissue Regeneration in Musculoskeletal Diseases, University Hospital Würzburg, Würzburg, GermanyBernhard-Heine-Center for Locomotion Research, University of Würzburg, Würzburg, GermanyInstitute of Human Genetics, Biocenter, University of Würzburg, Würzburg, GermanyIZKF Group Tissue Regeneration in Musculoskeletal Diseases, University Hospital Würzburg, Würzburg, GermanyBernhard-Heine-Center for Locomotion Research, University of Würzburg, Würzburg, GermanyInstitute for Medical Research, National Institute of the Republic of Serbia, University of Belgrade, Belgrade, SerbiaDepartment of Musculoskeletal Tissue Regeneration, Bernhard-Heine-Center for Locomotion Research and Orthopedic Hospital König-Ludwig-Haus, University of Würzburg, Würzburg, GermanyBernhard-Heine-Center for Locomotion Research, University of Würzburg, Würzburg, GermanyBernhard-Heine-Center for Locomotion Research, University of Würzburg, Würzburg, GermanyIZKF Group Tissue Regeneration in Musculoskeletal Diseases, University Hospital Würzburg, Würzburg, GermanyInstitute of Human Genetics, Biocenter, University of Würzburg, Würzburg, GermanyIntroductionBesides skeletal symptoms, dental abnormalities are a typical feature of the rare inherited disorder hypophosphatasia (HPP), which is caused by loss of function mutations in the ALPL gene (alkaline phosphatase, biomineralization associated) coding for tissue-nonspecific alkaline phosphatase (TNAP). Dental symptoms include premature loss of deciduous teeth, disturbance in dentin and cementum mineralization, and an increased risk for periodontitis. However, the underlying molecular mechanisms are not fully understood and experimental cell lines for in vitro analyses of these processes are missing.MethodsWe aimed to develop a physiologically relevant cellular model of dental origin with genetic ALPL variants to investigate the molecular consequences of TNAP deficiencies in vitro. For this purpose, we used immortalized periodontal ligament stem cells (PDL-hTERT cells) to establish five independent clonal cell lines via CRISPR/Cas9, harboring different ALPL genetic variants.ResultsDetailed investigation of their genetic properties revealed that four different genotypes were successfully established at two different positions within the ALPL gene locus. The detected variants either result in mis-splicing of ALPL mRNAs or in frameshift mutations. All determined variants implied severe consequences on TNAP function, as indicated by in silico modeling and comparison to reported human pathogenic variants. Subsequent detailed cell culture experiments demonstrated TNAP residual gene expression and altered TNAP activity in the newly established ALPLtg PDL-hTERT lines. Further assessment of cell line features showed significantly reduced cell growth, partly lower levels of intracellular ATP as well as mitochondrial function proteins. TNAP activity was furthermore investigated during in vitro osteogenic differentiation and strong suppression during this process in nearly all newly established lines was observed.DiscussionWe report the generation of a new set of immortalized ALPLtg PDL-hTERT cells for investigation of TNAP cellular function in PDL cells, which can be used in subsequent studies for deciphering molecular processes in dental cells affected by reduction of TNAP function.https://www.frontiersin.org/articles/10.3389/fcell.2025.1572571/fullhypophosphatasiaALPLTNAPPDL-hTERT cellsin vitro |
| spellingShingle | Jana Schiffmaier Jana Schiffmaier Sofia Rehling Sofia Rehling Katharina Marnet Katharina Marnet Angela Borst Drenka Trivanović Drenka Trivanović Drenka Trivanović Denitsa Docheva Franz Jakob Stephanie Graser Marietta Herrmann Daniel Liedtke Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia Frontiers in Cell and Developmental Biology hypophosphatasia ALPL TNAP PDL-hTERT cells in vitro |
| title | Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia |
| title_full | Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia |
| title_fullStr | Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia |
| title_full_unstemmed | Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia |
| title_short | Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia |
| title_sort | establishment of human periodontal ligament cell lines with alpl mutations to mimic dental aspects of hypophosphatasia |
| topic | hypophosphatasia ALPL TNAP PDL-hTERT cells in vitro |
| url | https://www.frontiersin.org/articles/10.3389/fcell.2025.1572571/full |
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