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Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

Prithiviraj, Sujeethkumar LU ; Garcia Garcia, Alejandro LU ; Linderfalk, Karin LU ; Yiguang, Bai LU ; Ferveur, Sonia LU ; Falck, Ludvig Nilsén LU ; Subramaniam, Agatheeswaran LU ; Mohlin, Sofie LU orcid ; Hidalgo Gil, David LU and Dupard, Steven J LU , et al. (2025) In eLife 13.
Abstract

Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred... (More)

Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knock out vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification, and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road toward the compositional eECMs editing and their exploitation in broad regenerative contexts.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Humans, CRISPR-Cas Systems, Mesenchymal Stem Cells/metabolism, Extracellular Matrix/metabolism, Animals, Gene Editing/methods, Tissue Engineering/methods, Rats, Cell Line, Vascular Endothelial Growth Factor A/genetics, Core Binding Factor Alpha 1 Subunit/genetics
in
eLife
volume
13
article number
RP96941
publisher
eLife Sciences Publications
external identifiers
  • pmid:40152921
ISSN
2050-084X
DOI
10.7554/eLife.96941
language
English
LU publication?
yes
additional info
© 2024, Prithiviraj et al.
id
27f0a458-1099-4ff7-baa7-cb8742c8df6d
date added to LUP
2025-09-29 12:53:33
date last changed
2025-09-29 13:33:38
@article{27f0a458-1099-4ff7-baa7-cb8742c8df6d,
  abstract     = {{<p>Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knock out vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification, and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road toward the compositional eECMs editing and their exploitation in broad regenerative contexts.</p>}},
  author       = {{Prithiviraj, Sujeethkumar and Garcia Garcia, Alejandro and Linderfalk, Karin and Yiguang, Bai and Ferveur, Sonia and Falck, Ludvig Nilsén and Subramaniam, Agatheeswaran and Mohlin, Sofie and Hidalgo Gil, David and Dupard, Steven J and Zacharaki, Dimitra and Raina, Deepak Bushan and Bourgine, Paul E}},
  issn         = {{2050-084X}},
  keywords     = {{Humans; CRISPR-Cas Systems; Mesenchymal Stem Cells/metabolism; Extracellular Matrix/metabolism; Animals; Gene Editing/methods; Tissue Engineering/methods; Rats; Cell Line; Vascular Endothelial Growth Factor A/genetics; Core Binding Factor Alpha 1 Subunit/genetics}},
  language     = {{eng}},
  month        = {{03}},
  publisher    = {{eLife Sciences Publications}},
  series       = {{eLife}},
  title        = {{Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines}},
  url          = {{http://dx.doi.org/10.7554/eLife.96941}},
  doi          = {{10.7554/eLife.96941}},
  volume       = {{13}},
  year         = {{2025}},
}