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

IF 6.4 1区 生物学 Q1 BIOLOGY
eLife Pub Date : 2025-03-28 DOI:10.7554/eLife.96941
Sujeethkumar Prithiviraj, Alejandro Garcia Garcia, Karin Linderfalk, Bai Yiguang, Sonia Ferveur, Ludvig Nilsén Falck, Agatheeswaran Subramaniam, Sofie Mohlin, David Hidalgo Gil, Steven J Dupard, Dimitra Zacharaki, Deepak Bushan Raina, Paul E Bourgine
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引用次数: 0

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 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.

利用CRISPR/Cas9技术编辑人间充质干细胞细胞系的细胞外基质
组织工程策略主要依赖于生产活体替代品,由此植入的细胞积极参与再生过程。除了成本和延迟移植的可用性外,工程组织的患者特异性表现对其临床转化能力提出了严重的担忧。一个更令人兴奋的范例是利用细胞铺设的工程细胞外基质(eecm),它可以用作现成的材料。在这里,再生能力仅依赖于eECM结构的保存和嵌入式信号来指导内源性修复。我们最近描述了利用定制人类干细胞系制造eECM的可能性。除了标准化之外,这些细胞系的可用性为应用专用遗传工具设计定制的eecm开辟了道路。在这项研究中,我们展示了CRISPR/Cas9作为编辑eecm的组成和功能的高精度系统的利用。对人间充质基质/干细胞(hMSC)系进行修饰,敲除血管内皮生长因子(VEGF)和runt相关转录因子2 (RUNX2),并评估其生成骨诱导软骨基质的能力。我们报道了对hMSCs的成功编辑,随后导致靶向VEGF和runx2敲除软骨eecm。尽管缺乏VEGF, eecm仍保留了指导异位软骨内成骨的全部能力。相反,runx2编辑的eecm在大鼠骨软骨缺损中表现出受损的肥大、减少的异位骨化和更好的软骨修复。总之,我们的方法可以用来识别驱动内源性修复的必要eECM因子。我们的工作为合成eecm编辑及其在广泛再生环境中的开发铺平了道路。
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来源期刊
eLife
eLife BIOLOGY-
CiteScore
12.90
自引率
3.90%
发文量
3122
审稿时长
17 weeks
期刊介绍: eLife is a distinguished, not-for-profit, peer-reviewed open access scientific journal that specializes in the fields of biomedical and life sciences. eLife is known for its selective publication process, which includes a variety of article types such as: Research Articles: Detailed reports of original research findings. Short Reports: Concise presentations of significant findings that do not warrant a full-length research article. Tools and Resources: Descriptions of new tools, technologies, or resources that facilitate scientific research. Research Advances: Brief reports on significant scientific advancements that have immediate implications for the field. Scientific Correspondence: Short communications that comment on or provide additional information related to published articles. Review Articles: Comprehensive overviews of a specific topic or field within the life sciences.
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