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Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation

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Abstract

Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPRBMP-9+, followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and  protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-β/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.

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Fig. 1: Use of CRISPRa to overexpress BMP-9 in immortalized bone marrow-derived MSCs (iMSCs).
Fig. 2: Effect of BMP-9 overexpression on osteoblastic differentiation of immortalized bone marrow-derived MSCs (iMSCs).
Fig. 3: Effect of immortalized bone marrow-derived MSCs (iMSCs) overexpressing BMP-9 on bone formation in rat calvarial defects: µCT analysis.
Fig. 4: Effect of immortalized bone marrow-derived MSCs (iMSCs) overexpressing BMP-9 on bone formation in rat calvarial defects: histological analysis.

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The corresponding author declares that the data are available if requested.

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Acknowledgements

This research was supported by São Paulo Research Foundation (FAPESP, grants # 2016/23850-8; 2017/12622-7; 2019/01346-4) and National Institutes of Health (NIH, grants # R01 AR039588 and R01 DE029311. The English language review was done by ENAGO (www.enago.com).

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  1. These authors contributed equally: Gileade P. Freitas, Helena B. Lopes

Authors and Affiliations

  1. Bone Research Lab, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

    Gileade P. Freitas, Helena B. Lopes, Alann T. P. Souza, Maria Paula O. Gomes, Georgia K. Quiles, Marcio M. Beloti & Adalberto L. Rosa

  2. Department of Biochemistry, University of Vermont School of Medicine, Burlington, VT, USA

    Jonathan Gordon, Coralee Tye, Janet L. Stein, Gary S. Stein & Jane B. Lian

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Contributions

GPF and HBL undertook the experiments, analyzed the data, and drafted the manuscript. ATPS, M.P.O.G., and GKQ performed the in vivo experiments, analyzed the data, and revised the manuscript. JG and CT performed the in vitro experiments, analyzed the data, and revised the manuscript. JLS, GSS, JBL, MMB, and ALR designed the study, analyzed the data, and drafted the manuscript.

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Correspondence to Adalberto L. Rosa.

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Freitas, G.P., Lopes, H.B., Souza, A.T.P. et al. Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation. Gene Ther 28, 748–759 (2021). https://doi.org/10.1038/s41434-021-00248-8

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