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

Author(s):Show less -

Freitas, Gileade P. ^[1] ; Lopes, Helena B. ^[1] ; Souza, Alann T. P. ^[1] ; Gomes, Maria Paula O. ^[1] ; Quiles, Georgia K. ^[1] ; Gordon, Jonathan ^[2] ; Tye, Coralee ^[2] ; Stein, Janet L. ^[2] ; Stein, Gary S. ^[2] ; Lian, Jane B. ^[2] ; Beloti, Marcio M. ^[1] ; Rosa, Adalberto L. ^[1]

Affiliation

^[1] Univ Sao Paulo, Sch Dent Ribeirao Preto, Bone Res Lab, Ribeirao Preto, SP - Brazil

^[2] Univ Vermont, Dept Biochem, Sch Med, Burlington, VT 05405 - USA

• Document type: Journal article
• Source: Gene Therapy; v. 28, n. 12, p. 748-759, DEC 2021.
• Web of Science Citations: 2

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-beta/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. (AU)

• FAPESP's process: 19/01346-4 - Evaluation of the angiogenic and bone regeneration potentials of BM-MSCs and AT-BMCs genetically edited by CRISP-Cas9 to overexpress VEGF-A and BMP-9
• Grantee: Gileade Pereira Freitas
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 16/23850-8 - Gene therapy for bone regeneration using CRISPR-Cas9 system
• Grantee: Gileade Pereira Freitas
• Support type: Scholarships abroad - Research Internship - Doctorate