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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.

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-VPR^BMP-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-VPR^BMP-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-VPR^BMP-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.

SUBMITTER: Freitas GP

PROVIDER: S-EPMC8423866 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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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.

Freitas Gileade P GP Lopes Helena B HB Souza Alann T P ATP Gomes Maria Paula O MPO Quiles Georgia K GK Gordon Jonathan J Tye Coralee C Stein Janet L JL Stein Gary S GS Lian Jane B JB Beloti Marcio M MM Rosa Adalberto L AL

Gene therapy 20210308 12

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-VPR<sup>BMP-9+</sup>, followed by in vitro evaluation of osteogenic potential and in vivo enhancement ...[more]

PMID: 33686254

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Project description:BackgroundOsteoporosis is characterized by low systemic bone mineral content and destruction of bone microarchitecture. Promoting bone regeneration and reversing its loss by infusion of exogenous bone marrow mesenchymal stem cells (BMSCs) is a potentially effective treatment for osteoporosis. However, their limited migration to target organs reduces the therapeutic effect of the cells. Stromal cell-derived factor 1 (SDF1) is a chemokine that induces targeted cell migration through the SDF1/CXCR4 (C-X-C chemokine receptor 4) axis and can induce migration of exogenous mesenchymal stem cells to sites of high SDF1 concentration. There are no studies on BMSCs overexpressing SDF1 (SDF1-BMSCs) in osteoporotic mice in vivo. We aimed to investigate if the increased SDF1 concentration facilitated cell migration to the bone.MethodsWe used lentivirus to construct BMSCs overexpressing SDF1 or knocking down CXCR4. We verified the proliferation ability of the cells in vitro using Cell Counting Kit-8 (CCK8) and 5-Bromodeoxyuridinc (BrdU), the migration ability of the cells using Transwell, and the osteogenic and lipogenic ability of the cells using osteogenic and lipogenic induction solutions. In in vivo experiments, we induced osteoporosis in 72 female mice by ovariectomy and injected different groups of cells via the tail vein. Femoral tissue samples were collected for a fixed time, and the osteogenic and homing abilities of the cells were verified by MicroCT and tissue section staining.ResultsWe successfully demonstrated that high expression of SDF1 promoted cell proliferation and migration in vitro, without affecting their cell differentiation ability. In an ovariectomized mouse model, SDF1-BMSCs were more likely to be home to the femur than the BMSCs, had a better pro-osteogenic ability, and had higher expression of Wnt-1. Blocking the SDF1/CXCR4 axis reduced the homing of exogenous mesenchymal stem cells (MSCs) to the femur and their osteogenic capacity.ConclusionsSDF1-BMSCs can further promote bone formation by increasing the number of cells homing to the femur in osteoporotic mice. Our study shows that stem cells can promote their proliferation and home to the femur via the SDF1/CXCR4 axis and further help bone formation via Wnt-1 signaling.

Dataset Information

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

Publications

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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