Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration

Hsu, Mu‐Nung; Huang, Kai-Lun; Yu, Fu-Jen; Lai, Po‐Liang; Truong, Anh Vu; Lin, Mingyao; Nguyen, Nuong Thi Kieu; Shen, Chih-Che; Hwang, Shiaw-Min; Chang, Yu‐Han; Hu, Yu‐Chen

doi:10.1016/j.ymthe.2019.11.029

Cited by 41 publications

(21 citation statements)

References 64 publications

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“…In contrast to the conventional gene therapy approach, the Bac-CRISPRa system, which is programmable and capable of activating multiple target genes, may be a promising technology in bone regeneration (Larouche & Aguilar, 2019). A recent study used CRISPRa-engineered BMSCs to activate Wnt10b and Foxc2, resulting in a remarkably improved calvarial bone healing (Hsu et al, 2020). Therefore, using cell markers to purified the primary PDCs and CRISPRs system therapy may further optimize bone healing.…”

Section: Discussionmentioning

confidence: 99%

Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

Jin

Sun

Fang

et al. 2020

PeerJ

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Background Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. Methods We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. Results Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. Conclusion Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

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Section: Discussionmentioning

confidence: 99%

Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

Jin

Sun

Fang

et al. 2020

PeerJ

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“…Because of their osteogenic potential, BMSCs are able to undergo self-renewal and differentiate into tissues of mesenchymal origin, making them significant contributors during bone repair [2,3]. Moreover, it was reported that BMSC osteogenesis promotes calvarial bone regeneration [4]. Based on the findings from a prior study, BMSCderived extracellular vesicles (EVs) assist osteogenic differentiation in steroid-induced femoral head necrosis [5].…”

Section: Introductionmentioning

confidence: 99%

Extracellular vesicle-encapsulated miR-22-3p from bone marrow mesenchymal stem cell promotes osteogenic differentiation via FTO inhibition

et al. 2020

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Background: Bone marrow mesenchymal stem cells (BMSCs) exhibit the capacity to self-renew and differentiate into multi-lineage cell types, including osteoblasts, which are crucial regulators of fracture healing. Thus, this study aims to investigate the effect of microRNA (miR)-22-3p from BMSC-derived EVs on osteogenic differentiation and its underlying mechanism. Methods: Extracellular vesicles (EVs) were isolated from BMSCs and taken up with BMSCs. Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-22-3p and FTO. Loss-and gain-of-function experiments were performed to determine the roles of EV-delivered miR-22-3p and FTO in osteogenic differentiation as well as their regulatory role in the MYC/PI3K/AKT axis. To determine the osteogenic differentiation, ALP and ARS stainings were conducted, and the levels of RUNX2, OCN, and OPN level were determined. In vivo experiment was conducted to determine the function of EV-delivered miR-22-3p and FTO in osteogenic differentiation, followed by ALP and ARS staining. Results: miR-22-3p expression was repressed, while FTO expression was elevated in the ovariectomized mouse model. Overexpression of miR-22-3p, EV-delivered miR-22-3p, increased ALP activity and matrix mineralization of BMSCs and promoted RUNX2, OCN, and OPN expressions in BMSCs. miR-22-3p negatively targeted FTO expression. FTO silencing rescued the suppressed osteogenic differentiation by EV-delivered miR-22-3p inhibitor. FTO repression inactivated the MYC/PI3K/AKT pathway, thereby enhancing osteogenic differentiation both in vivo and in vitro. Conclusion: In summary, miR-22-3p delivered by BMSC-derived EVs could result in the inhibition of the MYC/PI3K/ AKT pathway, thereby promoting osteogenic differentiation via FTO repression.

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“…BMSCs have osteogenic potential, and it can self‐renew and differentiate into mesenchymal‐derived tissues, making it an important contributor to the bone repair process 7 . In addition, it is reported that the osteogenesis of bone marrow mesenchymal stem cells promotes the regeneration of the skull 8 . In recent years, as a new means of intercellular communication, exosomes (exo) have attracted much attention because of their ability to carry a variety of biologically active molecules that regulate the activity of receptor cells 9 .…”

Section: Introductionmentioning

confidence: 99%

“…7 In addition, it is reported that the osteogenesis of bone marrow mesenchymal stem cells promotes the regeneration of the skull. 8 In recent years, as a new means of intercellular communication, exosomes (exo) have attracted much attention because of their ability to carry a variety of biologically active molecules that regulate the activity of receptor cells. 9 Exosomes are small extracellular vesicles with a diameter of 40-150nm that can be secreted by a variety of cells.…”

Section: Introductionmentioning

confidence: 99%

Obesity regulates miR‐467/HoxA10 axis on osteogenic differentiation and fracture healing by BMSC‐derived exosome LncRNA H19

Wang

Chen

Zhao

et al. 2021

J Cellular Molecular Medi

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This study explored the therapeutic effect of bone marrow mesenchymal stem cell‐derived exosomes on the treatment of obesity‐induced fracture healing. Quantitative real‐time PCR was used to detect the expression of lncRNA H19, miR‐467 and Hoxa10 and combined with WB detection to detect osteogenic markers (RUNX2, OPN, OCN). Determine whether exosomes have entered BMSCs by immunofluorescence staining. Alkaline phosphatase (ALP) and alizarin red staining (ARS) staining were used to detect ALP activity and calcium deposition. We found that high‐fat treatment can inhibit the secretion of BMSCs‐derived exosomes and affect the expression of H19 carried by them. In vivo and in vitro experiments show that high‐fat or obesity factors can inhibit the expression of osteogenic markers and reduce the staining activity of ALP and ARS. The treatment of exosomes from normal sources can reverse the phenomenon of osteogenic differentiation and abnormal fracture healing. Further bioinformatics analysis found that miR‐467 as a regulatory molecule of lncRNA H19 and Hoxa10, and we verified the targeting relationship of the three through dual luciferase report experiments. Further, we found similar phenomena in ALP and ARS staining. Bone marrow mesenchymal stem cell‐derived exosomes improve fracture healing caused by obesity.

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Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration

Cited by 41 publications

References 64 publications

Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

Extracellular vesicle-encapsulated miR-22-3p from bone marrow mesenchymal stem cell promotes osteogenic differentiation via FTO inhibition

Obesity regulates miR‐467/HoxA10 axis on osteogenic differentiation and fracture healing by BMSC‐derived exosome LncRNA H19

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