In Vitro and In Vivo Osteogenesis of Human Orbicularis Oculi Muscle-Derived Stem Cells

Abstract

BACKGROUND: Cell-based therapies for treating bone defects require a source of stem cells with osteogenic potential. There is evidence from pathologic ossification within muscles that human skeletal muscles contain osteogenic progenitor cells. However, muscle samples are usually acquired through a traumatic biopsy procedure which causes pain and morbidity to the donor. Herein, we identified a new alternative source of skeletal muscle stem cells (SMSCs) without conferring morbidity to donors. METHODS: Adherent cells isolated from human orbicularis oculi muscle (OOM) fragments, which are currently discarded during ophthalmic cosmetic surgeries, were obtained using a two-step plating method. The cell growth kinetics, immunophenotype and capabilities of in vitro multilineage differentiation were evaluated respectively. Moreover, the osteogenically-induced cells were transduced with GFP gene, loaded onto the porous b-tricalcium phosphate (b-TCP) bioceramics, and transplanted into the subcutaneous site of athymic mice. Ectopic bone formation was assessed and the cell fate in vivo was detected. RESULTS: OOM-derived cells were fibroblastic in shape, clonogenic in growth, and displayed phenotypic and behavioral characteristics similar to SMSCs. In particular, these cells could be induced into osteoblasts in vitro evidenced by the extracellular matrix calcification and enhanced alkaline phosphatase (ALP) activity and osteocalcin (OCN) production. New bone formation was found in the cell-loaded bioceramics 6 weeks after implantation. By using the GFP-labeling technique, these muscle cells were detected to participate in the process of ectopic osteogenesis in vivo. CONCLUSION: Our data suggest that human OOM tissue is a valuable and noninvasive resource for osteoprogenitor cells to be used in bone repair and regeneration.

Human Skeletal Muscle Cells Derived from the Orbicularis Oculi Have Regenerative Capacity for Duchenne Muscular Dystrophy

“…Our study and previous reports also show that growing myogenic stem cells are present in the muscle tissues of human orbicularis oculi derived from extra eyelid tissue, and could be successfully isolated and cultured without compromising their myogenic differentiation ability (Figure 2 and Figure 3) [12,13]. By merely plating dissociated eyelid tissue after enzymatic treatment, not only spindle shaped cells but fibroblastic cells formed adherent colonies.…”

High Therapeutic and Esthetic Properties of Extracellular Vesicles Produced from the Stem Cells and Their Spheroids Cultured from Ocular Surgery-Derived Waste Orbicularis Oculi Muscle Tissues

“…The isolation method for the OOM-derived cells was based on enzymatic digestion using collagenase type II after washing and chopping of the OOM tissues, followed by several centrifugations, and seeding of the obtained single cell onto a 0.2% gelatin-coated culture plate, as shown in Figure 1 A. From patients (n = 16) ( Supplementary Table S1 ), we could isolate the pretarsal parts of OOM tissues, which are composed of orbital, preseptal, and pretarsal parts [ 55 ] ( Figure 1 B,C). We also isolated OOFCs using the enzymatic digestion method.…”

Adult stem cells in the eye: Identification, characterisation, and therapeutic application in ocular regeneration – A review

“…142 However, the muscle samples are usually harvested through an invasive biopsy procedure. 143 Therefore, finding a new source without conferring morbidity to donors is necessary. Pieces of eyelid muscles, such as the orbicularis oculi and levator palpebrae superioris, are readily available from blepharoplasty patients.…”

“…Skeletal muscle stem cells have been extensively investigated in pre‐clinical and clinical trials for the treatment of muscular disorders 142 . However, the muscle samples are usually harvested through an invasive biopsy procedure 143 . Therefore, finding a new source without conferring morbidity to donors is necessary.…”