Isolation, cultivation and identification of skeletal muscle stem cells derived from human orbicularis oculi muscle / 中华整形外科杂志

Objective@#To establish the isolation and culture methods of skeletal muscle stem cells, derived from human orbicularis oculi muscle (OOMSCs), and to identify their multi-directional differentiation potential in vitro.@*Methods@#The cellswere isolated from pretarsal orbicularis oculi muscle (OOM), obtained in routine blepharoplasty surgery.The tissue was digested using collagenase type I combined with re-plating methods. Specific cell surface antigen markers were detected using flow cytometry analysis. OOMSCs were cultured under different inductive conditions, to identify their pluripotent differentiation ability.@*Results@#OOMSCs exhibited similar fibroblast-like morphology as mesenchymal stem cells with high expression of skeletal muscle-derived stem cell surface markers. OOMSCs were able to differentiate into adipocytes, osteoblasts and chondrocytes in vitro, in the presence of lineage-specific inductive media. Moreover, after myogenic induction, the differentiated cells were fused into multinucleated myotube-like structure, and positive for myogenic-related marks, Pax3, Pax7, Myf5 and MyoD.@*Conclusions@#Muscle-derived stem cells can be isolated from human OOM with myogenic differentiation properties, showing further applications potential intissue regeneration and medical therapies of muscle diseases.

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

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 β-tricalcium phosphate (β-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.

Effects of transplantation of autologous adipose-derived stem cells on osteoporosis in a rabbit ovariectomy model / 中华急诊医学杂志

Objective To observe the effects of transplantation of autologous adipose-derived stem cells (ASCs) on osteoporosis (OP) in a rabbit ovariectomy (OVX) model.Methods A total of fifteen 6-month-old female New Zealand white rabbits were randomly divided into two groups:ovariectomy group (group A,n =12) and sham operation group (group B,n =3).All rabbits were subjected to bilateral ovariectomy in the group A.Six months later,bone mineral density (BMD) of group A and group B were measured by dual energy X-ray absorptiometry (DXA) to check the result of OVX-OP.ASCs harvested from adipose of OP rabbits were cultured to be expanded and differentiated in osteogenic medium in vitro.Osteogenesis was evaluated by alizarin red staining,alkaline phosphatase (ALP) staining and quantitative assays of osteocalcin (OCN).Autologous osteo-induced ASCs were mixed in calcium alginate hydrogel (CAH) and then transplanted in the left distal femurs,while CAH was transplanted in the right distal femurs of OP rabbits.At 12 weeks after implantation,BMD,micro-CT and histomorphological analyses were performed on these rabbits.Results The BMD of femurs in group A rabbits were obviously lower than that of group B rabbits (P ＜ 0.05) at 6 months after OVX.Compared with control group,ASCs cultured in osteo-induction medium had similar proliferation rate as the non-induced cells,but displayed positive ALP and alizarin red staining and OCN contents.At 12 weeks after implantation,the cell-treated femurs displayed higher BMD,bone trabecula number,trabecula thickness and separation than those of control group,while the structure model index and porosity were lower (P ＜ 0.05).Histological examination indicated that the trabecular thickness increased with complete CAH resorption in cell-treated group,while CAH remained in control group.Conclusions Transplantation of autologous ASCs can help strengthen osteoporotic bone in OVX-OP rabbits,providing a novel approach to OP treatment.