Differentiation Potential Of Human Orbicularis Oculi Muscle Derived Stem Cells Towards Schwann Cell-like Cell Lineages

【Background】Peripheral nerve injuries are a common occurrence and represent a major economic burden for society. Clinical treatment usually involves surgical suture of the damaged nerves for minor defects whereas autologous nerve grafts are harvested for longer gaps. Despite such great surgical advance as microsurgery, functional recovery of treated nerve is often poor after careful surgical procedure. Experimentally, peripheral nerve regeneration can be enhanced by Schwann cell transplantation but the clinical application is limited by donor site morbidity and the inability to generate a sufficient number of cells quickly. Bone marrow and adipose tissue have been used as the main source of mesenchymal stem cells and adipose derived stem cells and under appropriate conditions some groups have shown they can be selectively differentiated into Schwann cell-like cell lineages. MDSCs (muscle derived stem cells) are newly explored from muscle and found distinct from satellite cells for their multiple lineages differentiation potency. Besides the capacity of renewal and multi-lineage differentiation, MDSCs have also been reported to exhibit long-term proliferation and immune-privileged behavior both in vitro and in vivo. Of more excitement is that MDSCs have also been isolated from human tissue. The apparent advantages of MDSCs and the new resource we found within human Orbicularis oculi muscle, which is commonly resected in blepharoplasty, have led the authors to investigate whether h-MDSCs (human muscle derived stem cells) isolated from human Orbicularis oculi muscle can be differentiated to a Schwann cell phenotype which could eventually provide functional benefits for peripheral nerve repair.【Objective】(1) Through the isolation and identification of MDSCs from murine muscle tissue, to master the correct method of enzyme digestion and pre-plating technique. (2) To discover a proper candidate as muscle tissue donor in human with acceptable rationale argument. (3) To isolate h-MDSCs from the Orbicularis oculi muscle with aid of pre-plating technique and have their identification surface markers stained. (4) To isolate and proliferate Schwann cell and prepare Schwann cell-conditioned medium, which would facilitate the transdifferention from h-MDSCs into Schwann cell-like cell lineages. (5) To establish a reliable method of transforming h-MDSCs into transdifferentiated Schwann cells which would serve as an alternative candidate for commonly studied Schwann cells in tissue engineering nerve graft. 【Methods】(1) Under the support of microscope, MDSCs were isolated from murine muscle tissue with tri-enzyme digestion and pre-plating technique, and identified by immunohistochemistry method. Also MDSC clone and sub-clone were prepared with limited dilution method. (2) Discarded human Orbicularis oculi muscle resected in the upper eyelid blepharoplasty were collected from three volunteers (aged 19-24 years old) during July to Nov, 2010. (3) We isolated h-MDSCs within collected Orbicularis oculi muscle with aid of with tri-enzyme digestion and pre-plating technique, and then identify the cells by immunohistochemistry method. (4) Schwann cells were isolated from Sprague-Dawley rats and identified by immunohistochemistry method. Through half-harvest method, we would like to prepare conditioned medium from Schwann cell culture. (5) We co-culture h-MDSCs with Schwann cell conditioned medium and morphology of the treated cells was investigated daily under microscope. The common used markers, S-100, GFAP and p75, to identify a Schwann cell phenotype were stained with use of immunohistochemistry method.【Results】(1) We successfully isolated MDSCs, and its clone as well as sun-clone from murine muscle tissue and have Desmin positively expressed and Sca-1 with a positive rate of 92.3±4.1%. (2) We collected human Orbicularis oculi muscle samples weighting 156mg - 241mg, which are sufficient for cell isolation, from three young female volunteers with their consensus. (3) H-MDSCs were isolated from Orbicularis oculi muscle and have their desmin positively stained and Sca-1 expressed with a positive rate of 81.8±2.4%. (4) Schwann cells were isolated and identified with S-100 stained with the positive rate of 97.4±0.7% and its conditioned medium was successfully prepared. (5) The isolated h-MDSCs were successfully transdifferentiated into Schwann cell-like cell lineages with a positive rate of 65.1±3.4% for S100, 23.3±1.7% for GFAP and 30.3±5.2% for p75, which would serve as an unanimous evidence of a Schwann cell phenotype.【Conclusions】(1) With application and modification of tri-enzyme digestion and pre-plating technique, MDSCs and its clone can be isolated from murine muscular tissue. (2) With application and modification of tri-enzyme digestion and pre-plating technique used in murine MDSCs isolation, h-MDSCs could be isolated from human Orbicularis oculi muscle sample, which serves as a new source of MDSCs. (3) Human-MDSCs could be transdifferentiated into Schwann cell-like cell lineages when co-cultured within Schwann cell conditioned medium, which would serve as an alternative candidate for commonly studied Schwann cells in tissue engineering nerve graft. (4) Esthetic and plastic surgery such as blepharoplasty can provide proper research candidate for tissue engineering and regenerative medicine with possible clinical and experimental purpose.