Allogenic Myoblast Transplantation in the Rat Anal Sphincter Jocelyn B. Craig, MD,* Felicia L. Lane,MD,* Gabriel Nistor, MD,Þ Saba Motakef, BS,Þ Quynh-Anh Pham, BS,Þ and Hans Keirstead, PhDÞ Objectives: To determine the feasibility of injecting rat myoblasts into the intact anal sphincter as a potential treatment for anal incontinence, and to detect transferred myoblast survival and integration. Study Design: A pilot study using nonpregnant female Sprague Dawley rodents of 8 to 10 weeks of age. A biopsy of skeletal muscle was harvested from a study animal and recovered myoblasts were ex- panded in vitro over 10 days. Myoblasts were then tagged with a cyto- megalovirus promoter to transduce green fluorescent protein (GFP) into the myoblasts. The cell aspirate was injected directly into the intact ex- ternal anal sphincter using an electromyographic guidance. The animals received 1.5 or 4.5 Â 10 6 cells of GFP-labeled myoblasts, dividing the dose between three injection sites. The remaining in vitro myoblasts were still viable 28 days post-harvest. Ten days after transplantation the anal sphincter complex was surgically extracted. Results: The presence of GFP-labeled myoblasts was confirmed within the external anal sphincter. Conclusions: This demonstrates that myoblasts can be successfully extracted, cultivated in vitro, transplanted and will integrate into the host tissue. Key Words: anal incontinence, allogenic myoblast, myoblast transplantation, rodent model (Female Pelvic Med Reconstr Surg 2010;16: 205Y208) A nal incontinence (AI) is the involuntary loss of feces or gas through the anal canal. It is estimated to affect 2%Y15% of the general population and is more common with increas- ing age. 1,2 In women AI is often the result of an injury to the anal sphincter sustained during childbirth. It is estimated that 0.7%Y19.3% of vaginal deliveries result in anal sphincter lac- eration. 2,3 These muscles can be repaired surgically but the long term success rates of primary or secondary sphincteroplasty are dismal with only 30% of patients demonstrating continence 5 years after surgery. 1 There are nonsurgical treatments for AI such as pelvic floor muscle training or medications to bulk the stool but current research provides limited evidence to support their long-term effectiveness. 2 Published animal studies and one study in human subjects have used tissue-engineered muscle cells to bulk the urethral sphincter for treatment of urinary incontinence. These studies have reported an increase in muscle volume detected by ultra- sound. Local muscular hypertrophy and regrowth of healthy skeletal muscle may possibly reverse the muscle loss that is associated with urinary stress incontinence. Experiments have been performed with many different cell types including those from human cord blood, muscle-derived stem cells (MDSCs) or undifferentiated fat cells taken from the body at the time of aesthetic liposuction procedures. 4Y7 Adult skeletal muscle regeneration begins with activation of satellite cells that lie be- neath the basal lamina of all adult skeletal muscle fibers. While normally dormant these precursor cells can be reactivated and begin myogenesis in response to muscle injury. Myoblasts are derived from activated satellite cells. Prior to entering apoptosis these cells will multiply and expand through several cell divi- sions and are capable of regenerating injured muscle. Autolo- gous transfer of one’s own healthy myoblast to an area of injury can allow injured muscle to grow new hybrid healthy muscle fibers 1,3,6,7 (as seen in Fig. 1). MATERIALS AND METHODS Animals All animal studies were carried out with the approval of the University of California-Irvine Animal Care and Use Com- mittee and the animals were maintained according to the stan- dards described in the American Physiological Society’s BGuide for the Care and Use of Animals.[ Nulliparous female Sprague Dawley rats (Charles River Lab- oratories Inc., Washington, Mass) of approximately 8Y10 weeks of age were used in this study. They had free access to food and water before and after surgery. The animals underwent a mini- mum of a 72-hour stabilization period in the vivarium animal holding room before surgical procedures were performed in or- der to allow them to acclimate. Cells Rat muscle fibers with associated satellite cells were ob- tained by harvesting a 1 Â 2 Â 2 mm biopsy of skeletal muscle from the hind limb of a study animal. The satellite cells were dissociated from the muscle fibers using trypsin-EDTA and collagenase IV mixture (Invitrogen). The dissociated cells were washed twice by centrifugation in Dulbecco modified Eagle medium Hams F-12 and plated in T-25 flasks pre-coated with 0.5% gelatin (Invitrogen). The proliferating medium consisted of 1:1 Dulbecco modified Eagle medium Hams F-12 supplemented with fetal bovine serum 10%, insulin 10 Kg/ml (Sigma-Aldrich), GlutaMax (Invitrogen), and penicillin-streptomycin (Invitrogen). Basic fibroblast growth factor, 10 ng/ml, and epidermal growth factor 20 ng/ml, were added every 2 days, at feeding. Cells were allowed to proliferate in vitro over 10 days, at 37.5 degrees and 5% CO 2 in a humidified incubator before the first passage, then continuous growth of the myoblasts allowed a passage at every 4Y5 days and was carried out for up to 8 weeks (Fig. 2). The surplus of cells was frozen for long-term storage. Fu- sion maturation was prevented by keeping cell cultures at low density and by the addition of growth factors. Prior to the transplantation, a third-generation replicationY deficient lentiviral vector was used to transduce green fluorescent ORIGINAL ARTICLE Female Pelvic Medicine & Reconstructive Surgery & Volume 16, Number 4, July/August 2010 www.fpmrs.net 205 From the *Division of Urogynecology, Department of Obstetrics and Gyne- cology, and †Department of Anatomy and Neurobiology, Reeve-Irvine Re- search Center, University of California-Irvine, Orange, CA. Reprints: Jocelyn B. Craig, MD, 118 3rd St, Seal Beach, CA 90740. E-mail: jcraig@uci.edu. This study is funded in part by a grant from Perinatal Resources Inc, Hilliard, OH. Presented at the 2008 American Urogynecologic Society 29th Annual Conference, Chicago, IL, September 4Y6, 2008. Awarded best overall oral presentation. Copyright * 2010 by Lippincott Williams & Wilkins DOI: 10.1097/SPV.0b013e3181ec1edd Copyright @ 20 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 10