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abstract

143 -URINE DERIVED PLURIPOTENT STEM CELLS FROM PATIENTS WITH STRESS URINE INCONTINENCE: A SOURCE FOR DERIVATION OF AUTOLOGOUS FIBROBLASTS FOR TREATMENT OF PELVIC FLOOR DISORDERS (PILOT PROJECT)

143

URINE DERIVED PLURIPOTENT STEMCELLS FROM PATIENTS WITH STRESS URINE INCONTINENCE: A SOURCE FORDERIVATION OF AUTOLOGOUS FIBROBLASTS FOR TREATMENT OF PELVIC FLOORDISORDERS (PILOT PROJECT)

M. KIBSCHULL1, T.NGUYEN 1, T. CHOW 1, S. LYE 1, H. P.DRUTZ 2, M. ALARAB 2, I. ROGERS 1,O. SHYNLOVA 1;
1Lunenfeld-TanenbaumResearch Institute, Sinai Hlth.System, Toronto, Canada, 2Obstetricsand Gynecology, Division of Urogynecology, Sinai Hlth.System,Toronto, Canada.

Introduction: Stress urineincontinence (SUI) is a common condition: up to 50% of older femaleshave SUI. In women, urinary continence relies on an intact urethralsphincter mechanism. Promising clinical results were achieved byperi- or intra-urethral injection of autologous cells (fibroblastsand myoblasts); however, this treatment was accompanied by sideeffects due to invasive method of muscle biopsy collection forderivation of autologous primary cells. Recent studies demonstratedthat human urine contains a mixture of cells that can be obtained insufficient numbers from a single void and grown in culture. Thesecells could be directly reprogrammed into induced pluripotent stemcells (iPSCs) and further differentiated into fibroblasts andmyoblasts. Thus, we hypothesized that urine-derived cells maypotentially be used in SUI therapy for urethral sphincterregeneration. In order to circumvent the problems associated withtissue biopsy and the establishment of primary cell cultures, we aimto generate autologous fibroblast cells from urine-derived iPSClines.
Objective: (1) To establish and characterize iPSClines generated from voided urine collected from young pre-menopausaland aged post-menopausal women with SUI and (2) to differentiateurine derived iPSC (uiPSC) into mesodermal fibroblasts underchemically defined, xeno-free culture conditions.
Methods:Void urine samples from pre- and post-menopausal women with SUI werecollected during regular office visits. Urine cells were immediatelypelleted and seeded into cell culture plates. Proliferating cultureswere transfected with an integration-free episomal reprogrammingsystem (Oct3/4, Sox2, KLF4, c-Myc) in defined stem cell medium. Theadvantage is that the vector is removed through passaging once theiPSC line is fully reprogrammed. iPSC colonies were isolated within21 days, expanded and tested for the presence of pluripotency markers(NANOG, OCT4, SOX2) by RT-PCR and immunocytochemistry. Fibroblastdifferentiation of uiPSC was achieved by embryoid body (EB) formationassay. Single uiPSC were aggregated in high-throughput microwellplates to form EBs, and maintained for 3 weeks in suspension culture.EBs were then allowed to attach to collagen IV-coated culture dishesand outgrowing cells were enzymatically dissociated and re-plated inchemically defined fibroblast culture medium supplemented with bFGF.Cells were cultured for up to 10 passages, characterized byimmunocytochemistry and cryopreserved.
Results: uiPSCDerivation. Our study of urine specimens from female SUI patients(n=10/group) demonstrated that 1) 6 out of 10 samples resulted iniPSC reprogramming using an integration-free episomal system and 2)there was no correlation with patients’ age or menopause status.iPSC lines can be maintained undifferentiated in stem cell conditionsfor more than 30 passages while expressing pluripotency markers OCT4,Sox2 and NANOG, and can be reconstituted from cryopreservation.
uiPSCDifferentiation. Fibroblast lines could be established from iPSClines in chemically defined, xeno-free culture conditions;cryopreservation did not influence the fibroblast phenotype. Cellsstain positive for specific markers (vimentin, alpha-smooth muscleactin, and fibroblast specific antibody TE-7) and negative forcytokeratins and surface markers NCAM1/CD56, revealing successfulmesodermal fibroblast (but not ectodermal/neural crest cell)differentiation. Cultures were also negative for pluripotency markersOCT4 and SOX2 and NANOG indicating the absence of adult stem cells.Expansion is possible for >10 passages in presence of bFGF withoutshowing reduction in proliferation.
Conclusions: We areable to derive and expand mesodermal fibroblasts from uiPSC cellsthat can serve as a source of autologous patient-specific cells fortreatment of SUI or other pelvic floor disorders (ie Pelvic OrganProlapse).The uiPSCs are free of the reprogramming vector andtherefore provide a safer source for potential applications in humanthan integrating reprogramming systems. Fibroblasts are of highinterest in the field of regenerative medicine as they play majorrole in wound healing processes and tissue regeneration. Our workdemonstrates the feasibility of this method of generating autologouscells from female patients using a xeno-free, defined culture systemto minimize the risk of pathogen transfer during celltransplantations.
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