Moza Al- Khulaifi1, Asma Al-Sulaiti1, Bella S. Guerrouahen1, Sara Al-Khawaga1, Chiara Cugno1, Zohreh Tatari Calderone1 1 Clinical Research Center, Research Division, Translational Medicine, Sidra Medicine, Qatar. Correspondence: Dr. Zohreh Tatari Calderone; Clinical Research Center, Research Division, Translational Medicine Sidra Medicine, Qatar. Email: [email protected] Keyword list: MSC, Foreskin, GMP, ISCT criteria. Abstract 1. Background: Mesenchymal stromal cells (MSCs) are known for their multipotent, immune-privileged and regenerative properties. MSCs can be obtained from different sources such as bone marrow, fat, umbilical cord, synovial fluid, tendons, muscles, periosteum, peripheral blood and nervous system. The International Society for Cellular Therapy (ISCT) has stated minimal characteristics for defining the MSCs as multipotent fibroblast-like cells highly adherent to plastic, positive for CD73, CD105 and CD90 but negative for CD45, CD34, CD14 or CD11b, CD19 or CD79a, and HLA-DR. The MSCs constitute a promising tool for regenerative medicine, due to their self-renewal capacity, multilineage differentiation potential, paracrine effects and immunosuppressive properties. They are extensively used in the treatment of several clinical settings including but not limited to, cardiovascular diseases, inflammatory bowel diseases, liver dysfunction, rheumatological disorders and diabetes. Bone Marrow-derived MSCs has been considered as the gold standard for cell-based therapy, but bone marrow aspiration is an invasive method that limits the access to a large number of MSC donor sources. Recently, the foreskin, which has been always retained as medical waste, has been recently shown as a source for MSCs. Sidra Medicine is performing 4000- 5000 circumcisions annually, therefore foreskin can be considered as a highly abundant and easily accessible source for the isolation of MSCs. Therefore, the aim of our study is: Specific Aim: To demonstrate that MSC isolated from foreskins (FSK-MSC) meet the high standard criteria of ISCT and to compare their phenotype, potency and multilineage potential with MSCs obtained from a classical source such as adipose tissue for the clinical application. 2. Method: Specimen Collection and MSC Isolation: Upon approval by the IRB committee, foreskins were collected after the circumcision from the Surgery Department at Sidra Medicine (age range: 1.5 -14 years old). Epidermis was manually separated from the dermis and was then subjected to enzymatic digestion by liberase. Cell suspension was cultured in T75 flasks containing low-Glucose media, complemented with 10% fetal bovine serum and 1% antibiotics for 3-5 days. After 3-5 days, the non-adherent cells were removed and fresh media was added to allow the adherent MSCs to grow. MSC Characterization:Cell Surface expression: Phenotypic characterization was determined using flow cytometry. Briefly cells were stained for the surface markers using fluorochrome conjugated antibodies for CD14, CD19, CD34, HLA-DR, CD45, CD73, CD105 and CD90 according to the ISCT criteria. Differentiation: FSK-MSCs were treated with osteogenic and adipogenic mediums in separate cultures for 7, 14 and 21 days and stained with Alizarin Red and Oil Red O staining respectively. The lineage differentiation was visualized using the inverted microscopy. Colony Forming Units (CFU): Colony formations Units measures the viable clonogenic cell numbers that is an important indication of the quality of cell preparation. The CFU assay was performed according to commonly used protocol and using crystal violet staining. 3. Results: FSK-MSC displayed a typical fibroblast-like morphology and an ISCT-compliant phenotype; FSK-MSCs were highly proliferative and had a marked clonogenic potential. FSK-MSCs displayed osteogenic and adipogenic differentiation capacities. 4. Conclusion: The isolated FSK-MSCs were shown to be compliant with ISCT criteria and can be considered as a reliable source of MSC. Their compliance allows considering the standardization of clinical grade FSK-MSCs production at Sidra Medicine GMP facility, and setting the ground for the potential establishment of a MSC biorepository.


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