Isolation and Characterization of Mesenchymal Stem Cells Derived from Human Amniotic Membrane by Explant Technique

Öz

Background: Mesenchymal stem cells (MSCs) are adult stem cell types. When examined by phase contrast microscopy, they appear spindle-shaped and fibroblast-like cell aggregates. Up to now, MSCs have been isolated from many tissues such as bone marrow, adipose tissue, cartilage, placenta, amniotic membrane and umbilical cord. Our aim in this study was to isolate mesenchymal stem cells from amniotic membrane by explant culture technique easily and without needing a large amount of equipment. Materials and Methods: In the cell culture laboratory, amniotic membranes were dissected into small pieces and explant culture medium was created. Then, flow cytometry analysis was performed at passage 3 (P3) to determine the characterization of proliferating cells. Differentiation experiments were conducted to show the changes in adipogenic, chondogenic and osteogenic direction. Results: In our study, mesenchymal stem cells were isolated from the human amniotic membrane by explant cell culture technique. Flow cytometry analysis showed that the cells expressed mesenchymal stem cell markers (CD73 and CD90) but not hematopoietic stem cell markers (CD34). In differentiation experiments, adipogenic, chondrogenic and osteogenic changes were observed. Conclusions: After the widespread use of mesenchymal stem cells isolated from the umbilical cord as a valuable source of regenerative and reparative medicine, studies on the isolation of mesenchymal stem cells from the amniotic membrane have intensified.

Anahtar Kelimeler

• Cell culture techniques
• amnion
• stem cells

İnsan Amniyon Zarından Eksplant Teknikle Elde Edilen Mezenkimal Kök Hücrelerin İzolasyonu ve Karakterizasyonu

Öz

Amaç: Mezenkimal kök hücreler (MKH) erişkin kök hücre tipidir. Faz kontrast mikroskobu ile incelendiğinde iğ seklinde ve fibroblast benzeri hücre toplulukları olarak görülürler. Şimdiye kadar MKH’ler, kemik iliği, adipoz doku, kıkırdak, plasenta, amniyon zarı, göbek kordonu, gibi birçok dokudan izole edilmişlerdir. Bu çalışmadaki amacımız amniyon zarından mezenkimal kök hücreleri eksplant kültür tekniğiyle kolay bir şekilde ve fazla miktarda ekipmana ihtiyaç olmadan izole etmektir. Materyal ve Metod: Hücre kültürü laboratuvarında amniyon zarları küçük parçalara ayrılarak eksplant hücre kültür ortamı oluşturuldu. Daha sonra prolifere olan hücrelerin karekterizasyonunu belirlemek amacıyla 3. pasajda (P3) flow sitometri analizleri yapıldı. Adipojenik, kondojenik ve osteojenik yöndeki değişimlerini göstermek için ise farklılaşma deneyleri yapıldı. Bulgular: Çalışmamızda insan amniyon zarından mezenkimal kök hücreler eksplant hücre kültür tekniğiyle izole edilmişlerdir. Yapılan flow sitometri analizinde hücrelerin mezenkimal kök hücre belirteçlerini (CD73 ve CD90) eksprese ettiklerini hematopoetik kök hücre belirtecini (CD34) ise eksprese etmedikleri gösterilmiştir. Farklılaşma deneylerinde ise adipojenik, kondojenik ve osteojenik yönde değişim gösterdikleri belirlenmiştir. Sonuç: Göbek kordonundan izole edilen mezenkimal kök hücrelerin rejeneratif ve reparatif tıpta değerli bir kaynak olarak yaygın bir şekilde kullanılmasından sonra amniyon zarından elde edilen mezenkimal kök hücreler üzerinde de çalışmalar yoğun bir şekilde devam etmektedir.

Anahtar Kelimeler

• Hücre kültürü teknikleri
• amniyon
• kök hücreler

Kaynakça

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