Differentiation Potential and Tumorigenic Risk of Rat Bone Marrow Stem Cells Are Affected By Long-Term In Vitro ExpansionErdal Karaöz1, Filiz Tepeköy21İstinye University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey; İstinye University Center for Stem Cell and Tissue Engineering Research and Practice, İstanbul, Turkey; Center for Regenerative Medicine and Stem Cell Research and Manufacturing (LivMedCell), İstanbul, Turkey
2İstinye University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey; Altınbaş University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey
Objective: Mesenchymal stem cells (MSCs) have the capacity for extensive expansion and adipogenic, osteogenic, chondrogenic, myogenic, and neural differentiation in vitro. The aim of our study was to determine stemness, differentiation potential, telomerase activity, and ultrastructural characteristics of long-term cultured rat bone marrow (rBM)-MSCs.
Materials and Methods: rBM-MSCs from passages 3, 50, and 100 (P3, P50, and P100) were evaluated through immunocytochemistry, reverse transcription-polymerase chain reaction, telomerase activity assays, and electron microscopy.
Results: A dramatic reduction in the levels of myogenic markers actin and myogenin was detected in P100. Osteogenic markers Coll1, osteonectin (Sparc), and osteocalcin as well as neural marker c-Fos and chondrogenic marker Coll2 were significantly reduced in P100 compared to P3 and P50. Osteogenic marker bone morphogenic protein-2 (BMP2) and adipogenic marker peroxisome proliferatoractivated receptor gamma (Pparγ) expression was reduced in late passages. The expression of stemness factor Rex-1 was lower in P100, whereas Oct4 expression was decreased in P50 compared to P3 and P100. Increased telomerase activity was observed in longterm cultured cells, signifying tumorigenic risk. Electron microscopic evaluations revealed ultrastructural changes such as smaller number of organelles and increased amount of autophagic vacuoles in the cytoplasm in long-term cultured rBM-MSCs.
Conclusion: This study suggests that long-term culture of rBMMSCs leads to changes in differentiation potential and increased tumorigenic risk.
Keywords: Bone marrow, Differentiation, Long-term culture, Mesenchymal stromal cells, Stemness, Telomerase
Sıçan Kemik İliği Kök Hücrelerinin Farklılaşma Potansiyeli ve Tümörojenik Riski Uzun Süreli İn Vitro Ekspansiyondan EtkilenmektedirErdal Karaöz1, Filiz Tepeköy21İstinye University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey; İstinye University Center for Stem Cell and Tissue Engineering Research and Practice, İstanbul, Turkey; Center for Regenerative Medicine and Stem Cell Research and Manufacturing (LivMedCell), İstanbul, Turkey
2İstinye University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey; Altınbaş University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey
Amaç: Mezenkimal kök hücreler (MKH) in vitro uzun süreli ekspansiyon, adipojenik, osteojenik, kondrojenik, miyojenik ve nöral farklılaşma potansiyeline sahiptir. Çalışmamızın amacı uzun süre kültüre edilen sıçan kemik iliği (sKİ)-MKH’lerinin kök hücre niteliklerini, farklılaşma potansiyellerini, telomeraz aktivitelerini ve ultrayapısal özelliklerini belirlemektir.
Gereç ve Yöntem: 3., 50. ve 100. pasajlardan (P3, P50 ve P100) elde edilen sKİ-MKH’leri, immünohistokimya, revers-transkriptaz polimeraz zincir reaksiyonu, telomeraz aktivite analizleri ve elektron mikroskopi ile değerlendirilmiştir.
Bulgular: P100’de miyojenik belirteçlerden aktin ve miyogenin seviyelerinde düşüş gözlemlenmiştir. Osteojenik belirteçler Coll1, osteonektin (Sparc) ve osteokalsin ile nöral belirteç c-Fos ve kondrojenik belirteç Coll2 P100’de P3 ve P50’ye kıyasla önemli ölçüde azalmıştır. Osteojenik belirteç kemik morfojenik protein-2 (BMP2) ve adipojenik belirteç peroksizom proliferatör ile aktive olan reseptör gamma (Pparγ) geç pasajlarda düşüş göstermiştir. Kök hücre belirteçlerinden Rex-1’in ekspresyonu P100’de düşüş gösterirken, Oct4 ekspresyonunun P50’de P3 ve P100’e göre düşüş gösterdiği belirlenmiştir. Uzun süre kültüre edilen hücrelerdeki artmış telomeraz aktivitesi tumorijenik riske işaret etmektedir. Elektron mikroskopik değerlendirmeler, uzun süre kültüre edilen sKİ-MKH sitoplazmasında düşük sayıda organel ve artmış oranda otofajik vaküol gibi utrayapısal değişiklikler ortaya koymuştur.
Sonuç: Bu çalışma, sKİ-MKH’lerinin uzun süreli kültüre edilmesinin bu hücrelerin farklılaşma potansiyelinde değişikliklere ve tümörijenik riskin artmasına neden olduğunu göstermiştir.
Anahtar Kelimeler: Kemik iliği, Farklılaşma, Uzun süreli kültür, Mezenkimal kök hücreler, Telomeraz
Erdal Karaöz, Filiz Tepeköy. Differentiation Potential and Tumorigenic Risk of Rat Bone Marrow Stem Cells Are Affected By Long-Term In Vitro Expansion. Turk J Hematol. 2019; 36(4): 255-265
Corresponding Author: Erdal Karaöz, Türkiye |
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