Mezenkimal Kök Hücrelerin Kardiyomiyositlere Farklılaştırılmasında Kimyasal ve Biyolojik İndükleyicilerin Kombine Etkisinin Değerlendirilmesi

Öz

Mezenkimal kök hücrelerden (MKH) elde edilen kardiyomiyositler, biyomedikal uygulamalar için önemli bir potansiyele sahiptir. Ancak, mevcut protokoller ile MKH'leriden yeterli sayıda olgun ve fonksiyonel kardiyomiyosit üretilememiştir. Bu çalışmada, sıçan kemik iliği kök hücrelerinin (rt-MKH'ler) kardiyomiyositlere trans-diferansiyasyonunu artırmada biyolojik ve kimyasal indükleyicilerin kombine etkisi araştırılmıştır. rt-MKH'ler, 5-Azasitidin (5-Aza), Dimetil sülfoksit (Me2SO) ve biyolojik molekül kokteyline (TGF-β1, BMP-2 ve FGF-2, topluca DM olarak adlandırılmıştır) tek başına veya kombinasyon halinde 14 gün boyunca maruz bırakılmıştır. Kardiyak transkripsiyon faktörleri ve kardiyak spesifik proteinler sırasıyla qPCR ve immünofloresan boyama yöntemleriyle analiz edilmiştir. Kardiyomiyosit oranı ise akış sitometri yöntemiyle değerlendirilmiştir. qPCR analizi, 5-Aza+Me2SO grubunda 7. günde GATA-4 ve NKX2.5 ekspresyon seviyelerinin sırasıyla 19 kat ve 8 kat arttığını göstermiştir (p<0,001). Kültür süresi 14 güne uzatıldığında, kardiyak transkripsiyon faktörlerinin ekspresyon seviyeleri 7. güne kıyasla azalmış olup, en yüksek GATA-4 ve NKX2.5 seviyeleri Me2SO grubunda gözlemlenmiştir. Özellikle, Me2SO grubundaki GATA-4 ekspresyonu, 5-Aza grubuna kıyasla 11 kat daha yüksek bulunmuştur (p<0,05). İmmünofloresan boyama, 5-Aza ve Me2SO ile muamele edilen rt-MKH'lerin, kontrol grubuna kıyasla yüksek seviyelerde cTnT, SAC, Cnx43 ve α-SMA eksprese ettiğini doğrulamıştır. Akış sitometri analizi ise, 5-Aza+Me2SO grubundaki indüklenmiş hücrelerin %42’sinin kardiyomiyosit olduğunu ortaya koymuştur. Sonuç olarak, kimyasal indükleyicilerin (5-Aza ve Me2SO) birlikte kullanımı, kimyasal ve biyolojik indükleyicilerin kombinasyonuna kıyasla rt-MKH’lerin kardiyomiyositlere farklılaşmasını teşvik etmede daha etkili olduğu tespit edilmiştir.

Anahtar Kelimeler

• Mezenkimal Kök Hücre
• Kardiyomiyojenik Farklılaşma
• 5-Azasitidin
• Dimetil Sulfoksit

Assessment of Combine Effect of Chemical and Biological Inducers to Differentiate Mesenchymal Stem Cells into Cardiomyocyte

Öz

Cardiomyocytes derived from mesenchymal stem cells (MSCs) hold significant potential for biomedical applications. However, existing protocols for differentiating MSCs into cardiomyocytes have not successfully generated a large number of mature, functional cells. This study aims to investigate the combined effects of biological and chemical inducers to enhance the trans-differentiation of rat bone marrow stem cells (rt-BMSCs) into cardiomyocytes. rt-BMSCs were treated with 5-Azacytidine (5-Aza), Dimethyl Sulfoxide (Me2SO), and biological molecule coctail (TGF-β1, BMP-2, and FGF-2, collectively referred to as DM) either individually or in combination for 14 days. Cardiac transcription factors and cardiac-specific proteins were analyzed using qPCR and immunofluorescence staining, respectively. The cardiomyocyte ratio was assessed by flow cytometry. qPCR analysis revealed that, after 7 days, GATA-4 and NKX2.5 expression levels increased 19-fold and 8-fold, respectively, in the 5-Aza+Me2SO group compared to the 5-Aza group (p<0.001). By day 14, the expression of cardiac transcription factors declined relative to day 7, with the highest GATA-4 and NKX2.5 levels observed in the Me2SO group. Notably, GATA-4 expression in the Me2SO group was 11 times higher than in the 5-Aza group (p<0.05). Immunofluorescence staining confirmed that rt-BMSCs treated with 5-Aza and Me2SO exhibited high expression of cTnT, SAC, Cnx43, and α-SMA compared to the control group. Flow cytometry analysis further revealed that 42% of the induced cells in the 5-Aza+Me2SO group were cardiomyocytes. In conclusion, the combination of chemical inducers (5-Aza and Me2SO) was more effective in promoting cardiomyocyte differentiation from rt-BMSCs than the combined use of chemical and biological inducers.

Anahtar Kelimeler

• Mesenchymal Stem Cells
• Cardiomyogenic Differentiation
• 5-azacytidine
• Dimethyl Sulfoxide

Kaynakça

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