The diferentiation of neuronal cells from Mouse embryonic stem cells

With new technologies emerging today, the importance of stem cells in the cell therapy of nervous system diseases is supported by recent studies. Therefore, the development of neuronal cell diferentiation protocols from stem cells is of great importance. In our study, the diferentiation of neuronal and neuroglial cells from mouse embryonic stem (ES) cell line and their analysis with neuronal cell markers are aimed. Mouse ES cells were diferentiated to neurogenic series cells by adding N2 and bFGF to the culture medium on coated Fibronectin dishes. For the identification of diferentiated cells, they were evaluated by light microscopy using immunhistochemistry techniques and by electron microscopy. Indirect immunohistochemical staining method was performed with SSEA-1 (mouse embriyonic stem cells marker), Nestin (neural precursor cells marker), βIII-Tubulin (neuronal cells marker), MAP-2 (neuronal cells marker), GFAP (astrocyte marker), and O4 (oligodendrocyte marker). After 1 week of diferentiation of cells, immunoreactivities of SSEA-1 and Nestin were detected to be negative and moderate, respectively. After 2 weeks culture time, the diferentiation was still continuing and especially positive immunoreactivities of β-III Tubulin and MAP-2 and weak immunoreactivities of O4 and GFAP were supported neuronal diferentiation. In conclusion, our results suggest that neuronal cell derived from mouse ES cells were diferentiated particularly to neuron using N2+bFGF+fibronectin culture condition. Therefore, these diferentiated cells may be used as a treatment method in degenerative diseases of the nervous system.

Fare embriyonik kök hücrelerden nöronal hücrelerin farklılaşması

Günümüzde gelişen yeni teknolojiler sayesinde sinir sistemi hastalıklarının hücresel tedavisinde kök hücrelerinin önemi son yıllardaki çalışmalar ile desteklenmektedir. O nedenle kök hücrelerden nöronal hücrelerin farklılaştırılması protokollerinin oluşturulması büyük önem taşımaktadır. Çalışmamızda, fare embriyonik kök (EK) hücre hattından, nöron ve nöroglial hücrelerin farklılaştırılması ve nöronal hücre belirteçleri ile analizi amaçlanmıştır. Fare EK hücreler, fibronektin kaplı petrilerde kültür ortamına N2 ve bFGF ilavesi ile nörojenik seri hücrelerine farklılaştırıldı. Farklılaşmış hücrelerin tanımlanması için hücreler, immünohistokimya tekniği kullanılarak ışık mikroskobu ile ve elektron mikroskobu ile değerlendirildi. İndirek immünohistokimyasal boyama yöntemi SSEA-1 (fare ES hücre belirteci), Nestin (nöron öncülü hücre belirteci), βIII-Tubulin (nöron hücre belirteci), MAP-2 (nöron hücre belirteci), GFAP (astrosit belirteci) ve O4 (oligodendrosit belirteci) için uygulandı. Hücrelerin farklılaşmasının 1. haftasından sonra SSEA-1 ve Nestin immünoreaktivitesi sırasıyla negatif ve orta saptandı. Kültürün 2. haftasından sonra farklılaşmanın hala devam etmesi ve özellikle β-III Tubulin ve MAP-2 immünoreaktivitesinin güçlü pozitif ve O4 ve GFAP immünoreaktivitesinin zayıf olması nöronal farklılaşmayı desteklemiştir. Sonuç olarak, bizim sonuçlarımız göstermiştir ki fare EK hücrelerinden kaynaklanmış nöronal hücreler, N2+bFGF+fibronektin kullanılan kültür koşullarında özellikle nörona farklılaşmıştırlar. Bu nedenle, farklılaştırılmış bu hücreler dejeneratif sinir sistemi hastalıklarının hücresel tedavisinde kullanılabilir.

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