Changes in pattern of DNA methylation in DE promoter region of Oct3/4 gene before and after neural differentiation of HUCB-NSC

• Published in: Acta neurobiologiae experimentalis Vol. 71; no. 1; p. 171
• Main Authors: Habich, A, Zayat, V, Buzanska, L, Domanska-Janik, K
• Format: Journal Article
• Language: English
• Published: 01.01.2011
• ISSN: 0065-1400

Umbilical cord blood is considered as a promising source of stem cells capable of self-renewal and differentiation into different cell types, including neural. Differentiation processes are governed by microenvironmental cues and by unique molecular mechanisms, where epigenetic changes of the chromatin play an important role. Emerging evidence suggests, that changes in expression of so called "stemness" gene, like Oct3/4, are associated with the specific epigenetic modifications of gene promoter. Methylation status of Oct3/4 and Nanog promoters correlates strongly with their ability to be expressed. The promoters are unmethylated in pluripotent stem cells, where those genes are expressed, and almost fully methylated in differentiated cells, where Oct3/4 and Nanog are silenced. The aim of the study was to analyze the DE (Distal Enhancer) promoter region's methylation pattern in Oct3/4 gene in HUCB-NSC (Human Umbilical Cord Blood - Neural Stem Cells) line comparing to hESC (Human Embryonic Stem Cells) and also changes caused by neural differentiation of HUCB-NSC. Materials and Methods. HUCB-NSC were cultured in serum free, low serum (2% FBS) and in differentiating medium containing dBcAMP (300 mu M) in the density of 5x10 super(4) cells per cm super(2) in standard conditions. After 14 DIV DNA from harvested cells was isolated. Methylation status of gene DE promoter region was analyzed by sodium bisulfite reaction. To analyze sequence of obtained PCR fragment subcloning into pGEM-T easy vector and sequencing was performed (at least 10 individual clones). DNA of hESC was received from Prof. Dvorak laboratory in Brno. Results. Methylation pattern of Oct3/4 DE promoter region was changing along differentiation process. HUCB-NSC after neural differentiation revealed higher methylation status in promoter region than in undifferentiated cells. Those changes correlate with the expression of Oct3/4 gene.