In vitro induction of human embryonal carcinoma differentiation by a crude extract of Rhazya stricta.

BACKGROUND: Rhazya stricta Decne. is a medicinal plant that is widespread in Saudi Arabia and desert areas of the Arabian Peninsula. Its extract contains alkaloids, tannins, and flavonoids that are involved in different biological activities. The study aim was to evaluate the effects of Rhazya stricta plant extracts on the proliferation and differentiation of NTERA-2 (NT2) pluripotent embryonal carcinoma cells. METHODS: Soxhlet extraction was carried out using different solvents to extract stems, leaves and fruit parts of this plant. Cytotoxicity was evaluated by an MTS cell viability assay. The ability of the plant extract to induce cell differentiation was examined phenotypically using an inverted light microscope. The expression of pluripotency markers was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Phytochemical screening of chloroform stem extracts was carried out and a chromatographic fingerprint was generated using gas chromatography - mass spectrometry (GC-MS). RESULTS: Chloroform stem extract induced differentiation of NT2 cells at 5 µg/ml, and the differentiated cells exhibited neurite formation. Following induction of differentiation, there was significant down-regulation of the pluripotency marker genes Oct4 and Sox2. In addition, the surface antigen pluripotency marker, TRA-1-60, was strongly down-regulated. Phytochemical analysis of the extract showed the presence of alkaloids and saponins. The chromatogram revealed the presence of fifteen compounds with different retention times. CONCLUSION: Our results demonstrate for the first time that chloroform stem extract of R. stricta can induce neuronal differentiation of stem cells at an early stage and may contain potential therapeutic agent that can be used in neurodegenerative diseases.

Phylogenetic and Structural Analysis of the Pluripotency Factor Sex-Determining Region Y box2 Gene of Camelus dromedarius (cSox2).

Although the sequencing information of Sox2 cDNA for many mammalian is available, the Sox2 cDNA of Camelus dromedaries has not yet been characterized. The objective of this study was to sequence and characterize Sox2 cDNA from the brain of C. dromedarius (also known as Arabian camel). A full coding sequence of the Sox2 gene from the brain of C. dromedarius was amplified by reverse transcription PCRjmc and then sequenced using the 3730XL series platform Sequencer (Applied Biosystem) for the first time. The cDNA sequence displayed an open reading frame of 822 nucleotides, encoding a protein of 273 amino acids. The molecular weight and the isoelectric point of the translated protein were calculated as 29.825 kDa and 10.11, respectively, using bioinformatics analysis. The predicted cSox2 protein sequence exhibited high identity: 99% for Homo sapiens, Mus musculus, Bos taurus, and Vicugna pacos; 98% for Sus scrofa and 93% for Camelus ferus. A 3D structure was built based on the available crystal structure of the HMG-box domain of human stem cell transcription factor Sox2 (PDB: 2 LE4) with 81 residues and predicting bioinformatics software for 273 amino acid residues. The comparison confirms the presence of the HMG-box domain in the cSox2 protein. The orthologous phylogenetic analysis showed that the Sox2 isoform from C. dromedarius was grouped with humans, alpacas, cattle, and pigs. We believe that this genetic and structural information will be a helpful source for the annotation. Furthermore, Sox2 is one of the transcription factors that contributes to the generation-induced pluripotent stem cells (iPSCs), which in turn will probably help generate camel induced pluripotent stem cells (CiPSCs).

Generation of human iPS cell line SKiPSc1 from healthy Human Neonatal Foreskin Fibroblast cells.

The SKiPSc1 induced pluripotent stem (iPS) cell line was generated from Human Neonatal Foreskin Fibroblasts (HNFFs) obtained from a healthy donor infant that were reprogrammed using non-integrating Sendai viral vectors expressing Oct3/4, Sox2, c-Myc, and Klf4.

Molecular Modeling and Phylogeny of the Krüppel-like Factor 4 (cKLF4) Protein from the Arabian Camel, Camelus dromedarius.

Krüppel-like factor 4 (KLF4) is a pluripotency transcription factor that helps in generating induced pluripotent stem cells (iPSCs). We sequenced for the first time the full coding sequence of Camelus dromedarius KLF4 (cKLF4), which is also known as the Arabian camel. Bioinformatics analysis revealed the molecular weight and the isoelectric point of cKLF4 protein to be 53.043 kDa and 8.74, respectively. The predicted cKLF4 protein sequence shows high identity with some other species as follows: 98% with Bactrian camel and 89% with alpaca KLF4 proteins. A three-dimensional (3D) structure was built based on the available crystal structure of the Mus musculus KLF4 (mKLF4) of 82 residues (PDB: 2 WBS) and by predicting 400 residues using bioinformatics software. The comparison confirms the presence of the zinc finger domains in cKLF4 protein. Phylogenetic analysis showed that KLF4 from the Arabian camel is grouped with the Bactrian camel, alpaca, cattle, and pig. This study will help in the annotation of KLF4 protein and in generating camel-induced pluripotent stem cells (CiPSCs).