| In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a fully differentiated cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of tissues and cell types. Cellular differentiation process can be understood as the result of a gene regulatory network. A few evolutionarily conserved types of molecular processes, involving the cell signaling, are often involved in the cellular mechanisms that control these switches and differentiation. In spite of the advanced researches that have been done, the mechanism of differentiation and specialization way during development is still unclear. One way to explain this process is to study it from the opposite side and so study the dedifferentiation of the cells to be returned back again to its origin and so it is thought to determine the controller of this process.For long time before, it was thought that when a cell differentiates, it loses chromosomes or permanently inactivates genes that it no longer needs so it was thought that these differentiated adult cells generally have no ability to switch fates. But, the evidences resulted from the experimental approaches of cell fusion, somatic cell nuclear transfer, and whole cell extracts have shown that the cell fate can be reversed and so returning the cell to the characters of the embryonic state. This suggested that soluble trans-acting factors must exist in the cells that can confer the cell from one state to another one and through discovering and identification of these factors, it is considered to be the main responsible for the specialization and differentiation. This clarified that the development imposes reversible epigenetic rather than irreversible genetic changes on the genome during cellular differentiation.Any way at the level of reprogramming understanding, it is considered as a good example and tool to understand the mechanism of the normal differentaition process during normal development.Great efforts have been achieved in reprogramming of somatic cells to generate cells with greater pluripotency that help in understanding the mechanisms of both differentiation and dedifferentiation and high valiable important for science and medicine in place of ES cells overcoming the challenges of using its non-ethical sources. In addition, reprogramming to cells have the pluripotent state avoids the risk of immunological rejection as it is derived from a patient’s own cells. Moreover, it is able to participate in the field of tissue engineering, regenerative medicine, cell replacement therapy and drug development.The reprogramming of somatic cells to the pluripotent state has been successfully achieved through transferring somatic cell nuclear material into oocytes (SCNT) and through creation of cell hybrids by fusion of somatic cells with pluripotent cells and recently by ectopic expression of defined specific transcription factors (TFs) through viral transduction of transgenes. From that time, numerous refinements of this method have been done as the transgene-based approaches has been hampered by its integration of DNA sequences that may be a cause of proto-oncogenes expression leading to malignancy and undesired results in addition to the dangerous use of the viral methods. In spite of using the non-integrative DNA- based approaches as adenovirus and sendai virus or using virus free approaches as plasmids, minicircles and episomal vectors, the integration problem of DNA is difficult to be completely excluded. Pluripotency induction have been derived through DNA free methods as protein transduction of recombinant transcription factors but this method faced some problems as the compromised in vivo functionality of the bacteria-produced proteins in addition to its cost and low efficiency. Also, overexpression and transfection microRNAs (miRNAs) associated to pluripotency were demonstrated in non integrating reprogramming to pluripotency but a clear picture of this method still unobvious.Recently, a safer method for cellular reprogramming was performed through introduction of modified mRNA molecules encoding the reprogramming factors into somatic cells (mRNA-mediated gene delivery). This way promoted highly efficient protein expression when used in hematopoietic progenitor cells, mesenchymal stromal cells, dendritic cell and lymphocytes and used to derive astrocytes from neurons; and fibroblasts as well as astrocytes have been reprogrammed into cardiomyocytes. This technique has been validated recently for human somatic cell reprogramming to generate pluripotent cells that showed successful activation of the pluripotency genes in the transfected somatic cells. Reprogramming upon ectopic expression of the related factors is linked up with a major genetic, epigenetic modification to can overcome the transcriptional barriers during reprogramming.Due to few reports concerned to utilize mRNAs of reprogramming factors to induce pluripotency in murine species, our research aimed to dedifferentiate or reprogram the mouse embryonic fibroblast to be returned back in its developmental way to be the cells have the pluripotent characteristics using the mRNA transfection method. To achieve this main goal, we planned to, firstly, clone the genes or factors associated to the pluripotency stage in one of the expressing eukaryotic vectors downstream to T7 promotor region. Secondly, these newly constructed vectors containing the interested factors used for in vitro synthesize of their corresponding mRNAs which would be introduced in the donor fibroblast cells that goal to its reprogramming to be with the properties of embryonic stem cells, these properties were examined by specialized morphological, molecular and functional analyses. Finally, looking for understanding the mechanism of reprogramming, we check the onset of some genetic and epigenetic alterations of pluripotency markers through following up their expressional and promotor methylation changes during reprogramming and also under the effect of the epigenetic molecules. This research will provide the basis for better understanding of regulation of the reprogramming process and aid in discovering additional mechanisms of early embryonic developmental processes.Our results revealed the following points;1. Cloning and analysis of the four pluripotency transcription factors Oct4, Sox2, c-Myc, and Klf4 (OSCK) were performed in referring to their sequences in Gene Bank. Reverse transcription PCR (RT-PCR) was used to get the open reading frame or the coding sequences (CDS) of OSCK from testis, small intestine, and colon of mouse. The results showed that, the CDS length of OSCK were 1059 bp,960 bp,1320 bp, and 1452 bp, respectively. Concerning to, NCB1 BLAST of the resulted sequences showed that each gene has a high percent of sequence homology to its corresponding reference sequence of mouse as follow; 100%,100%,100%. and 99% for Oct4, Sox2, c-Myc. and Klf4. All these findings indicated that the obtained cloning sequences of these four genes were correct.2. The in vitro transcription template of each factor was constructed through cloning its open reading frame (ORF) of the in an eukaryotic expressing vector downstream to T7 promotor and the in vitro transcription reaction was incorporated with synthetic cap analog and also provided with poly (A) tailing reagents. The synthesized mRNA were successfully delivered into the cellular cytoplasm of the mouse embryonic fibroblast cells through a cationic lipid vehicle as it is cleared from the results of RT-PCR that revealed high expression in the next day of transfection in addition to translation to their correlated proteins that were rightly localized in the nuclei of the transfected cells as shown in the result of immuno-staining.3. Our results of transfection condition optimization, through fluorescence activated cell sorting (FACS) of the transfected cells with mRNA encoding GFP, revealed that the most suitable amount of mRNA was 1 μg per 1 × 105 MEF cells and also detected its expression for several days with high level that followed by its decline due to the degradation of the protein and mRNA, the result indicated the required several repeated transfections of mRNAs to preserve high level of protein expression for prolonged time that is necessary for cell reprogramming. After five consecutive transfections, There were cellular morphological changes from the mesenchymal appearance of fibroblasts to compact, round epithelial cell shape were observed and increased during transfection until observation of small colony-like structures at day 8 of reprogramming which increased in size with time and exhibited tightly defined borders and a high nuclear/cytoplasm ratio by day 15 with number of 100-130 colonies. The newly formed colonies were positive for alkaline phosphatase, expressed ES cell specific markers either through RT-PCR or the immuno-staining techniques. The newly converted cells showed also promoter methylation pattern near that of ESCs and also its ability of differentiation to the three primary developmental germ layers in vitro showing positive immune-staining for the specific markers; βⅢ tubulin (ectoderm), smooth muscle actin (SMA) (mesoderm) and Sox 17 (endoderm). And in vivo through teratoma formation which comprised of tissues of all three germ layers including cartilage, muscle, fat (mesoderm), pigmented epidermal tissue (ectoderm), and epithelium (endoderm). Therefore, activation of mouse pluripotency genes and induced pluripotent stem cells generation can be achieved in a safe manner when using the mouse specific synthesized mRNAs for transfection.4. The molecular events occur in the period of reprogramming were examined through detection of the expression level and promoter methylation status of some pluripotency markers during this period. There was up-regulation of epithelial genes such as E-cadherin (Cdhl) as well as the concomitant down-regulation of key mesenchymal genes such as Snail and Thy 1 that accompanied by morphological changes in the process which known as a mesenchymal-to-epithelial transition (MET). While the expression of the transfected and non-transfected pluripotency genes showed a potent up-regulation of the transferred factors (OSCK) during the transfection (first 6 days) then followed a period of down-regulation for all these factors after transfection cessation. Our study revealed a second up-regulation phase began at day 12 and suspected to be resulted from the activated endogenous genes of these factors at a time when most of the transferred factors were degraded. Upon transfection in our experiment, a gradual decreasing in the methylation percent was observed during the reprogramming timeline in the promoter region of the checked pluripotency associated factors (Oct4, Nanog, Rexl, and N-Mycl) so it was demonstrated the ability to reactivate the inactive pluripotency genes, which may be due to progressive demethylation of promoter regions and/or enhancer regions, resulting in turning on these factors.5. In this study, we demonstrated that small molecules (Valporic acid, Vitamin C and 5-Aza-cytidine) could promote an efficient expression of the pluripotency associated genes during reprogramming of mouse embryonic fibroblast into the pluripotency way using the mRNAs of OKSM factors with more attention for Vitamin C and also Valporic acid. |
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