Induction of pluripotency by defined factors.

Somatic cells can be reprogrammed into pluripotent stem cells by introducing a combination of several transcription factors. The induced pluripotent stem (iPS) cells from a patient’s somatic cells could be useful source of cells for drug discovery and cell transplantation therapies. However, most human iPS cells are made by viral vectors, such as retrovirus and lentivirus, which integrate the reprogramming factors into host genomes and may increase the risk of tumor formation. Studies of the mechanisms underlying the reprogramming and establishment of non-integration methods contribute evidence to resolve the safety concerns associated with iPS cells. On the other hand, patient-specific iPS cells have already been established and used for recapitulating disease pathology.


Introduction
Cell differentiation is a process of limiting their differentiation potential.

iPS induction methods
The original method of iPS induction used a retrovirus vector for transgene expression.
MMLV (Moloney murine leukemia virus)-based vectors with the infection efficiency over 5×10 6 TU/ml were employed [5]. The vector can robustly infect a variety of cell types and introduce their coding genes into the host genome by reverse transcriptase which thereby enables constant transgene expression during reprogramming. The inactivation of the retroviral promoter is observed in ES cells as well as in iPS cells maybe due to epigenetic modifications, such as histone methylation [6]. Therefore the expression of retroviral transgenes continues until the cells become iPS cells. This guided reprogramming and automatic silencing mechanism is thought to provide effective iPS induction in somatic cells. Most patient-specific iPS cells have been established with retrovirus vectors. However, the retrovirally derived iPS cells have numerous transgene integrations in the genome, and the integrations may results in leaky expression which could disturb endogenous transcription factor network and lead to failure of differentiation. Another important problem of transgene integration is tumorigenic risk after transplantation. In particular, c-Myc, one of the reprogramming factors, is a well-known oncogene, and its reactivation could give rise to transgene derived-tumor formation in chimeric mice [7]. There have been several reports of improvements of the transduction method for making safe iPS cells. Removal of the c-Myc transgene from reprogramming cocktail is one important approach. Human and mouse iPS cells can be established from fibroblasts with only Oct3/4, Sox2, and Klf4, although the efficiency is significantly reduced [8]. The chimeric mice produced with c-Myc-free iPS cells did not show enhanced of tumor formation during the observation period (6 months) in comparison to control mice. However, the overexpression of Oct3/4 and Klf4 can cause tumor formation, and various human tumors express OCT3/4, SOX2 and KLF4. In addition, the retroviral insertion to the genome itself may disturb endogenous gene structure and increase the risk of tumors [9]. Another approach is the reduction of integration sites by putting the reprogramming factors into a single vector with IRES or 2A self-cleavage peptide. This reprogramming cassette was used with a lentivirus system containing a loxP sequence in the LTR and produced iPS cells with only single insertions [10]. The expression of Cre recombinase successfully cut out the cassette. Although it left an incomplete LTR in the iPS genome, this method minimizes the genomic alteration. A transposon system encoding a reprogramming cassette has also been used for iPS induction [11,12]. reprogramming factors, such as Lin28 and shRNA for p53, mainly regulate the reprogramming efficiency through the control of cell proliferation [14]. In contrast, Nanog is seemed to enhance the efficiency of reprogramming through affecting the process itself. Tbx3 would also affect the process because it improves the germ line transmission efficiency of mouse iPS cells [15].

Molecular mechanisms of reprogramming
ES cells are maintained with strict regulation of the transcription factors network, which A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp includes Oct3/4, Sox2, Nanog, and Klf family. Some of these factors have direct protein-protein interactions, and they also bind promoter regions of a lot of common genes and control them. Oct3/4, Sox2, and Nanog induce the expression of stemness genes, such as STAT3 and ZIC3, with RNA polymerase II in human ES cells [16]. On the other hand, these factors are thought to have the opposite function, to repress differentiation related genes like PAX6 and ATBF1 with SUZ12 [17]. Therefore forced expression of some members of the network enables both induction and suppression of genes for pluripotency. The inactivation of a differentiation related gene, Thy-1, and the activation of SSEA-1, a stemness marker gene, occur at early time point of reprogramming during iPS induction from mouse embryonic fibroblasts (MEF) [18].
More specific pluripotent markers like endogenous Oct3/4 and Sox2, and activation of telomerase subsequently became detectable. Genome wide analysis of transcription factor binding sites reveals the importance of activation of genes co-occupied with Oct4, Sox2, and Klf4 for full reprogramming [19]. Live cell imaging allows a detailed analysis of the reprogramming steps. Retroviral transduction of reprogramming factors stimulates MEF to divide several times in a morphologically symmetric manner, while maintaining a fibroblastic shape [20]. Most descendant cells fail to reprogram and undergo cell death. Only a small portion of cells are gradually transformed into an Mouse iPS cells were also established from pancreatic -cells [24]. Terminally

Possible medical application
The first cell transplantation model of iPS cells used a humanized mouse model of sickle cell anemia [37]. It is a blood disorder which makes abnormal, sickle shaped red blood cells. A mutation in the  -globin gene causes the disease. Homozygous model mice for  -globin genes shows characteristic symptoms including severe anemia due to erythrocyte sickling, splenic infarcts, urine concentration defects and poor health. Better systems for reprogramming, differentiation, and purification of required cells is required to make better therapeutic cells. In addition, new methods are also needed to guarantee the quality and the safety of such cell transplantation. Drug inducible suicide genes can therefore be employed to achieve this aim.  Yamanaka, Induction of pluripotent stem cells from adult human fibroblasts by A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp