Generation of pluripotency in somatic cells became possible three years ago, when Japanese scientists S.Yamanaka and K.Takahashi described pluripotent cells derived from mouse fibroblast cells as a result of retroviral transfection of four genes - Oct4, Klf4, Sox2 and c-Myc (OKSM) and called them induced pluripotent stem cells (iPS cells) [2]. For generation of pluripotency in somatic cells initiating the expression of ‘pluripotent’ self-genes - Oct4, Sox2 and Nanog is the principal event. Being transcription factors the products of these genes are *bound to/ attached to target genes inhibiting the expression of linear-specific genes and activating the expression of the genes responsible for maintaining cell pluripotency and self-renewal. Moreover, employing the positive feedback mechanism these transcription factors maintain the stable expression level of each other. It was these peculiarities that enabled S. Yamanaka and Takahashi to generate pluripotency in somatic cells due to temporal expression of OKSM exogenous genes that were inserted into cell nuclei by means of virus- mediated transduction.
The principal impediment of the developed method for clinical use is employment of viral vectors integrating into the cell genome as well as proto-oncogenes as transgenes (c-Myc, Klf4). This may result in malignant transformation of iPS cells or their committed derivatives that prevents therapeutic applications of iPS cells. Significant advances in solving this problem have been made by deriving iPS cells without viral integration into human and mouse cells, using instead adenoviruses, plasmids, transposones or recombinant proteins [3, 4].
The journal Stem Cells published an article of the American investigators, where an alternative approach to induce pluripotency in somatic cells was described. This strategy applied an induction of the expression of “pluripotency” self-genes without introducing any genetic material or proteins. The investigators supposed that the self-gene expression might be initiated by placing somatic cells into a specific microenvironment. This microenvironment has to be a source of stimuli, which eventually are to initiate the expression of required genes by means of intracellular signal pathways.
In their investigation as somatic cells the authors used neurosphere cells obtained in culturing eye limbal epithelial cells of adult rats. Limbal epithelium, a thin layer of circular epithelium, separating the cornea from the conjunctiva, is a source of stem cells/ progenitors in its basal layer, by means of which the cornea renewal occurs. When these cells are derived from their niche and cultured in the presence of Noggin they originate the progenitors of nervous tissue which differentiate into the functional neurons under the influence of extracellular stimuli [5]. Somatic cells of this type were chosen owing to a high constitutive expression of three of four OKSM factors - Klf4, Sox2 and c-Myc.
As a specific microenvironment the investigators used a mouse ESC conditioned medium. The cells of neurospheres were cultured in this medium for 10-14 days then the presence of iPS cells in the cultures was evaluated. As the control the neurosphere cells cultured in a fibroblast conditioned medium and mouse ESCs were used.
The limbal epithelial cells derived from the eyes of adult Sprague-Dawley rats were cultured in a Nogin-containing medium. In 7 days the neurospheres wereisolated and cultured in a mouse ESC conditioned medium for 10-14 days. A colony formation was observed in five doublings that did not occur when neurospheres were cultured in a fibroblast-conditioned medium. A karyotype analysis of the colony cells showed that they were derivatives of neurosphere cells (42 chromosomes as in a normal mouse chromosome set), but not of mouse ESCs (40 chromosomes). There may be mouse ESCs in primary cultures due to improper purification of an ESC conditioned medium. The cells of resulting colonies could passage every 3-4 days. At the time of 10 doublingsin these cells the Oct4 and Nanog transcription factors were observed, as well as a number of other ESC specific markers - alkaline phosphatase and stage-specific embryonic antigen-1.
An expression analysis of a number of markers showed that there were practically no markers of the progenitor-cells of limbal epithelium - p63 and α- enolase – in the colony cells resulting from culturing neurospheres in mouse ESC conditioned media. At the sate time transcriptors of OKSM, Nanog and Lin28 genes were revealed, though in less amounts than in mouse ESCs. These findings give evidence of the obtained cells being similar but not identical to the ESCs.
To confirm the findings on formation of pluripotent cells obtained in the study, the investigators transduced the rat limbal epithelial cells with a genetic construction where a gene of the green fluorescent protein (GFP) was inserted under the control of the Nanog gene promoter. Thus, the GFP could be revealed in these cells only in case of the Nanog gene expression [6]. The efficacy of reprogramming was evaluated by scoring the GFP+-colonies obtained while culturing Nanog-GFP lentivirus transduced rat limbal progenitors in mouse ESC conditioned media. 0.0025% of primarily transduced limbal progenitors in culture with an ESC conditioned medium produced GFP+-colonies. Introduction of the valporic acid increased approximately four times the efficacy of reprogramming.
The differentiation potential of rat iPS cells obtained was similar to that of mouse ESCs. When removing a LIF factor from the culture iPS cells as well as ESCs formed embryoid bodies where the expression of markers of embryonic ectoderm (OTX2), entoderm (SOX17) and mesoderm BRACHYURY) was detected. Subcutaneous introduction of isolated rat iPS cells and mouse ESCs to NOD SCID immunodeficient mice resulted in teratoma formation within 3 weeks which were morphologically identical. All the teratomas contained the derivatives of all there germ layers. iPS cells obtained were differentiated in vitro into neurons, cardiomyocytes and hepatocytes according to the protocols previously developed for mouse ESCs.
Thus, the investigators achieved the generation of pluripotency in somatic cells without introducing any foreign genetic material or proteins into the nuclei, but by means of simple manipulations in the microenvironment. The investigators demonstrated the differentiation potential of obtained rat iPS cells to be similar to that of mouse ESCs. The investigation revealed that the colonies of pluripotent cells were not derivatives of mouse ESCs which could contaminate an ESC-conditioned medium used to culture neurosphere cells. The method developed could be easily introduced into a clinical practice as biopsy of limbal epithelium is an easy and safe procedure. The cells obtained could be reprogrammed within a relatively short time and used for autologous cell-based therapy.
Nevertheless, the method developed by the investigators to induce pluripotency in somatic cells cannot be referred to as a revolutionary one for some reasons. First of all, the results obtained have to be proved by other investigation groups, with, ideally, more detailed characteristics of iPS cells provided by the comparative analysis of the global gene expression pattern, analysis of chromatin configuration, analysis of differentiation potential when inserted into a blastocyst cavity and others. Furthermore, the developed method has to be reproduced for human cells. Moreover, what appears most important must be detected signaling pathways responsible for initiating the expression of endogenous genes of pluripotency. Only on the basis of certain mechanisms the development and introduction of actually effective methods of generation pluripotency in somatic cells for clinical purposes might be performed. In addition, a real interest in this investigation of the Invitrogen company which tends to take place in iPS cell industry causes some awareness.