Two-Phase Analysis of Molecular Pathways Underlying Induced Pluripotent Stem Cell Induction
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Induced pluripotent stem cells (iPSCs) can be reprogrammed from adult somatic cells by transduction with Oct4, Sox2, Klf4, and c-Myc, but the molecular cascades initiated by these factors remain poorly understood. Impeding their elucidation is the stochastic nature of the iPS induction process, which results in heterogeneous cell populations. Here we have synchronized the reprogramming process by a two-phase induction: an initial stable intermediate phase following transduction with Oct4, Klf4, and c-Myc, and a final iPS phase following overexpression of Sox2. This approach has enabled us to examine temporal gene expression profiles, permitting the identification of Sox2 downstream genes critical for induction. Furthermore, we have validated the feasibility of our new approach by using it to confirm that downregulation of transforming growth factor β signaling by Sox2 proves essential to the reprogramming process. Thus, we present a novel means for dissecting the details underlying the induction of iPSCs, an approach with significant utility in this arena and the potential for wide-ranging implications in the study of other reprogramming mechanisms.Keywords:
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In 2006 Yamanaka and colleagues succeeded to convert somatic cells into induced pluripotent stem cells. To do so, they introduced four defined transcription factors, Oct-3/4, Sox2, Klf4 and c-Myc, by retroviral infection.
This methodology opens a new field in stem cell research, not only in terms of future medical applications but also for new approaches in gene targeting in animals where no stable ES cells are yet available like the rat.
The three goals of this work were: Firstly, the determination of differences in reprogramming efficiency in dependence of the genetic background of the rat cells used. Secondly, the generation efficiency of rat iPS (riPS) cells from wild type rat embryonic fibroblasts (REFs) before and after gene targeting. The third goal was to clarify the role of Pramel7, a protein that was recently shown to stabilize the pluripotent state of embryonic stem cells, in terms of reprogramming efficiency. For all reprogramming rounds three factors (Oct-3/4, Klf4, Sox2) were used.
By reprogramming REFs of the different genetic backgrounds no difference in the reprogramming efficiency could be obtained. The generation efficiency of riPS before and after gene targeting was similar, indicating that gene targeting does not affect the reprogramming potential. In this work we also could show that Pramel7 overexpression is sufficient to drive reprogramming in a LIF independent manner.
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A 45-year-old Han Male from China contributed peripheral blood mononuclear cells induced by reprogramming human OKSM transcription factors (OCT3/4, KLF4 SOX2 and C-MYC) with a non-integrated additional vector system. Immunological markers confirmed the pluripotent nature of IPSC. Spontaneous tridermal differentiation confirmed the differentiation ability of IPSC with normal karyotype.
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Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here, we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network, even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs and thereby bear major implications for further development and application of iPSC technology.
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Skin punch biopsy was donated by a healthy 51-year-old Caucasian male and the dermal fibroblasts were reprogrammed into human induced pluripotent stem cell (hiPSC) lines by using non-integrative Sendai viruses expressing OCT4, SOX2, KLF4 and c-MYC. Three iPSC lines (NUIGi046-A, NUIGi046-B, NUIGi046-C) highly expressed the pluripotent markers and were capable of differentiating into cells of endodermal, mesodermal, and ectodermal origin. These iPSCs can be offered as controls and in combination with genome-editing and three-dimensional (3D) system. They may be used for human disease modelling and drug screening.
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Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) as a powerful tool for regenerative medicine has gained wide attention in recent years. However, there are certain concerns regarding the efficiency of this reprogramming. Partially reprogrammed iPSCs (piPSCs) are stable cell lines originating from cells that have exited the normal reprogramming route at an early time point. Analysis of the associated global gene expression changes between iPSCs and piPSCs may help understand the barriers to reprogramming. In our study, human fibroblasts were transduced with the four classic transcription factors, OCT4, SOX2, KLF4, and C-MYC. Only a few cells were completely reprogrammed to a fully pluripotent state. Instead, we obtained more number of intermediate standstill clones than human-induced pluripotent stem cells (hiPSCs) during reprogramming. We studied the genome-wide expression profiles of two different fibroblasts, five intermediate standstill clones, and two iPSCs derived from the two fibroblasts. Hierarchical clustering and principal component analysis demonstrated that intermediate standstill clones were on the way to becoming hiPSCs. A remarkable difference in the expression of genes related to cancer and cell adhesion pathway was observed between the intermediate standstill clones and iPSCs. These observations suggest that some cells may become trapped in partially reprogrammed states.
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Current advances in cellular reprogramming technology has demonstrated that the identity of a cell can be converted by the use of master transcription factors to reprogram the transcriptome.Notably, this allows us to convert somatic cells into induced pluripotent stem cells (iPSCs), providing a feasible method to generate patient-specific pluripotent stem cells.This technology was firstly discovered by Shinya Yamanaka's group in 2006.The initial iPSCs were formed by the induction of dedifferentiation in mouse fibroblasts using transcription factors: Oct4, Sox2, Klf4 and c-Myc.This approach has tremendous medical potentials to revolutionize the way we study and develop treatment for ocular diseases.Here we reviewed the potential of using patient-specific iPSCs for 3D disease modeling and various types of retinal disease modeling, cell replacement therapy and clinical trials, high-throughput screening test and drug toxicity testing.We also discussed the recent development of direct reprogramming and the future direction for utilising iPSCs and cellular reprogramming technology for eye research.
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Induced pluripotent stem cells; Cellular reprogramming; Retina; Disease modeling; Cell therapy; Drug screening
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The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp) vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system) is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.
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MicroRNA (miRNAs) are short noncoding RNA molecules involved in many cellular processes and shown to play a key role in somatic cell induced reprogramming. We performed an array based screening to identify candidates that are differentially expressed between dermal skin fibroblasts (DFs) and induced pluripotent stem cells (iPSCs). We focused our investigations on miR-145 and showed that this candidate is highly expressed in DFs relative to iPSCs and significantly downregulated during reprogramming process. Inhibition of miR-145 in DFs led to the induction of "cellular plasticity" demonstrated by: (a) alteration of cell morphology associated with downregulation of mesenchymal and upregulation of epithelial markers; (b) upregulation of pluripotency-associated genes including SOX2, KLF4, C-MYC; (c) downregulation of miRNA let-7b known to inhibit reprogramming; and (iv) increased efficiency of reprogramming to iPSCs in the presence of reprogramming factors. Together, our results indicate a direct functional link between miR-145 and molecular pathways underlying reprogramming of somatic cells to iPSCs.
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