Efficiency Of iPS Cell Generation Research Articles

Pramef12 enhances reprogramming into naïve iPS cells

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Somatic cell nuclear transfer can also be utilized to reprogram somatic cells into totipotent embryos, suggesting that factors present in oocytes potentially enhance the efficiency of iPS cell generation. Here, we showed that preferentially expressed antigen of melanoma family member 12 (Pramef12), which is highly expressed in oocytes, enhances the generation of iPS cells from mouse fibroblasts. Overexpression of Pramef12 during the early phase of OKSM-induced reprogramming enhanced the efficiency of iPS cell derivation. In addition, overexpression of Pramef12 also enhanced expression of naïve pluripotency-associated genes, Gtl2 located within the Dlk1–Dio3 imprinted region essential for full pluripotency, glycolysis-associated genes, and oxidative phosphorylation-associated genes, and it promoted mesenchymal-to-epithelial transition during iPS cell generation. Furthermore, Pramef12 greatly activated β-catenin during iPS cell generation. These observations suggested that Pramef12 enhances OKSM-induced reprogramming via activation of the Wnt/β-catenin pathway.

MiR-6539 is a novel mediator of somatic cell reprogramming that represses the translation of Dnmt3b.

Global DNA hypomethylation has been shown to be involved in the pluripotency of induced pluripotent stem (iPS) cells. Relatedly, DNA methyltransferases (DNMTs) are believed to be a substantial barrier to genome-wide demethylation. There are two distinct stages of DNMT expression during iPS cell generation. In the earlier stage of reprogramming, the expression of DNMTs is repressed to overcome epigenetic barriers. During the late stage, the expression of DNMTs is upregulated to ensure iPS cells obtain the full pluripotency required for further development. This fact is strongly reminiscent of microRNAs (miRNAs), critical regulators of precise gene expression, may be central to coordinate the expression of DNMTs during reprogramming. Using a secondary inducible system, we found that miR-6539 had a unique expression dynamic during iPS cell generation that inversely correlated with DNMT3B protein levels. Enforced upregulation of miR-6539 during the early stage of reprogramming increased the efficiency of iPS cell generation, while enforced downregulation impaired efficiency. Further analysis showed that Dnmt3b mRNA is the likely target of miR-6539. Notably, miR-6539 repressed Dnmt3b translation via a target site located in the coding sequence. Our study has therefore identified miR-6539 as a novel mediator of somatic cell reprogramming and, to the best of our knowledge, is the first to demonstrate miRNA-mediated translation inhibition in somatic cell reprogramming via targeting the coding sequence. Our study contributes to understand the mechanisms that underlie the miRNA-mediated epigenetic remodeling that occurs during somatic cell reprogramming.

E-Ras improves the efficiency of reprogramming by facilitating cell cycle progression through JNK–Sp1 pathway

We have previously shown that pluripotent stem cells can be induced from adult somatic cells which were exposed to protein extracts isolated from mouse embryonic stem cells (mESC). Interestingly, generation of induced pluripotent stem (iPS) cells depended on the background of ES cell lines; possible by extracts from C57, but not from E14. Proteomic analysis of two different mES cell lines (C57 and E14) shows that embryonic Ras (E-Ras) is expressed differently in two mES cell lines; high level of E-Ras only in C57 mESC whose extracts allows iPS cells production from somatic cells. Here, we show that E-Ras augments the efficiency in reprogramming of fibroblast by promoting cell proliferation. We found that over-expression of E-Ras in fibroblast increased cell proliferation which was caused by specific up-regulation of cyclins D and E, not A or B, leading to the accelerated G1 to S phase transition. To figure out the common transcription factor of cyclins D and E, we used TRANSFAC database and selected SP1 as a candidate which was confirmed as enhancer of cyclins D and E by luciferase promoter assay using mutants. As downstream signaling pathways, E-Ras activated only c-Jun N-terminal kinases (JNK) but not ERK or p38. Inhibition of JNK prevented E-Ras-mediated induction of pSP1, cyclins D, E, and cell proliferation. Finally, E-Ras transduction to fibroblast enhanced the efficiency of iPS cell generation by 4 factors (Oct4/Klf4/Sox2/C-myc), which was prevented by JNK inhibitor. In conclusion, E-Ras stimulates JNK, enhances binding of Sp1 on the promoter of cyclins D and E, leading to cell proliferation. E-Ras/JNK axis is a critical mechanism to generate iPS cells by transduction of 4 factors or by treatment of mESC protein extracts.

Proliferation Rate of Somatic Cells Affects Reprogramming Efficiency

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.

Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation

Induced pluripotent stem (iPS) cells generated using Yamanaka factors have great potential for use in autologous cell therapy. However, genomic abnormalities exist in human iPS cells, and most mouse iPS cells are not fully pluripotent, as evaluated by the tetraploid complementation assay (TCA); this is most likely associated with the DNA damage response (DDR) occurred in early reprogramming induced by Yamanaka factors. In contrast, nuclear transfer can faithfully reprogram somatic cells into embryonic stem (ES) cells that satisfy the TCA. We thus hypothesized that factors involved in oocyte-induced reprogramming may stabilize the somatic genome during reprogramming, and improve the quality of the resultant iPS cells. To test this hypothesis, we screened for factors that could decrease DDR signals during iPS cell induction. We determined that Zscan4, in combination with the Yamanaka factors, not only remarkably reduced the DDR but also markedly promoted the efficiency of iPS cell generation. The inclusion of Zscan4 stabilized the genomic DNA, resulting in p53 downregulation. Furthermore, Zscan4 also enhanced telomere lengthening as early as 3 days post-infection through a telomere recombination-based mechanism. As a result, iPS cells generated with addition of Zscan4 exhibited longer telomeres than classical iPS cells. Strikingly, more than 50% of iPS cell lines (11/19) produced via this "Zscan4 protocol" gave rise to live-borne all-iPS cell mice as determined by TCA, compared to 1/12 for lines produced using the classical Yamanaka factors. Our findings provide the first demonstration that maintaining genomic stability during reprogramming promotes the generation of high quality iPS cells.

MiR‐138 Promotes Induced Pluripotent Stem Cell Generation Through the Regulation of the p53 Signaling

Induced pluripotent stem (iPS) cells, especially those reprogrammed from patient somatic cells, have a great potential usage in regenerative medicine. The expression of p53 has been proven as a key barrier limiting iPS cell generation, but how p53 is regulated during cell reprogramming remains unclear. In this study, we found that the ectopic expression of miR-138 significantly improved the efficiency of iPS cell generation via Oct4, Sox2, and Klf4, with or without c-Myc (named as OSKM or OSK, respectively), without sacrificing the pluripotent characteristics of the generated iPS cells. Exploration of the mechanism showed that miR-138 directly targeted the 3' untranslated region (UTR) of p53, significantly decreasing the expression of p53 and its downstream genes. Furthermore, the ectopic expression of p53 having a mutant 3'-UTR, which cannot be bound by miR-138, seriously impaired the effect of miR-138 on p53 signaling and OSKM-initiated somatic cell reprogramming. Combined with the fact that miR-138 is endogenously expressed in fibroblasts, iPS cells, and embryonic stem cells, our study demonstrated that regulation of the p53 signaling pathway and promotion of iPS cell generation represent an unrevealed important function of miR-138.

Human-Induced Pluripotent Stem Cells: In Quest of Clinical Applications

In the field of regenerative medicine, the development of induced pluripotent stem (iPS) cells may represent a potential strategy to overcome the limitations of human embryonic stem cells (ESCs). iPS cells have the potential to mimic human disease, since they carry the genome of the donor. Hypothetically, with iPS cell technology it is possible to screen patients for a genetic cause of disease (genetic mutation), develop cell lines, reprogram them back to iPS cells, finally differentiate them into one or more cell types that develop the disease. Although the creation of multiple lineages with iPS cells can seem limitless, a number of challenges need to be addressed in order to effectively use these cell lines for disease modeling. These include the low efficiency of iPS cell generation without genetic alterations, the possibility of tumor formation in vivo, the random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. The establishment of protein or RNA-based reprogramming strategies will help generate human iPS cells without permanent genetic alterations. Finally, direct reprogramming strategies can provide rapid production of models of human "diseases in a dish", without first passing the cells through a pluripotent state, so avoiding the challenges of time-consumming and labor-intensive iPS cell line generation. This review will overview methods to develop iPS cells, current strategies for direct reprogramming, and main applications of iPS cells as human disease model, focusing on human cardiovascular diseases, with the aim to be a potential information resource for biomedical scientists and clinicians who exploit or intend to exploit iPS cell technology in a range of applications.

Autophagy in Stem Cell Maintenance and Differentiation

Autophagy is a lysosome-dependent degradation pathway that allows cells to recycle damaged or superfluous cytoplasmic content, such as proteins, organelles, and lipids. As a consequence of autophagy, the cells generate metabolic precursors for macromolecular biosynthesis or ATP generation. Deficiencies in this pathway were associated to several pathological conditions, such as neurodegenerative and cardiac diseases, cancer, and aging. The aim of this review is to summarize recent discoveries showing that autophagy also plays a critical role in stem cell maintenance and in a variety of cell differentiation processes. We also discuss a possible role for autophagy during cellular reprogramming and induced pluripotent stem (iPS) cell generation by taking advantage of ATP generation for chromatin remodeling enzyme activity and mitophagy. Finally, the significance of autophagy modulation is discussed in terms of augmenting efficiency of iPS cell generation and differentiation processes.

The Art of Human Induced Pluripotent Stem Cells: The Past, the Present and the Future

In 2006, Yamanaka and Takahashi electrified the scientific community by discovering that mouse somatic cells can be converted into embryonic stem cell-like cells by retroviral transduction of four transcription factors: Oct4, Sox2, Klf4, and c-Myc (OSKM). The first generation of mouse induced pluripotent stem (iPS) cells was incompletely reprogrammed, and failed to contribute to germline transmission. Nearly one year later, three groups, including Yamanaka's, improved the reprogramming methodology and generated iPS cells that were in many respects, indistinguishable from ES cells, and also contributed to chimera formation and germline transmission. Shortly thereafter, the successful reprogramming of human somatic cells opened the gate for the development of patient-specific iPS cells for biomedical research and clinical application. Though human iPS cells resemble human ES cells in many aspects, the current iPS cell technologies showed several limitations for clinical usage. First, the efficiency of iPS cell generation is still low and the reprogramming process takes at least two weeks. Second, the virus-delivery of reprogramming factors introduces inconceivable risks of insertional mutagenesis in the genome. Third, given the various strategies for direct reprogramming, it remains difficult to assess the quality of iPS cells generated in each lab and for each patient. These issues should be addressed properly before any iPS cells could be translated into clinic. Here, we review recent progress in human iPS cell technologies, with a focus on the virus-free and integration-free iPS cell generation, which may lead towards the eventual goal of clinical applications. Keyword: Human induced pluripotent stem cells (hiPSCs), reprogramming strategy, hiPSC efficiency.

Butyrate Promotes Induced Pluripotent Stem Cell Generation

Recent studies have demonstrated that embryonic stem cell-like induced pluripotent stem (iPS) cells can be generated by enforced expression of defined transcription factors. The fact that cell fate change is accompanied by changes in epigenetic modifications prompted us to investigate whether chemicals known to modulate epigenetic regulators are capable of enhancing the efficiency of iPS cell generation. Here, we report that butyrate, a natural small fatty acid and histone deacetylase inhibitor, significantly increases the efficiency of mouse iPS cell generation using the transcription factors Oct4, Sox2, Klf4, and c-Myc. We show that butyrate not only changes the reprogramming dynamics, but also increases the ratio of iPS cell colonies to total colonies by reducing the frequency of partially reprogrammed cells and transformed cells. Detailed analysis reveals that the effect of butyrate on reprogramming appears to be mediated by c-Myc and occurs during an early stage of reprogramming. Genome-wide gene expression analysis reveals up-regulation of ES cell-enriched genes when mouse embryonic fibroblasts are treated with butyrate during reprogramming. Thus, our study identifies butyrate as a chemical factor capable of promoting iPS cell generation.

Activation of the Imprinted Dlk1-Dio3 Region Correlates with Pluripotency Levels of Mouse Stem Cells

Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.

The Ink4/Arf locus is a barrier for iPS cell reprogramming

The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood. The Ink4/Arf locus comprises the Cdkn2a-Cdkn2b genes encoding three potent tumour suppressors, namely p16(Ink4a), p19(Arf) and p15(Ink4b), which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of 'stemness'. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.

Roles of Sall4 in the generation of pluripotent stem cells from blastocysts and fibroblasts

Pluripotency of embryonic stem (ES) cells is maintained by a network consisting of multiple transcription factors, including Oct3/4, Sox2, Nanog, Klf4 and Sall4. Among these factors, the forced expressions of Oct3/4, Sox2 and Klf4 are sufficient to reprogram fibroblasts into induced pluripotent stem (iPS) cells. The current study analyzed the role of Sall4 during the generation of ES cells and iPS cells. The mouse Sall4 gene was deleted by homologous recombination. Sall4-null embryos died shortly after implantation, as has been reported. ES-like cell lines can be established from Sall4-null blastocysts, albeit with a lower efficiency and a slower time course. The knockdown of Sall4 significantly decreased the efficiency of iPS cell generation from mouse fibroblasts. Furthermore, retroviral transduction of Sall4 significantly increased the efficiency of iPS cell generation in mouse and some human fibroblast lines. These results demonstrated that Sall4 plays positive roles in the generation of pluripotent stem cells from blastocysts and fibroblasts.