Efficient Generation of Rat Induced Pluripotent Stem Cells Using a Non-Viral Inducible Vector

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.     

Induced pluripotent stem cells

Induced pluripotent stem cells (iPS) result from a reprogramming of somatic cells via transduction with viral vectors expressing the Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28 genes, which are essential for the establishment and maintenance of the pluripotent state. In properties, iPS are almost fully similar to embryonic stem cells (ESC). To date, iPS have been obtained from various differentiated cells of mice and humans. Along with ESC, iPSs are highly promising for research and medicine.     

A novel strategy to derive iPS cells from porcine fibroblasts

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors. This technology has created an interest in deriving iPS cells from domesticated animals such as pigs, sheep and cattle. Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells. However, this retrovirus system infects only mouse and rat cells, which limits its use in establishing iPS cells from other mammals. In our study, we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts. We transfected four human reprogramming factors (Oct4, Sox2, Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells. We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF. Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies. Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.     

Direct Reprogramming of Rat Neural Precursor Cells and Fibroblasts into Pluripotent Stem Cells

Background

Given the usefulness of rats as an experimental system, an efficient method for generating rat induced pluripotent stem (iPS) cells would provide researchers with a powerful tool for studying human physiology and disease. Here, we report direct reprogramming of rat neural precursor (NP) cells and rat embryonic fibroblasts (REF) into iPS cells by retroviral transduction using either three (Oct3/4, Sox2, and Klf4), four (Oct3/4, Sox2, Klf4, and c-Myc), or five (Oct3/4, Sox2, Klf4, c-Myc, and Nanog) genes.

Methodology and Principal Findings

iPS cells were generated from both NP and REF using only three (Oct3/4, Sox2, and Klf4) genes without c-Myc. Two factors were found to be critical for efficient derivation and maintenance of rat iPS cells: the use of rat instead of mouse feeders, and the use of small molecules specifically inhibiting mitogen-activated protein kinase and glycogen synthase kinase 3 pathways. In contrast, introduction of embryonic stem cell (ESC) extracts induced partial reprogramming, but failed to generate iPS cells. However, when combined with retroviral transduction, this method generated iPS cells with significantly higher efficiency. Morphology, gene expression, and epigenetic status confirmed that these rat iPS cells exhibited ESC-like properties, including the ability to differentiate into all three germ layers both in vitro and in teratomas. In particular, we found that these rat iPS cells could differentiate to midbrain-like dopamine neurons with a high efficiency.

Conclusions/Significance

Given the usefulness of rats as an experimental system, our optimized method would be useful for generating rat iPS cells from diverse tissues and provide researchers with a powerful tool for studying human physiology and disease.     

Cell signalling pathways underlying induced pluripotent stem cell reprogramming

Induced pluripotent stem(i PS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3 K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and c Myc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few "stabilisation competent" cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-i PS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, i PS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As i PS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.     

Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells

In vitro reprogramming of somatic cells into a pluripotent embryonic stem cell-like state has been achieved through retroviral transduction of murine fibroblasts with Oct4, Sox2, c-myc and Klf4. In these experiments, the rare 'induced pluripotent stem' (iPS) cells were isolated by stringent selection for activation of a neomycin-resistance gene inserted into the endogenous Oct4 (also known as Pou5f1) or Nanog loci. Direct isolation of pluripotent cells from cultured somatic cells is of potential therapeutic interest, but translation to human systems would be hindered by the requirement for transgenic donors in the present iPS isolation protocol. Here we demonstrate that reprogrammed pluripotent cells can be isolated from genetically unmodified somatic donor cells solely based upon morphological criteria.     

体细胞重编程为诱导多能干细胞

诱导多功能性干细胞（induced pluripotent stem cells,iPS细胞）是通过导入特定的转录因子（如Oct3/4、Sox2、c-Myc和Klf4等）将体细胞诱导重编程为多能性干细胞,其功能与胚胎干细胞相似.iPS细胞的建立,在生命科学领域引起了新的轰动.目前,iPS细胞的研究领域在转录因子的优化、iPS细胞的筛选、载体的运用、体细胞种类的选择和iPS细胞的应用等方面取得突破进展,但仍然存在致癌性、效率低等一系列急需解决的问题.     

Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells. The low efficiency with which iPS cells are derived hinders studies on the molecular mechanism of reprogramming, and integration of viral transgenes, in particular the oncogenes c-Myc and Klf4, may handicap this method for human therapeutic applications. Here we report that valproic acid (VPA), a histone deacetylase inhibitor, enables reprogramming of primary human fibroblasts with only two factors, Oct4 and Sox2, without the need for the oncogenes c-Myc or Klf4. The two factor-induced human iPS cells resemble human ES cells in pluripotency, global gene expression profiles and epigenetic states. These results support the possibility of reprogramming through purely chemical means, which would make therapeutic use of reprogrammed cells safer and more practical.     

Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells

We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.     

Cyclin-dependent Kinase-mediated Sox2 Phosphorylation Enhances the Ability of Sox2 to Establish the Pluripotent State

Sox2 is a key factor in maintaining self-renewal of embryonic stem cells (ESCs) and adult stem cells as well as in reprogramming differentiated cells back into pluripotent or multipotent stem cells. Although previous studies have shown that Sox2 is phosphorylated in human ESCs, the biological significance of Sox2 phosphorylation in ESC maintenance and reprogramming has not been well understood. In this study we have identified new phosphorylation sites on Sox2 and have further demonstrated that Cdk2-mediated Sox2 phosphorylation at Ser-39 and Ser-253 is required for establishing the pluripotent state during reprogramming but is dispensable for ESC maintenance. Mass spectrometry analysis of purified Sox2 protein has identified new phosphorylation sites on two tyrosine and six serine/Threonine residues. Cdk2 physically interacts with Sox2 and phosphorylates Sox2 at Ser-39 and Ser-253 in vitro. Surprisingly, Sox2 phosphorylation at Ser-39 and Ser-253 is dispensable for ESC self-renewal and cell cycle progression. In addition, Sox2 phosphorylation enhances its ability to establish the pluripotent state during reprogramming by working with Oct4 and Klf4. Finally, Cdk2 can also modulate the ability of Oct4, Sox2, and Klf4 in reprogramming fibroblasts back into pluripotent stem cells. Therefore, this study has for the first time demonstrated that Sox2 phosphorylation by Cdk2 promotes the establishment but not the maintenance of the pluripotent state. It might also help explain why the inactivation of CDK inhibitors such as p53, p21, and Arf/Ink4 promotes the induction of pluripotent stem cells.     

Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes

In this study, we created porcine‐induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c‐Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA‐Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537–553, 2017. © 2016 Wiley Periodicals, Inc.