BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone

Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based reprogramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ～1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells.     

Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy

Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11-12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm.     

Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.     

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.     

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.