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.     

Epigenetic changes to human umbilical cord blood cells cultured with three proteins indicate partial reprogramming to a pluripotent state

We have previously reported the existence of a subpopulation of cells from human umbilical cord blood capable of differentiating into oligodendrocytes, Schwann cells, bone, muscle, and endothelial cells despite their origins as CD45-positive cells. These stem cells (called FSFl cells) arise only after a period in vitro in medium containing FGF4, SCF, and Flt-3 ligand (FSFl medium) during which they express the pluripotency genes Oct4 and Nanog. The objective of this study was to determine if the novel expression of these pluripotency genes coupled with the newly acquired ability of these cells to differentiate into all three germ layers was the result of epigenetic changes to these cells after reprogramming in FSFl medium. We confirm that CD45-derived FSFl cells express Oct4 protein at levels similar to that observed among undifferentiated embryonic stem cells, during which time acetylated histones H3 and H4 display increased binding at the promoter region of Oct4. Changes to binding of acetylated histones at Oct4 when these cells are in a differentiated state (either prior to FSFl culture or after in vitro differentiation into neural cells) and when they are undifferentiated suggest that this is one way by which these cells acquire their pluripotency. While DNA hypermethylation at this gene region as well as the continued H3 and H4 acetylation at the CD45 promoter region among FSFl cells indicate this is only a partial reprogramming event, this is a significant step toward non-transgene reprogramming of somatic cells.     

Pluripotency,Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation

Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood. Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics. Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development.