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

Abstract

BackgroundGiven 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 FindingsiPS 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/SignificanceGiven 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.

Inhibition of Protein Kinase C Signaling Maintains Rat Embryonic Stem Cell Pluripotency*

“…Later, cells were maintained without Dox in the PKCi culture condition. We also used the 2i/LIF culture condition as a positive control because earlier studies reported the successful establishment of riPSC cells with the 2i/LIF culture condition from differentiated rat cells (16,18). We found that the efficiency to derive rat iPSCs in the 2i/LIF condition is very low (two of five attempts were successful in the Weiss laboratory and zero of four attempts in the Paul laboratory, data not shown).…”

Generation of Rat Induced Pluripotent Stem Cells Using a Plasmid Vector and Possible Application of a Keratan Sulfate Glycan Recognizing Antibody in Discriminating Teratoma Formation Phenotypes

“…Chang et al reported differentiation of rat iPS cells into neurons of which approximately 10% were dopamine neurons, but the nature of the remaining population was not demonstrated. 10) To our knowledge, this is the first work identifying a population of glutamate neurons from riPSCs and furthermore demonstrating the neurons were responsive to agonist mediated stimulation. Indeed, it would be of great interest to study pharmacological glutamate neurotransmitter signaling using riPSCs derived neurons.…”

“…We subjected riPSCs to a multistep protocol 10) with modifications for converting iPSCs into neurons (Fig. 4A).…”

Direct Comparison of DNA versus mRNA on the Efficiency of Induced Pluripotent Stem Cells Generation from Human Primary Fibroblasts

“…As is has been previously proved that OSK combination is sufficient to successfully induce pluripotency on human fibroblasts [6,12,13] the aim of this work is only to compare the efficiencies obtained with the three proposed induction methods, and demonstrate that IRES structures in between the genes arrangement allows different levels of protein production in either version of ribonucleic acid used. Then, cells that are easily obtained from patients and have reliable dedifferentiation capabilities are desirable for a non-invasive patient-specific approach.…”

“…However, many different combinations have been analyzed [2][3][4][5][6][7][8][9][10][11]. Even though efficiencies are still low, promising results have been obtained with the Oct4, Sox2 and Klf4 (OSK) combination [6, 12,13]. It is known that TFs stoichiometry influences dedifferentiation…”