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. 2017 Jun 21;63(3):311-318.
doi: 10.1262/jrd.2016-164. Epub 2017 Mar 23.

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions

Affiliations

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions

Masafumi Katayama et al. J Reprod Dev. .

Abstract

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

Keywords: Cellular senescence; Immortalized cells; Induced pluripotent stem cell (iPSC); Pluripotency; Prairie vole.

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Figures

Fig. 1.
Fig. 1.
Reprogramming vector schematics and their expression in primary and immortalized prairie vole cells. (A) Schematics of the six vectors used in this study. EF1: elongation factor 1 synthetic promoter; IRES: internal ribosomal entry site; GFP: green fluorescent protein; LTR: long terminal repeat; CMV: cucumber mosaic virus promoter; MCS: multicloning site; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; CR4: CR4 enhancer; ETn: early transposon element; TAD: transactivation domain; CAG: synthetic promoter composed of the cytomegalovirus (CMV) early enhancer element and the chicken beta-actin promoter; PGK: phosphoglycerate kinase promoter. (B) Infection of primary prairie vole-derived cells with the lentiviral STEMCCA and pCDH-CMV-MCS-EF1-copGFP (copGFP) vectors. Upper panels: STEMCCA vector (negative control). Lower panels: copGFP vector (surrogate marker). Scale bar = 300 μm. (C) Infection of immortalized prairie vole VMF-K4DT cells with pCDH-CMV-MCS-EF1-copGFP (copGFP) vector. Scale bar = 300 μm. (D) Detection of EOS reporter expression in immortalized prairie vole cells following the introduction of four reprogramming factors via the STEMCCA vector. Upper panels: infection-free controls. Lower panels: STEMCCA vector infected cells. Arrows indicate EOS reporter-positive cells. Scale bar = 300 μm. (E) Western blots showing protein expression of the indicated exogenous reprogramming factors, following transfection of 293T cells with the PB-EF1-6F vector. Lanes 1 and 2: infection-free controls; lanes 3 and 4: PB-EF1-6F vector-infected cells. (F) Generation of murine iPSCs. Scale bar = 50 μm.
Fig. 2.
Fig. 2.
Establishment of iPSCs from immortalized prairie vole-derived cells using the PB-CAG-6F reprogramming vector. (A) Schematic representation of the reprogramming vectors, showing the replacement of the EF1 promoter with a CAG promoter for the expression of reprogramming factors. (B) Schematic representation of the experimental time course using the PB-CAG-6F reprogramming vector. (C) Morphology of primary iPSC colonies derived from immortalized prairie vole cells transformed with the PB-CAG-6F reprogramming vector. a: GFP-positive iPSC colony. b: GFP-negative iPSC colony. Scale bar = 300 μm. (D) Low levels of GFP expression in late passage iPSCs derived from immortalized prairie vole cells transformed with the PB-CAG-6F reprogramming vector. Scale bar = 300 μm. (E) Upper panels: morphology of iPSC colonies obtained from immortalized prairie vole cells using the PB-CAG-6F vector. Lower panels: AP staining of iPSCs established from immortalized prairie vole-derived cells using the PB-CAG-6F reprogramming vector. Left panels: low magnification. Scale bar = 500 μm. Right panels: high magnification. Scale bar = 100 μm.
Fig. 3.
Fig. 3.
Establishment of iPSCs from immortalized prairie vole-derived cells using the PB-R6F reprogramming vector. (A) Schematic representation of the experimental time course using the PB-R6F reprogramming vector. (B) Morphology of primary iPSC colonies established from immortalized prairie vole cells using the PB-R6F vector. a: merged images; b: green fluorescent images using GFP; c: bright field images. Scale bar = 300 μm. (C) Morphology of established iPSC colonies generated from immortalized prairie vole cells using the PB-R6F vector. a: merged images; b: green fluorescent images using GFP; c: bright field images. Scale bar = 300 μm. (D) Upper panels: morphology of iPSC colonies established from immortalized prairie vole cells using the PB-R6F vector. Lower panels: AP staining of these iPSC colonies. Left panels: low magnification. Scale bar = 500 μm. Right panels: high magnification. Scale bar = 50 μm.
Fig. 4.
Fig. 4.
Formation of teratomas from iPSCs derived from immortalized prairie vole cells transformed with the PB-6F reprogramming vector. (A) Photographs of teratomas derived from iPSCs that were established from immortalized prairie vole cells using the PB-6F vector. a: gross appearance of the teratoma tissue; b: dissected teratoma. (B) Histological analysis of the teratomas shown in (A). a: gastrointestinal tract-like tissue (endoderm, scale bar = 50 μm); b: adipose-like tissue (mesoderm, scale bar = 100 μm); c: neural tube-like tissue (ectoderm, scale bar = 50 μm).

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