A reprogrammable mouse strain from gene-targeted embryonic stem cells

Abstract

The derivation of induced pluripotent stem cells (iPSCs) usually involves the viral introduction of reprogramming factors into somatic cells. Here we used gene targeting to generate a mouse strain with a single copy of an inducible, polycistronic reprogramming cassette, allowing for the induction of pluripotency in various somatic cell types. As these 'reprogrammable mice' can be easily bred, they are a useful tool to study the mechanisms underlying cellular reprogramming.

Figure 1. An ES-cell based reprogramming system

(a) Genomic configuration of Collagen-OKSM ESCs with the cDNA of the optimized reverse tetracycline-dependent transactivator (M2-rtTA, protein product represented by dark triangles) targeted to the constitutively active ROSA26 locus and a polycistronic cassette encoding Oct4 (O), Klf4 (K), Sox2 (S) and c-Myc (M) targeted to the Col1a1 locus under control of a tetracycline-dependent minimal promoter (tetOP). In the presence of doxycycline (dox) M2-rtTA binds to tetOP, thereby inducing expression of the four reprogramming factors. (b) Outline for a secondary reprogramming system based on Collagen-OKSM ESCs or Lenti-OKSM iPSCs. Chimeric animals are generated by blastocyst injection and ESC-derived or iPSC-derived somatic cells are isolated and reprogrammed into iPSCs by culture in dox-containing media. (c) Brightfield images of a chimera-derived MEF-iPSC clone before picking (P0, left) and after passaging (P3, right). (d) Alkaline phosphatase staining of colonies derived from Collagen-OKSM MEFs or MEFs carrying two copies of a dox-inducible lentiviral vector encoding the same polycistronic cassette (Lenti-OKSM). (e) Graph showing the efficiency of iPSC formation from Collagen-OKSM MEFs (blue bar) and Lenti-OKSM MEFs (green bar), respectively. (f) Fluorescent images of Collagen-OKSM and Lenti-OKSM MEFs cultured for two days in the presence of doxycycline and stained with an antibody against Oct4 (red) and DAPI (blue). Numbers indicate the percentage of Oct4⁺ cells. Scale bars = 100 μm. (g) Histological section through a teratoma derived from Collagen-OKSM MEF-iPSCs showing differentiation in ectodermal (EC, keratinized epithelium), endodermal (EN, glandular structures) and mesodermal (MS, muscle) derivatives. (h) Image of an adult coat color chimera (red arrow) derived from MEF-iPSC. Also shown are two non-chimeric BDF1 mice. (i) Image of an E13.5 embryo derived after injection of MEF-iPSCs into tetraploid blastocysts (“all iPSC” fetus). Scale bars, 500 μm (c), 10 mm (d), 100 μm (f), 50 μm (g), 2 mm (i).

Figure 2. Derivation and characterization of reprogrammable mice

(a) Images of two representative adult chimeras derived from Collagen-OKSM ESCs. (b) Outline for the derivation of reprogrammable mice by crossing ESC chimeras with Oct4-GFP reporter mice. Acquisition of pluripotency in somatic cells isolated from these animals is revealed by GFP fluorescence. (c) Brightfield and GFP fluorescence image of an iPSC colony derived from postnatal tail-tip fibroblasts (TTFs) isolated from a reprogrammable mouse. (d) Effect of serum (FBS) and serum replacement (SR) on the efficiency of iPSC formation from ROSA26-rtTA heterozygous (het, yellow bars) or homozygous (homo, green bars) TTFs. GFP⁺ transgene-independent colonies with ESC-like morphology were scored. Error bars indicate one standard deviation. (e) Effect on cell density on the reprogramming efficiency of homozygous TTFs cultured in either serum (FBS, dotted lines) or serum replacement (SR, solid lines). (f) Efficiencies of reprogramming after culture of ROSA26-rtTA heterozygous and homozygous TTFs in dox-containing medium for different lengths of time. Dox was withdrawn on the indicated days and colonies scored on day 20. (g) Reprogramming efficiencies of mature hematopoietic cell types isolated from the peripheral blood of reprogrammable mice heterozygous (yellow bars) or homozygous (green bars) for ROSA26-M2rtTA. Cells were cultured either in the presence (+) or absence (−) of cell type-specific cytokines (see methods section). (h) Reprogramming efficiencies of hematopoietic stem cells (HSCs) and granulocyte/macrophage progenitor cells (GMPs) from bone marrow. Scale bar, 500 μm (c).