Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector - PubMed (original) (raw)

Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector

Federico Gonzalez et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Induced pluripotent stem (iPS) cells have generated keen interest due to their potential use in regenerative medicine. They have been obtained from various cell types of both mice and humans by exogenous delivery of different combinations of Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28. The delivery of these transcription factors has mostly entailed the use of integrating viral vectors (retroviruses or lentiviruses), carrying the risk of both insertional mutagenesis and oncogenesis due to misexpression of these exogenous factors. Therefore, obtaining iPS cells that do not carry integrated transgene sequences is an important prerequisite for their eventual therapeutic use. Here we report the generation of iPS cell lines from mouse embryonic fibroblasts with no evidence of integration of the reprogramming vector in their genome, achieved by nucleofection of a polycistronic construct coexpressing Oct4, Sox2, Klf4, and c-Myc.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Generation of iPS cells by transfection of a single nonviral polycistronic construct. (A and B) Maps of the polycistronic constructs used in this study. (C) Expression of transgene mRNA relative to GAPDH in transfected (T) and nontransfected (NT) fibroblasts by real-time RT-PCR. (D) Western blot analysis of transgene factor expression in T and NT cells. (E and F) Timeline of iPS production with either 1 (E) or 2 (F) nucleofections.

Fig. 2.

Fig. 2.

Integration analysis of iPS cell lines. (A) Linear representation of pCAG-OSKM showing the location of the probes used for Southern blot (red bars, i–iv) and the approximate position and length of the amplicons generated by PCR (gray bars 1–22). (B) Southern blot analysis of clones 6, 11, 16, 36, 38, and 41, and mouse ES cells using probes against Oct4, Sox2, Klf4, and c-Myc. Black and gray arrowheads point respectively to specific and nonspecific endogenous bands present in the genomic DNA of control ES cells. Extra bands are highlighted by a red asterisk in clones 6, 16, and 36, indicating variable degrees of insertion of the transgene. (C) PCR analysis of clones 6, 11, 16, 36, 38, 41. Consistent with Southern blot, clones 6 and 36 are positive for almost the full set of primer pairs, whereas clone 16 is not positive for the backbone-specific primers 2–9. Clone 11 shows a weak signal for primer pairs 3, 9, 12, and 20, whereas clones 38 and 41 are negative for all primer pairs tested except faint bands for primer pairs 17 and 20.

Fig. 3.

Fig. 3.

Characterization of integrative and NiPS cell clones: comparison with MEFs and mouse ES cells. (A Upper) Real-time RT-PCR showing up-regulation of endogenous expression of the pluripotency markers Oct4, Sox2, Nanog, Utf1, and Zfp41 (Rex1). (Lower Left) Real-time RT-PCR showing down-regulation of expression of the fibroblast markers Col6a2, Grem2, and Thy1. (Lower Right) Real-time RT-PCR showing level of silencing of the transgene, if present. (B) Oct4 promoter methylation analysis. Percentages of methylation of the Oct4 promoter are indicated in red. (C) Immunofluorescence analysis showing expression of the ES cell markers NANOG, SOX2, OCT4, and SSEA1.

Fig. 4.

Fig. 4.

Differentiation potential of iPS cell lines. (A) In vitro differentiation toward ectoderm (i–iii) (TuJ1-positive neuronal cells, green), mesoderm (iv–vi) (α-actinin-positive cardiac myocytes, red), and endoderm (vii–ix) (α-fetoprotein-positive, green; FOXA2-positive, red). Blue nuclear staining is DAPI. Scale bars, 25 μm. (B) In vivo differentiation (teratomas) toward ectoderm (i–iii) (TuJ1-positive neuronal cells, green; glial fibrillary acidic protein-positive cells, red), mesoderm (iv–vi) (α-actinin-positive cardiac myocytes, green), and endoderm (vii–ix) (α-fetoprotein-positive, green; FOXA2-positive, red). Scale bars: iv, 25 μm; all others, 50 μm; (C) In vivo differentiation (blastocyst injection) of chimeric (Top and Middle) and control (Bottom) pups obtained by injecting NiPS cell clone 41 in a ICR host blastocysts. Black hair is from iPS cells.

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