Telomere dynamics in human cells reprogrammed to pluripotency - PubMed (original) (raw)

Telomere dynamics in human cells reprogrammed to pluripotency

Steven T Suhr et al. PLoS One. 2009.

Erratum in

Abstract

Background: Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell "rejuvenation."

Methodology/principal findings: We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres.

Conclusions/significance: While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Lentiviral vectors used in IPSC production.

LTR: viral LTRS, EF1A-IN: elongation factor 1-alpha promoter, PGK: PGK promoter, M: cMyc, O: Oct4, S: Sox2, K: KLF4, L: Lin28, N: Nanog transgenes. 2A: translation interruption element, IRES: internal ribosomal entry site, Puro: puromycin resistance gene.

Figure 2

Figure 2. Phase-contrast images of FIB and IPSC lines used in this report (as labeled).

FIBA and FIBB displayed a flat stellate cytoplasm with irregular edges characteristic of fibroblast cell types (200X). IPSC lines displayed round colonies with regular edges evident in low magnification 40X images that were composed of tightly packed cells with prominent nucleoli (200X, lower panels of IPSC lines) that appeared morphologically homogenous within the center of the colony and flattened toward the edges where they were bounded by the MEF feeder layer. IPSCB1 and IPSB2 are shown only at 40X magnification.

Figure 3

Figure 3. Indicators of cell phenotype and pluripotency in IPSC lines.

(A) Immunofluorescent analysis of pluripotency markers as labeled. Label color corresponds to marker color in images. Blue staining in all panels is DAPI labeling of nuclei. All IPSC lines displayed strong positive expression of pluripotency markers that were not observed at high levels in input FIB cells with the exception of the Lin28 antibody that reproducibly produced faint fluorescence in the cytoplasm of fibroblasts. (B) Representative QPCR analysis of FIB lines and reprogrammed IPSCs for markers of pluripotency and reprogramming factors relative to factor levels in hESCs. Input fibroblasts express very low levels of most factors whereas IPSCs display levels very similar to hESCs.

Figure 4

Figure 4. Indicators of pluripotency and differentiation in IPSC lines.

(A) Representative sections from teratomas generated from IPSC lines as labeled. Arrows in ectodermal (ECTO) tissue indicate neural rosettes, in mesodermal tissue (MESO) indicate cartilage, and in endodermal tissue (ENDO) indicate glandular columnar epithelium. mag. 100X. (B) Morphology of FIB lines and TER lines, as labeled, in phase-contrast images (top row) and stained for fibronectin (red, bottom row). mag. 400X. Blue is DNA stain. (C) Genome-wide methylation heat map and cluster analysis of representative input fibroblasts, IPSCs, TER, and hESC lines indicating that the overall pattern of methylation of IPSCs closely matches HES lines while differentiated TER lines cluster with input fibroblasts. Value indicates the level of methylation ranked from 0 (hypomethylated) to 1 (hypermethylated).

Figure 5

Figure 5. Analysis of telomeres in input, pluripotent, and re-differentiated cell lines.

(A) Blue-stained nuclear DNA with punctate red fluorescence indicating telomeres hybridized to the PNA FISH telomere probe for input fibroblasts, IPSCs, or TER cells from lines A and B as labeled. mag. 400X. (B) Quantification of TRFs from cell lines as labeled. Yellow shading indicates significance compared to line FIBA and red shading significance compared to line FIBB. TRF indicates mean length in kilobase pairs (Kb). * = P<0.05, ** = P<0.01, *** = P<.001. Error bars indicate SEM. (C) Example of changes in TRF size in cell lines as labeled. Numbers at left are fragment lengths in kilobases. (D) Mean TRF lengths for all cell lines combined by type. Labeling as in part B. Significance calculated relative to FIB.

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