Different telomere-length dynamics at the inner cell mass versus established embryonic stem (ES) cells

Abstract

Murine embryonic stem (ES) cells have unusually long telomeres, much longer than those in embryonic tissues. Here we address whether hyper-long telomeres are a natural property of pluripotent stem cells, such as those present at the blastocyst inner cell mass (ICM), or whether it is a characteristic acquired by the in vitro expansion of ES cells. We find that ICM cells undergo telomere elongation during the in vitro derivation of ES-cell lines. In vivo analysis shows that the hyper-long telomeres of morula-injected ES cells remain hyper-long at the blastocyst stage and longer than telomeres of the blastocyst ICM. Telomere lengthening during derivation of ES-cell lines is concomitant with a decrease in heterochromatic marks at telomeres. We also found increased levels of the telomere repeat binding factor 1 (TRF1) telomere-capping protein in cultured ICM cells before telomere elongation occurs, coinciding with expression of pluripotency markers. These results suggest that high TRF1 levels are present in pluripotent cells, most likely to ensure proficient capping of the newly synthesized telomeres. These results highlight a previously unnoticed difference between ICM cells at the blastocyst and ES cells, and suggest that abnormally long telomeres in ES cells are likely to result from continuous telomere lengthening of proliferating ICM cells locked at an epigenetic state associated to pluripotency.

Fig. 1.

Blastocyst ICM bears the longest telomeres which further lengthen upon expansion of ICM-derived ES cells. (A) Quantification of telomere length by telomapping analysis of embryo sections at the indicated stages of development, ES cells (passage 9) and primary MEFs (passage 2). Telomere-length quantification is given in arbitrary units of fluorescence (a.u.). n = number of embryos or independent cell cultures. (B) (Top) Representative image of a telomapping of a blastocyst section. Nuclei are colored according to their telomere length and normalized by the telomere length of ES cells. Because ES cells are derived from the ICM we reasoned that they should have equivalent telomere length. The division of the CY3 intensity value of each blastocyst cell by the mean CY3 intensity value of ES cells should render the blastocyst cells with the longest telomeres (values around or equal to 1). For the blastocyst map we grouped intensity values in three fractions to simplify the identification of the cells with the longest telomeres. Note that the longest telomeres localize to the ICM of the blastocyst. (Scale bar, 10 μm.) (Lower) Quantification of telomere length of blastocyst, ES cells and MEFs, as indicated. n = number of embryos or independent ES and primary MEF cultures. (C) Telomere-length frequency histograms of blastocysts, ES cells at the specified passages, and primary MEFs and mean telomere length for the same samples. Note that the telomere length of ES at early passages is similar to that found in the ICM. n = number of embryos or independent ES or primary MEFs cultures. (D) Scheme of the process of isolation of ES cells from blastocysts. In brief, zona pellucida is removed from blastocysts and they are transferred to a 60-mm dish. After 4 to 6 d the ICM has divided to ∼1,000 cells. Individual ICM colonies are transferred to a 96-well plate. At this step, ES colonies emerge and are transferred to a 24-well plate for expansion. From the 24-well plate, cells are transferred to 25-mm plates and are considered passage 1. Further passages are plate colonies or ES and primary MEF cultures. (E) Mean telomere length and telomere-length frequency histograms in ICM from the blastocyst, in vitro cultured ICM, emerging ES from the 96-well plate, established ES cells (passages 5, 9, and 12), iPS cells (passage 1 and 29), and primary MEFs determined by telomapping. Note that telomeres of the ICM at the blastocyst are longer than those of the cultured ICM.

Fig. 2.

Telomere-length dynamics during establishment and expansion of ES cell lines as well as in vivo aggregation of ES cells in morulae. (A) Mean telomere length for ICM cultivated from the 60-mm tissue-culture plate, and successive passages of ES cells. Telomere length was analyzed by metaphase Q-FISH. n = number of ICM colonies or independent ES and primary MEF cultures. (B) Scheme of the aggregation experiments. Established ES cells at passage 16 expressing GFP were microinjected in eight-cell morulae. Blastocyst from injected and noninjected morulae were fixed for the analysis of telomere length by telomapping. (C) Mean telomere length for primary MEFs (passage 2), noninjected and injected blastocysts, as well as GFP-ES cells before injection (passage 16) and ES cells at passage 9. n = number of blastocysts or independent clones of ES cells or primary MEFs. (D) Representative images of an injected blastocyst.

Fig. 3.

The loss of heterochromatic marks accompanies telomere lengthening. (A) Mean H4k20me3 intensity for primary MEFs (passage 2), in vitro cultured ICM, cells from the 96-well plate, and established ES cells at passage 9. (Lower graphs) The H4k20me3 histograms for the same samples. Note that in the ICM as well as in the 96-well plate there are cells with low H4k20me3 signals. (B) Percentage of cells with less than 7 arbitrary units of H4k20me3 fluorescence. Note the portion of cells with low methylation signal in both the cultured ICM and the 96-well plate. (C) Colocalization of the H4k20me3 heterochromatic mark with telomeres in percentage for the samples described in A. (D) Representative images of telomeres and H4k20me3 signals for the samples described in A. (E) Mean H3k9me3 intensity and histograms for the samples described in A. (F) Percentage of cells with less than 7 arbitrary units of H3k9me3 fluorescence. Note the portion of cells with low methylation signal in the cultured ICM and the 96-well plate. (G) Percentage of colocalization of the H3k9me3 heterochromatic mark with telomeres for the samples described in A. (H) Representative images of telomeres and H3k9me3 signals for the samples described in A. n = number of ICM or 96-well plate colonies or independent ES, and primary MEF cultures. Arbitrary units of H4k20me3 fluorescence is plotted. (Scale bars, 10 μm.)

Fig. 4.

Analysis of TRF1 and Oct4 expression during isolation of ES cells. (A) Mean TRF1 intensity for primary MEFs (passage 2), in vitro cultivated ICM, 96-well plate emerging ES cells, and established ES-cell lines at passage 9 analyzed by telomapping. (B) (Left) Mean Oct3/4 intensity for the same samples described in A. (Right) Percentage of cells with Oct3/4 intensity bigger than 20 a.u. Note that at the cultured ICM stage, a 38% of cells are Oc3/4 positive. (C) Representative images of TRF1 and Oct3/4 expression for the same samples described in A. (Scale bars, 10 μm.) n = number of ICM or 96-well plate colonies or ES and primary MEF cultures. (D) Mean TRF1 intensity values for primary MEFs, the cell line L5178Y-R or R cells, cultured ICM, and ES passage 9. (E) Mean Oct3/4 intensity for the samples described in D. (F) TRF1 expression frequency histograms corresponding for the samples described in D. (G) Oct3/4 expression frequency histograms corresponding to the samples described in D. (H) Representative images of TRF1 and Oct3/4 expression for the samples described in D. (Scale bars, 10 μm.) (I) TRF1 intensity values plotted against Oct3/4 intensity values to analyze correlation. Primary MEFs, cultured ICM, and established ES cells at passage 9 are shown in the Upper panels. (Lower) Cells from the cultivated ICM were divided in high or low TRF1 intensity for the analysis. n = number of ICM colonies or L5178Y-R, ES, primary MEF cultures.