Telomere elongation in parthenogenetic stem cells

Dear Editor, 
 
Parthenogenetic embryonic stem (pES) cells, generated from oocytes by artificial activation without involvement of fertilization, show differentiation and pluripotency as evidenced by their capacity to generate germline chimeras and all pES pups by tetraploid embryo complementation, indicating the ability of pES cells to form all cell types in the body (Chen et al., 2009; Liu et al., 2011). Indeed, pES cells can repair injured muscle (Koh et al., 2009) and cardiomyocytes with reduced risk of tumorigenesis (Liu et al., 2013), and contribute to long-term hematopoiesis (Eckardt et al., 2007), supporting the potential applications of pES cells in cell transplantation therapy and tissue engineering (Koh et al., 2009). Furthermore, successful derivation of human pES cells provides important pluripotent stem cell sources alternative to ES cells (or fES, ES cells derived from fertilized embryos) for future clinic therapeutic uses (Mai et al., 2007). Telomere length maintenance is critical for genomic stability, unlimited self-renewal, and developmental pluripotency of ES cells. It remains elusive whether telomeres are sufficiently reprogrammed in pES cells. 
 
We thought to analyze the telomere lengths of pES cells, characteristic of ES cells in morphology (Fig. 1A), in comparison with those of ES cells at similar passages. ES and pES cells were depleted off mouse embryonic fibroblasts (MEF) as feeder prior to harvest for analysis in subsequent experiments. We show that telomeres elongated, and were even slightly longer in pES than in ES cells. Two different pES cell lines (C3 and 1116) exhibited longer telomeres than did ES cells with identical genetic background estimated initially by telomere qPCR analysis (Fig. 1B), and also by quantitative telomere FISH (QFISH) method (Fig. 1C and ​and1D).1D). Moreover, telomeres of pES cells elongated slightly during passages, like those of ES cells (BF10). The telomere QFISH data were generally consistent with relative telomere length expressed as T/S ratio by qPCR. Also, two other pES cell lines generated from oocytes of inbred young C57BL/6 mice displayed telomere maintenance or elongation during passages, like fES cells (N33) (Fig. 1E). Together, telomeres are reprogrammed and sufficiently elongated in pES cells. 
 
 
 
Figure 1 
 
Telomere length and genome-wide gene expression of pES cells versus ES (fES) cells. (A) Colony morphology of pES cells (1116, C3) and fES cells (BF10) at passages 13–15. (B) Relative telomere length expressed as T/S ratio measured by quantitative ... 
 
 
 
To investigate the molecular bases of differential telomere elongation, we performed global gene expression analysis of pES cells, compared with fES cells by microarray. Genes important for development and differentiation showed no or only minimal differences in their expression between pES and fES cells, and were not enriched in the differentially expressed gene lists (Tables S1 and S2). Expression of genes associated with pluripotency of ES cells, such as Pou5f1 (Oct4), Nanog, Sox2, and Rex1 did not differ among these three cell lines. Major telomerase genes Tert and Terc also did not show differential expression between pES and ES cells. 
 
Interestingly, most of the up-regulated genes in both pES cell lines were enriched in 2-cell embryo state, including Tcstv1/3, Dub1, Eif1α, Gm4340, and Zscan4 (Zalzman et al., 2010; Macfarlan et al., 2012). Differential gene expression profile also was found between two pES cell lines, but pES cells 1116 closely resembled ES cells more than did pES cells C3 (Fig. 1F). For instance, Lefty1 and Stella (also known as Dppa3), expressed at reduced levels in pES cells C3, compared with pES 1116 or ES cells. Coincidently, chimeras generated from pES 1116 but not C3 showed germline competency (Liu et al., 2011). The microarray data were validated by qPCR analysis of selected genes, although the fold in relative expression levels showed some differences for a few genes (Fig. 1G). 
 
By immunofluorescence microscopy, Zscan4 was expressed sporadically in only small proportion (1%–5%) of ES cell cultures, consistent with the reports (Zalzman et al., 2010; Macfarlan et al., 2012). While some of Zscan4 positive ES cells were excluded from Oct4 expression, a few others showed weak positive staining for Oct4 (Fig. 2A). In addition, the proportion of Zscan4 positive cells was increased in pES cells, with higher ratio in pES C3 by both flow cytometry and immunofluorescence microscopy quantification (Fig. 2A–C). The protein levels of Zscan4 also were higher in pES cells than in ES cells, and highest in pES cell C3 (Fig. 2D), consistent with flow cytometry and immunofluorescence quantification data. Moreover, murine endogenous virus element (MERV) expressed at higher levels in pES than in ES cells by qPCR (Fig. S1A and S1B). 
 
 
 
Figure 2 
 
Involvement of Zscan4 in telomere elongation of pES cells. (A) Immunofluorescence images of Zscan4 (red) and Oct4 (green) in pES (1116 and C3) versus fES cells (BF10) at passage 15–16. Nuclei stained with Hoechst 33342 (blue). (B) Analysis of ... 
 
 
 
Zscan4 was shown to lengthen telomeres of ES cells presumably via recombination based mechanism (Zalzman et al., 2010). To examine whether elevated levels of Zscan4 also are implicated in telomere elongation of pES cells, we knocked down Zscan4 in two pES cell lines by two independent shRNAs effectively targeting Zscan4 (Fig. 2E). Depletion of Zscan4 shortened telomeres in both pES cell lines estimated by QFISH as well as qPCR (Fig. 2F and ​and2G),2G), suggesting that Zscan4 might involve in telomere elongation of pES cells. However, various telomere lengths of pES cells (Fig. 1B–D), did not completely correlate with absolute Zscan4 protein levels (Fig. 2D), suggesting that genes other than Zscan4 may also play roles in telomere elongation and self-renewal of pES cells. 
 
Moreover, epigenetic modifications at telomeres and subtelomeres regulate telomere lengths (Blasco, 2007). Active histones H3K4me3, H3K9Ac, H3Ac and repressive histone H3K27me3 mostly enriched in euchromatin did not show noticeable differences in their protein levels between pES and ES cells, whereas heterochromatic repressive H3K9me3 levels were reduced in pES cells compared with ES cells (Fig. S1C). Lower levels of H3K9me3 may de-repress Zscan4 and Tcstv1 located at subtelomeres. 
 
Reduced MAPK and increased Wnt Signaling are implicated in derivation, self-renewal, and pluripotent state of mouse ES cells. Parthenogenetic ‘blastocysts’ display reduced MAPK and increased Wnt signaling (Liu et al., 2010) and this may contribute to more efficient derivation of pES cells in mice (Chen et al., 2009) as well as in human (Mai et al., 2007). Membrane β-catenin was found in both pES and ES cells (Fig. S2A), and nuclear β-catenin protein expressed at relatively higher levels in pES than in ES cells (Fig. S2B), although total β-catenin levels seemed not to differ between the two cell lines (Fig. S2C). Erk levels appeared slightly lower in pES 1116, relative to ES cells, but much lower in pES C3 (Fig. S2C), which showed highest Zscan4 levels. 
 
Factors in oocytes or early cleavage embryos presumably contribute to both rapid telomere reprogramming and epigenetic reprogramming during early embryo development. These critical factors can be exploited to improve reprogramming induction of iPS cells. Notably, telomeres elongate slowly during iPS cell induction and gradually acquire ES cell telomere lengths during continued passages (Wang et al., 2012), different from rapid telomere lengthening in early cleavage embryos (Liu et al., 2007). Our data suggest that increased expression of 2-cell genes particularly Zscan4 may partially explain telomere elongation of pES cells. Also, Zscan4 proves to rapidly lengthen telomeres and greatly enhance genomic stability and quality of iPS cells (Jiang et al., 2013). Multiple factors can influence pluripotency and germline competency of ES/iPS/pES cells. The functional significance of telomere elongation in pES cells or slightly longer telomeres in pES cells relative to ES cells remains unclear, but speculatively the robust telomere maintenance may help maintenance of self-renewal of pES cells in vitro. 
 
In summary, pES cells generated from parthenogenetically activated oocytes exhibit telomere elongation or even slightly longer telomeres compared with fES cells. Without complication of sperm factors, parthenogenetically activated oocytes themselves can effectively elongate telomeres during early cleavage development (Liu et al., 2007). Consistently, somatic cell nuclear transfer (SCNT) using oocytes further improves chromatin remodeling and telomere elongation of iPS cells (Liu et al., 2012). SCNT efficiency in cloning human embryos has been remarkably improved recently with success at long last, such that human SCNT ES cells now are effectively achieved (Tachibana et al., 2013). It would be interesting to further explore whether telomeres are effectively elongated to maintain genomic stability of SCNT ES cells achieved using oocyte factors.

mean centering and normalization of genes and arrays before average linkage clustering.

Gene expression analysis by quantitative real-time PCR
Total RNA was isolated using TRIzol Reagent (Invitrogen). 2µg of RNA was subject to cDNA synthesis using M-MLV Reverse Transcriptase (Invitrogen). PCR reaction was set up in duplicates using the FastStart Universal SYBR Green Master (ROX, Roche) and run on the iCycler real-time PCR detection system (Bio-Rad) using primer sets specific for each gene (Supplementary Table 3).
For nuclear protein extraction, 2×10 6 cells were harvest and treated with 200 μl ice-cold lysis buffer A (10 mM Hepes-NaOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , added with 1,0 mM protease inhibitor cocktail and 1.0 mM PMSF prior to use) and incubated on ice for 15 min, then added with 10 μl buffer B (10% NP-40), vortex and centrifuged. The supernatant was discarded and the pellet washed with ice-cold buffer A and add 50 μl nuclei lysis buffer C (20 mM Hepes-NaOH (pH 7.9), 420 mM NaCl, 3 mM EDTA, 1 mM DTT 2mM MgCl 2 , 25% Glycerol, added with 1,0 mM protease inhibitor cocktail and 1.0 mM PMSF prior to use). The pellet dispersed and put on ice for 30 min, vortex for 15 s every 3 min, centrifuged at 13000g for 15 min, and the nuclear proteins were extracted.

Zscan4 RNAi
For stable knockdown of Zscan4, shRNA sequences (Zalzman et al., 2010) were synthesized and cloned into pSIREN-retroQ RNAi vector according to manufacturer's instructions. The negative control shRNA without sequence homology to mouse genes served as negative control. The RNAi retrovirus was packaged using Plat-E cells and then infected pES cells. 48h after infection, the cells were selected using 2 µg/ml puromycin for 7 days, and clones were picked.

Telomere QFISH
Telomere length and function (telomere integrity and chromosome stability) was estimated by telomere quantitative FISH. Cells were incubated with 0.5 μg/ml nocodazole for 1.5 h to enrich cells at metaphases (Liu et al., 2007). Chromosome spreads were made by standard method. Metaphase-enriched cells were exposed to hypotonic treatment with 75 mM KCl solution, fixed with methanol: glacial acetic acid (3:1) and spread onto clean slides. FITC-labeled (CCCTAA) peptide nucleic acid (PNA) probe was used in this study. Telomeres were denatured at 80 ºC for 3 min and hybridized with telomere PNA probe (0.5μg/ml) (Panagene, Korea). Chromosomes were stained with 0.5μg/ml DAPI. Fluorescence from chromosomes and telomeres was digitally imaged on a Zeiss microscope with fluorescein isothiocyanate (FITC)/DAPI filters, using AxioCam and AxioVision software 4.6. Telomere length shown as telomere fluorescence intensity was integrated using the TFL-TELO program (a gift kindly provided by Peter Lansdorp, Terry Fox Laboratory).

Telomere measurement by quantitative real-time PCR
Cells were washed in PBS and stored at -20ºC until subsequent DNA extraction. Genome DNA was prepared using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). Average telomere length was measured using real-time PCR assay, as previously described, but 5 modified for measurement of mouse telomeres (Liu et al., 2007). PCR reactions were performed using the iCycler iQ real-time PCR detection system (Bio-Rad), using telomeric primers, primers for the reference control gene (mouse 36B4 single copy gene).
For each PCR reaction, a standard curve was made by serial dilutions of known amounts of DNA. The telomere signal was normalized to the signal from the single copy gene to generate a T/S ratio indicative of relative telomere length. Equal amounts of DNA were used for each reaction.

Statistics
Data were analyzed by analysis of variance and means compared by Fisher's protected least-significant difference using the StatView software from SAS Institute Inc. (Cary, NC), a value of P<0.05 was considered statistical significance.  Nuclear extracts were prepared for the analysis at passage 15~16. H3 served as loading control. (C) Protein levels of total ß-catenin, Erk and p-Erk in fES cells (BF10) and pES cells (1116 and C3) at passage 15~16. Whole cell lysis was used for the western-blot analysis. ß-Actin served as loading control.