Shuai Gao

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of “4N-ON” and the corresponding “4N-OFF” iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.

The Combination of Tet1 with Oct4 Generates High‐Quality Mouse‐Induced Pluripotent Stem Cells

The DNA dioxygenase Tet1 has recently been proposed to play an important role in the reprogramming of somatic cells to pluripotency. Its oxidization product 5‐hydroxymethylcytosine, formerly considered an intermediate in the demethylation of 5‐methylcytosine, has recently been implicated as being important in epigenetic reprogramming. Here, we provide evidence that Tet1 (T) can replace multiple transcription factors during somatic cell reprogramming and can generate high‐quality mouse induced pluripotent stem cells (iPSCs) with Oct4 (O). The OT‐iPSCs can efficiently produce viable mice derived entirely from iPSCs through tetraploid complementation; all 47 adult OT‐iPSC mice grew healthily, without tumorigenesis, and had a normal life span. Furthermore, a new secondary reprogramming system was established using the OT all‐iPSC mice‐derived somatic cells. Our results provide the first evidence that the DNA dioxygenase Tet1 can replace multiple pluripotency transcription factors and can generate high‐quality iPSCs with Oct4. Stem Cells 2015;33:686–698

Melatonin improves the reprogramming efficiency of murine‐induced pluripotent stem cells using a secondary inducible system

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all‐iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10−6, 10−7, 10−8, 10−9, or 10−10 m) or with vitamin C (50 μg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10−8 (0.81 ± 0.04%), and 10−9 m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT2 was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT1 and MT2 receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis‐related genes p53 and p21 was lower in the group treated with 10−9 m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53‐mediated apoptotic pathway.

Unique features of mutations revealed by sequentially reprogrammed induced pluripotent stem cells

Although viable mice can be generated from induced pluripotent stem cells (iPSCs), the impact of accumulated mutations on the developmental potential of the resulting iPSCs remains to be determined. Here, we demonstrate that all-iPSC mice generated through tetraploid blastocysts complementation can tolerate the accumulation of somatic mutations for up to six generations using a Tet-on inducible reprogramming system. But, the viability of the all-iPS mice decreased with increasing generations. A whole-genome sequencing survey revealed that thousands of single-nucleotide variations (SNVs), including 44 non-synonymous ones, accumulated throughout the sequential reprogramming process. Subsequent analysis provides evidence that these accumulated SNVs account for the gradual reduction in viability of the resultant all-iPSC mice. Unexpectedly, our present reprogramming system revealed that pluripotent stem cells are heterogeneous in terms of possessing a set of copy-number alterations (CNAs). These CNAs are unique for pluripotent cells and subsequently disappear in the differentiating progenies.

Xist Repression Shows Time‐Dependent Effects on the Reprogramming of Female Somatic Cells to Induced Pluripotent Stem Cells

Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X‐inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer studies. In this study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the downregulation of Xist by an isopropyl β‐d‐1‐thiogalactopyranoside (IPTG)‐inducible short hairpin RNA (shRNA) system can greatly impair the mesenchymal‐to‐epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre‐iPSCs to iPSCs in the late phase. Furthermore, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was released from the inactivated X chromosome (Xi). This enables the X chromosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist repression enables the transition from pre‐iPSCs to iPSCs. Stem Cells 2014;32:2642–2656

Impaired imprinted X chromosome inactivation is responsible for the skewed sex ratio following in vitro fertilization.

Significance Sex ratio is an important indicator of reproductive health, and its skewing reflects disturbed embryonic development. This study focused on sex skewing, which has recently identified in human in vitro fertilization (IVF) babies. We reported herein that the skewed sex ratio in mouse IVF offspring was due to the impaired imprinted X chromosome inactivation via suppressing the ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) pathway; the sex skewing can be corrected by overexpressing Rnf12 or by supplementation of retinoic acid in embryo culture medium. Hence, our study not only identified a major epigenetic error responsible for sex skewing in IVF offspring, but also implicated a potential strategy for preventing sex skewing and IVF-associated complications by targeting erroneous epigenetic modifications induced by IVF. Dynamic epigenetic reprogramming occurs during normal embryonic development at the preimplantation stage. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, the skewed sex ratio, an indicator of reproductive hazards, was reported in bovine and porcine embryos and even human IVF newborns. However, since the first case of sex skewing reported in 1991, the underlying mechanisms remain unclear. We reported herein that sex ratio is skewed in mouse IVF offspring, and this was a result of female-biased peri-implantation developmental defects that were originated from impaired imprinted X chromosome inactivation (iXCI) through reduced ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) expression. Compensation of impaired iXCI by overexpression of Rnf12 to up-regulate Xist significantly rescued female-biased developmental defects and corrected sex ratio in IVF offspring. Moreover, supplementation of an epigenetic modulator retinoic acid in embryo culture medium up-regulated Rnf12/Xist expression, improved iXCI, and successfully redeemed the skewed sex ratio to nearly 50% in mouse IVF offspring. Thus, our data show that iXCI is one of the major epigenetic barriers for the developmental competence of female embryos during preimplantation stage, and targeting erroneous epigenetic modifications may provide a potential approach for preventing IVF-associated complications.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers

BackgroundNucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells.ResultsWe generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs’ tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs.ConclusionsThe re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

Generation of Fully Pluripotent Female Murine-Induced Pluripotent Stem Cells

ABSTRACT The high quality of induced pluripotent stem cells (iPSCs) has been determined to be high-grade chimeras that are competent for germline transmission, and viable mice can be generated through tetraploid complementation. Most of the high-quality iPSCs described to date have been male. Female iPSCs, especially fully pluripotent female iPSCs, are also essential for clinical applications and scientific research. Here, we show, for the first time, that a gender-mixed induction strategy could lead to a skewed sex ratio of iPSCs. After reprogramming, 50%, 70%, and 90% female initiating mouse embryonic fibroblasts at different male ratios resulted in 14.1 ± 6.8% (P < 0.05), 31.8 ± 5.4% (P < 0.05), and 80.1 ± 2.8% (P < 0.05) female iPSCs, respectively. Furthermore, these female iPSCs had pluripotent properties typical of embryonic stem cells. Importantly, these fully pluripotent female iPSCs could generate viable mice by tetraploid complementation. These findings indicate that high-quality female iPSCs could be derived effectively, and suggest that clinical application of female iPSCs is feasible.

miR-6539 is a novel mediator of somatic cell reprogramming that represses the translation of Dnmt3b

Global DNA hypomethylation has been shown to be involved in the pluripotency of induced pluripotent stem (iPS) cells. Relatedly, DNA methyltransferases (DNMTs) are believed to be a substantial barrier to genome-wide demethylation. There are two distinct stages of DNMT expression during iPS cell generation. In the earlier stage of reprogramming, the expression of DNMTs is repressed to overcome epigenetic barriers. During the late stage, the expression of DNMTs is upregulated to ensure iPS cells obtain the full pluripotency required for further development. This fact is strongly reminiscent of microRNAs (miRNAs), critical regulators of precise gene expression, may be central to coordinate the expression of DNMTs during reprogramming. Using a secondary inducible system, we found that miR-6539 had a unique expression dynamic during iPS cell generation that inversely correlated with DNMT3B protein levels. Enforced upregulation of miR-6539 during the early stage of reprogramming increased the efficiency of iPS cell generation, while enforced downregulation impaired efficiency. Further analysis showed that Dnmt3b mRNA is the likely target of miR-6539. Notably, miR-6539 repressed Dnmt3b translation via a target site located in the coding sequence. Our study has therefore identified miR-6539 as a novel mediator of somatic cell reprogramming and, to the best of our knowledge, is the first to demonstrate miRNA-mediated translation inhibition in somatic cell reprogramming via targeting the coding sequence. Our study contributes to understand the mechanisms that underlie the miRNA-mediated epigenetic remodeling that occurs during somatic cell reprogramming.

Identification of the new gene Zrsr1 to associate with the pluripotency state in induced pluripotent stem cells (iPSCs) using high throughput sequencing technology.

Finding the markers to predict the quality of induced pluripotent stem cells (iPSCs) will accelerate its practical application. The fully pluripotent iPSCs has been determined as viable all-iPSC mice can be generated through tetraploid (4N) complementation. The activation of the imprinted Dlk1-Dio3 gene cluster was reported to correlate with the pluripotency of iPSCs. However, recent studies demonstrated that the loss of imprinting at the Dlk1-Dio3 locus does not strictly correlate with the reduced pluripotency of iPSCs. In our study (ref [1]), iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was well characterized using tetraploid (4N) complementation assay. The gene expression and global epigenetic modifications of “4N-ON” and the corresponding “4N-OFF” iPSC lines were compared through deep sequencing analysis of mRNA expression, small RNA profiling, histone modifications (H3K4me3, H3K27me3 and H3K4me2) and DNA methylation. Very few differences were detected in the iPSC lines that were investigated. However, an imprinted gene, Zrsr1 was disrupted in the “4N-OFF” iPSC lines. Here we provide more detail about the dataset and the R script with additional data for others to repeat the finding.