Effects of Cellular Extract on Epigenetic Reprogramming

Abstract

Functional reprogramming of a differentiated cell toward pluripotent cell may have long-term applications in numerous aspects, especially in regenerative medicine. Evidences accumulating from recent studies suggest that cellular extracts from stem cells or pluripotent cells can induce epigenetic reprogramming and facilitate pluripotency in otherwise highly differentiated cell types. Epigenetic reprogramming using cellular extracts has gained increasing attention and applied to recognize the functional factors, acquire the target cell types, and explain the mechanism of reprogramming. Now, more and more researches have proved that cellular extract treatment is an important strategy of cellular reprogramming. Thus, this review mainly focused on the progresses and potential mechanisms in epigenetic reprogramming using cellular extracts.

Introduction

The process of inducing differentiated cells to a pluripotent status or to another unrelated cell type is referred to as cellular reprogramming. This can be achieved through the approaches of cell fusion, somatic cell nuclear transfer (SCNT), specific transcription-factor transduction (such as Oct4, Sox2, and Klf4), and treatment with kinds of cellular extracts. Successful cloning by SCNT, regarded as a reliable tool for studying nuclear reprogramming, involves a maternal-zygotic transition in gene expression from a differentiated somatic cell pattern to an embryonic form during the early development stage.

Such epigenetic reprogramming of gene expression is thought to require the modification of somatic cells chromatin, and often appear faulty and/or incomplete chromatin remodeling. That may contribute to the aberrant genes' expression and abnormal development in SCNT embryos and individuals. Furthermore, the SCNT method has several defects for studying the potential mechanisms of epigenetic reprogramming, and requires complicated processes of embryonic manipulation.

A cell-free system of oocyte extracts is an attractive way to mimic epigenetic reprogramming in vitro, as a large number of cells could be treated and analyzed simultaneously. Such cell-free system has been established in Xenopus laevis (Miyamoto et al., 2008; Yang et al., 2012), Drosophila melanogaster (Ulitzur and Gruenbaum, 1989), and other nonmammalian and mammalian species (Betthauser et al., 2006; Liu et al., 2014).

This cell-free system is beneficial for studying the corresponding mechanisms of embryonic development and epigenetic reprogramming (Sangalli et al., 2012), especially for these samples that are not enough for biological analysis, as this system can treat a large number of cells at the same time. Cumulative evidences suggested that cellular extracts derived from stem cells or pluripotent cells would be beneficial for understanding the molecular mechanisms of epigenetic reprogramming in mammalian species. Thus, this review mainly focused on the kinds of cellular extract, the effects of cellular extract, and the potential mechanism in cellular reprogramming using cellular extracts.

Kinds of Cellular Extract

Cellular extract of oocytes

Since the first successful production of the cloned sheep, lots of factors, which are necessary for the nuclear reprogramming of somatic cells in oocytes, have been explored in previous reports. The birth of live animals after SCNT has demonstrated that the genome of differentiated cells can be induced modification by mammalian oocytes. The main reason for the current speculation is that some factors in the oocyte can be induced epigenetic reprogramming of differentiated cells. However, these factors and mechanisms responsible for epigenetic reprogramming remain unclear despite numerous attempts.

In SCNT of mammals, matured MII (metaphase II) oocytes were usually used as recipient cells, which suggested that oocytes may be a reasonable source for extracts to induce epigenetic reprogramming. In contrast, germinal vesicles (GVs) stage oocytes were considered to be inadequate for using as recipient cells (Gao et al., 2002). However, there is a little evidence that Oct4 in the nuclear of mouse somatic cells was induced expression by components within GV stage Xenopus oocytes (Byrne et al., 2003), which suggesting that GV oocytes also as a potential cell type to induce epigenetic reprogramming.

Previous studies showed that the reprogramming efficient of oocytes extracts have distinctive and advance to reprogram differentiated cells, which are different with the defined transcription factors reprogram in induced pluripotent stem (iPS) cells (Farthing et al., 2008; Morgan et al., 2005). Bui et al. (2008) treated somatic cells with mouse GV stage oocyte extracts, and found that histone H3K9 of somatic cells was completely demethylated, H3K9 and H3K14 partially deacetylated, and significantly promoted the subsequent development potential of cloned embryos. Thus, it is believed that there are also many factors in GV stage oocytes that play an important role in reprogramming.

Cellular extract of stem cells and cancer cells

As the sources of oocytes are extremely limited and oocyte cannot proliferate in vivo or in vitro, other cells were used for extract. The pluripotency cell lines might be as a good material for preparing cellular extract to analyze its mechanism associated with differentiation and epigenetic reprogramming. NIH/3T3 cells can be dedifferentiated by the extracts derived from teratocarcinoma cells and made modified express transcription factors associated with totipotent (Zhang et al., 2012). It has also shown that exposed T cells extract has altered the cell fate of 293T (Håkelien et al., 2004). Taranger et al. (2005) treated 293T cells with an extract from human NCCIT carcinoma cells, and found that they form more defined colonies and maintained for >23 passages in vitro.

Furthermore, the 293T cells after treatment express embryonic stem cell-specific markers Oct4, Klf4, Sox2, and Nanog, and have multidirectional differentiation potential, which can be differentiated into nerve cells, endothelial cells, fat cells, and so on under appropriate conditions. In 2008, researchers used mouse embryonic stem cell extracts to treat NIH3T3 cells, then dedifferentiated and successfully differentiated into skeletal muscle cells, epithelial cells, and cardiomyocytes under specific induction conditions (Rajasingh et al., 2008). Han et al. (2010) found that fetal fibroblasts cell dedifferentiated more effectively after human embryonic stem cell extract treatment assisted with DNA methyltransferase and histone deacetylase inhibitors.

All these observations demonstrated that reprogramming of nuclear function can be elicit partial or complete after exposure a differentiated cell to factors from pluripotent or undifferentiated cell. Mesenchymal stem cells derived from mice adipose tissue (ADSC), which the genome is inherently less differentiated than other cells, has an equal potential to differentiate into cells and tissues of mesodermal origin, and ADSC has potential impact on regenerative cell therapy for ischemic diseases (Schäffler and Büchler, 2007).

Exposing to extracts of ADSC may drive them further toward lower undifferentiated status. Using ADSC extract treatment could be a potential promising and plausible method for the purposes of cells therapeutic, as well as to provide suitable donor nuclei for SCNT. In our previous study (Xiong et al., 2014), fibroblast cells of yak were permeabilized with streptolysin O and pretreated in the ADSC extracts. The results showed that colony formation was promoted after pretreatment with ADSC extracts. Also, we found that the expression of pluripotent gene was significantly upregulated, and associated with increased global demethylation and loss of repressive histone modifications.

Cellular extract of others

Except oocytes and stem cells, other cells can also be used to prepare extracts (shown in Table 1). Qin et al. (2005) used mouse type II pneumocytes extracts to treat embryonic stem cells, and the result showed enhanced differentiation of pneumocytes, increased expression of surfactant protein C and mRNA. The research team of Liu et al. (2009) extracted human fetal liver tissue cell extracts from the hematopoietic developmental period (15 weeks) to treat human embryonic stem cells.

Table 1.

A Part of the Cellular Extract and Effects Reported in Previous Studies

Extract sources	Donor nuclei	Effects	References
Xenopus egg	293T cells	Upregulation of pluripotency markers Oct-4	Hansis et al. (2004)
Xenopus egg	Porcine cells	Facilitate embryos development	Miyamoto et al. (2006)
Xenopus egg	Somatic cells	Upregulation of OCT4, SOX2, and NANOG expression	Miyamoto et al. (2008)
Xenopus oocyte	Ovine fibroblasts	Improved live birth and survival rates, increase in cloning efficiency	Rathbone et al. (2010)
Xenopus oocyte	Embryonic stem cells	Loss of somaticH1 linker histone	Jullien et al. (2010)
Xenopus egg	Fetal fibroblasts	Colony formation, increased blastocyst formation	Liu et al. (2011)
Xenopus oocyte	Breast cancer cells	DNA demethylation, removal of repressive histone marks, and reduction growth	Allegrucci et al. (2011)
Xenopus oocyte	Pig fibroblasts	Decrease of DNA methylation, enhanced the SCNT embryo development	Yang et al. (2012)
Axolotl oocytes	Mouse embryo fibroblast	H3K9Me3, HP1a and DNA methylation reduced, Oct4 and Nanog activation	Bian et al. (2009)
Mouse GV stage oocyte	Cumulus cells	Demethylate histone H3K9 and deacetylate H3K9/14, facilitate cloning efficiency	Bui et al. (2008)
Porcine oocyte	Somatic cells	Histone deacetylation	Miyamoto et al. (2009)
Bovine oocyte	Bovine fibroblasts	Detectable expression of Oct4 and global deacetylation, improved cloning	Tang et al. (2009)
Yak/bovine oocyte	Yak fibroblasts	TBP1 and Mash2 upregulated, demethylation, increase cloning efficiency	Xiong et al. (2012)
Embryonic stem cell	GV oocytes	Pseudopronucleus formation, cloning embryos development	Gao et al. (2002)
Mouse embryonic stem cells	293T cells	Loss of repressive histone H3 modifications, repression of Lamin A/C, dedifferentiation	Bru et al. (2008)
Embryonic stem cell	NIH3T3 cells	Hyper-acetylation of histones 3/4, decreased H3K9 demethylation	Rajasingh et al. (2008)
Human embryonic stem cell	Somatic cells	Colony formation, pluripotency genes upregulated, epigenetic states changed	Han et al. (2010)
Spermatozoa	Sea-urchin eggs	Exocytosis of cortical granules	Dale et al. (1985)
Sperm cytosolic extract	Horse somatic cells	Facilitate cloning embryos development	Choi et al. (2002)
Porcine sperm	Bovine oocytes	Facilitate embryos development	Knott et al. (2002)
Porcine sperm	Porcine oocytes	Activation oocytes	Tan et al. (2009)
Primary human T cell	293T fibroblasts	A chromatin remodeling complex, histone acetylation, and activation of lymphoid cell-specific genes	Håkelien et al. (2002)
Rat insulin-producing beta cell line	Fibroblasts	Detected Pdx-1 and insulin expression, dedifferentiation	Håkelien et al. (2004)
Rat pancreatic extract	Mesenchymal cells	Differentiate rat mesenchymal cells into insulin-producing cells	Choi et al. (2005)
Embryonal carcinoma cell	293T cells	Reprogramming of DNA methylation and histone modifications	Freberg et al. (2007)
Fetal liver cell	Human embryonic stem cells	Hematopoietic differentiation	Liu et al. (2009)
Human NCCIT carcinoma cell	293T cells	Upregulation of OCT4, SOX2, NANOG, induced to differentiate	Taranger et al. (2005)
Adipose-derived stem cell	Yak fibroblast cells	Colony formation, histone modifications, and increased global demethylation	Xiong et al. (2014)

GVs, germinal vesicles; SCNT, somatic cell nuclear transfer.

It was found that the treated embryonic stem cells no longer expressed endodermal-specific genes during differentiation, but expressed hematopoietic development-related genes, and can be efficiently differentiated into hematopoietic cells without adding any cytokines. Then the hematopoietic cells were cocultured with low-dose cytokines, and it was found that these cells can be efficiently induced to differentiate into red blood cells. The red blood cells differentiated are similar to fetal liver red blood cells and have oxygen carrying capacity (Liu et al., 2010).

Sperm cell extracts have also made some breakthroughs and progresses. Previous study has shown that cytoplasmic extracts of sea urchin spermatozoa injected into unfertilized oocytes can initiate cleavage (Dale et al., 1985). The rabbit sperm extract was injected into the oocytes of rabbits and mice, and as a result, prokaryotic formation and cleavage were observed. Choi et al. (2002) injected horse sperm extract into oocytes and found that significantly improved the rate of cleavage.

Moreover, different doses of pig sperm extract were injected into bovine oocytes, and the pronuclear formation rates of bovine oocytes were 30%, 82%, and 40%, respectively. Among them, the 5 mg/mL group had the best parthenogenetic effects (Knott et al., 2002). Tan et al. (2009) also acquainted the same results in buffalo research. The aforementioned indicates that the sperm extract can activate the oocyte and initiate genes related to embryo development.

Effects of Cellular Extract

In recent years, studies on effects of cellular extract to induce cell reprogramming mainly focus on changing the gene expression patterns, modifying the epigenetic status, improving cloning efficiency, and inducing cell reverse differentiation and transdifferential (shown in Table 1).

Changing the gene expression profiles

Previous studies reported that the expression of pluripotent gene (such as Oct-4, Sox-2, c-Myc, and Klf-4) in differentiated cells were upregulated after treated with embryonic stem cells extracts (Boland et al., 2009; Bru et al., 2008; Freberg et al., 2007; Taranger et al., 2005; Zhang et al., 2012). Unlike these studies, our previous study showed that these genes were upregulated, including Klf-4 (∼10-fold) and c-Myc (∼5-fold) compared with that of ADSC cells, but the expression of Oct-4 (∼4-fold) was still significantly lower compared with that of ADSC cells, and there was no significant difference in Sox-2 expression levels between untreated and treated fibroblast cells with ADSC extracts (Xiong et al., 2014).

Hakelien et al. (2004) found that 293T cells reprogrammed with NCCIT cell extracts were formed clone and maintained for many generations during culture, whereas 293T cell extracts or Jurkat T cell extracts could not form clone or clear morphology. After 1 week of treatment with embryonic stem cell extract, 60%–70% of the cells expressed intracellular Oct4 protein, accompanied by the expression of other pluripotency markers (such as Sox2), and the expression of lamin A/C was significantly decreased. The gene expression profile of cells was changed after treatment with embryonic stem cell extracts, ∼1800 genes upregulated and ∼1700 genes downregulated, with 70% and 34% of genes being embryonic stem cell genes, respectively.

Taranger et al. (2005) found that transient exposure of permeable NIH3T3 cells to undifferentiated murine embryonic stem cell extracts resulted in reprogramming cells into other type with activation of Oct4 and repression of A-type Lamins (Oct4⁺ lamin A⁻). Hundreds of NCCIT genes were involved in a dynamic upregulation among the transition from a 293T to a pluripotent cell phenotype by microarray and quantitative analyses. Simultaneously, 293T genes and of indicators of differentiation (such as lamin A) were significantly downregulated, whereas embryonic and stem cell markers, including Oct4, Sox2, Nang, and Oct4-responsive genes, were upregulated. The Oct4 promoter was significant demethylation and associated with Oct4 activation and nuclear targeting of Oct4 protein.

Modification the epigenetic state

Cell extract-induced reprogramming alters the apparent state of modification, including DNA and histone levels. First, the DNA methylation of the reprogrammed cells has changed, such as cancer cell extracts that can induce the expression of Oct4 and Nanog in epithelial cells. Human lung cancer cells (H460) were pretreated with the extract of bovine parthenogenetic oocytes after reversibly permeabilized by streptolysin O. The results of bisulfite sequencing showed that the promoters of the tumor suppressor genes (Runx3 and Cdh1) were significant demethylation after pretreated with bovine parthenogenetic oocyte extracts, while no significant change at the promoter of pluripotency gene Sox2.

The results of chromatin immunoprecipitation (CHIP) showed that the status histone modifications in the promoters of Runx3 and Cdh1 were activated, but that in the promoter of Sox2 were significantly repressive. Moreover, pretreated with bovine parthenogenetic oocytes extract reversed the epigenetic changing of Runx3 and Cdh1, and downregulated the expression of Sox2. Rathbone et al. (2010) showed that after ovine somatic cells pretreated with X. laevis oocyte extract, the global methylation pattern was significantly decreased, and the epigenetic reprogramming was significantly improved. In addition, the DNA methylation of NIH/3T3 fibroblasts were significantly modified after pretreated with teratoma cellular extract (Zhang et al., 2012).

Second, the histones acetylation of reprogrammed cells was also modified. Freberg et al. (2007) found that treatment of epithelial cells with cancer cell extracts resulted in increased acetylation of histone 3 lysine 9 (H3K9) on the Oct4 promoter/enhancer, and the demethylation of H3K9 and H3K4. It is consistent with the phenomenon that normal cells occur during gene transcriptional activation. Likewise, we also observed that acetylation in H3K9 of yak fibroblasts was significantly increased after pretreated with the extracts of ADSC, and reduced obviously the global DNA methylation level (Xiong et al., 2014). Taken together, the aforementioned data suggested that there were many necessary regulatory components in the cellular extracts, which provide the required for inducing cell nuclear reprogramming and altering the epigenetic status of differentiated cells.

Improve cloning efficiency

Since the birth of Dolly, mammalian SCNT has a history of >20 years, but the efficiency is still extremely low. At present, it is generally considered the main factor that the highly differentiated somatic cells are incompletely reprogrammed in the oocyte cytoplasm after nuclear transfer, and there are a large number of epigenetic modification abnormalities, which make the embryo difficult to develop normally and affect the cloning efficiency. The application of cellular extract induces somatic cell reprogramming and restores to a relatively low differentiation state, which can be used as a nuclear donor to improve SCNT efficiency. This is the main focus of current research.

Liu et al. (2012) pretreated porcine fetal fibroblasts with Xenopus oocyte extracts, and then used as nuclear donors to prepare cloned embryos. It was found that the blastocyst formation rate of the cloned embryo was significantly increased as compared with the control group. Because the biochemical activity and quality of the extract are different from batch to batch, they aimed to evaluate three different batches of extracts prepared by the same method, based on the colony formation and subsequent cloning efficiency using pretreated cells as nuclear donors.

The result showed that the number of forming colonies was significantly different among the different treatment groups, and blastocyst formation rates were significantly increased after the donor cells were treated with extract, especially from a batch showing higher colony formation. Therefore, it is speculated that the number of colony formation may be a selection marker of the quality of the extract (Liu et al., 2011).

However, there are also inconsistent conclusions. Miyamoto et al. (2006) hypothesized that differentiated cells that had been reprogrammed to dedifferentiated status before SCNT might support higher developmental ability of SCNT embryos. Although Xenopus oocyte extracts restored donor cells reprogramming to a lower differentiation state and increased the number of the inner cell mass (ICM), it did not improve cloning efficiency. Furthermore, Rathbone et al. (2010) found that donor cells pretreated with Xenopus oocyte extract did not increase the blastocyst rate and pregnancy rate of cloned embryos, but only significantly increased the survival rate of cloned animals.

This study is the first time to report that Xenopus GV stage oocyte extract can increase the birth of cloning live offspring. The same findings were confirmed in mice (Bui et al., 2008). Only extracts of the matured bovine oocyte, not immature or parthenogenetic oocyte, could induce cells to express Oct4 gene and significantly increased the blastocyst formation rate and embryo quality of the SCNT embryos (Tang et al., 2009). Similar, our group also has obtained the same result in yak iSCNT (Xiong et al., 2012).

Induced cell reverse differentiation and transdifferential

Returning differentiated cells to undifferentiated status is a hot topic in scientific research, and cell extract treatment is one of the ways to dedifferentiate cells. As we all know, differentiated cells are induced by embryonic stem cells. Once the differentiation is initiated, it is irreversible to an original undifferentiated status in normal development. However, recent research reveals differentiated somatic cells can be induced to an undifferentiated status after transfer into oocytes. This method breaks the traditional concept, achieves the transition from highly differentiated cells to pluripotent cells, which could be differentiated into any expected type of cells for clinical therapy (Park et al., 2015).

Induction of cell extracts can also directly transdifferentiate one cell type into another type, omitting the cell dedifferentiation step, which will increase cell transformation efficiency. Håkelien et al. (2002) treated 293T cells with T cell extracts and found that the transcription factors of the receptor cells were enriched, and the chromatin remodeling complex was activated, which caused the epigenetic modification of 293T cells to express lymphocyte-specific genes and surface markers. Choi et al. (2005) reported that rat pancreatic extracts treated mesenchymal cells, the cells showed insulin-like growth and expressed four insulin-specific expression hormones, and in response to glucose. These transdifferentiated insulin-like cells will provide a large amount of material for the study of diabetes. However, there are few studies on the induction of somatic cell transdifferentiation by extracts, which needs further study.

Potential Mechanism

The mechanism of cell extracts on reprogramming has been discussed in depth, and it has been found that extract processing may reprogram target cells in several ways. Reprogramming factor is considered to be the main reason, which has been mainly identified in Xenopus. Compared with mammalian oocytes, Xenopus eggs are extremely large and easily accessible in vitro. It is because of these characteristics that the Xenopus egg or oocyte extract system has been established. In this extracts system, a large number of cells can induce reprogramming events at the same time (Kikyo et al., 2000), providing sufficient material for studying reprogramming mechanisms.

In Xenopus eggs and oocytes, lots of proteins have been identified as reprogramming factors, including FRGY2a/b (Gonda et al., 2003), ISWI (Kikyo et al., 2000), nucleoplasmin (NPL) (Tamada et al., 2006), BRG1 (Hansis et al., 2004), and histone B4 (Jullien et al., 2010). It has not been demonstrated that these factors are crucial and necessary for epigenetic reprogramming in cloned embryos during early embryonic development, although the roles of these factors on epigenetic remodeling and transcriptional reprogramming have been verified. Betthauser et al. (2006) injected NPL, purified from X. laevis eggs, into bovine oocytes with different concentrations, either before or after SCNT.

It was found that there were >200 genes were upregulated after post-NT NPL injection in cloned embryos compared with in vitro fertilized embryos, several of these genes were previously shown to be significantly downregulated in SCNT embryos. Thus, they speculated that NPL may be a key factor in oocyte extract for improving cloning efficiency by facilitating epigenetic reprogramming of the somatic nucleus.

Recently, Miyamoto et al. (2011) identified 25 proteins in porcine oocyte extracts from the metaphase II stage by mass spectrometry, including a multifunctional protein, DJ-1. They revealed that DJ-1 is an oocyte-specific factor, which is necessary for the development of SCNT embryos. However, the research on the mechanism of extracting somatic cell reprogramming is still not deep enough. Only the effect of individual substances or/and factor on cell reprogramming can be found. How these factors form the reprogramming regulatory network needs further research.

Some researchers believe that it is induced reprogramming by changing the apparent state of the recipient cells. The cell extract can change the degree of histone acetylation on the chromatin of the recipient cell, thereby changing the chromatin structure of the cell and establishing a new gene expression pattern. The degree of activation of downstream genes is closely related to the degree of histone-4 acetylation in the promoter region. Studies have shown that T cell extracts can make the IL-2 promoter of different types of recipient cells (HeLa cells, NT2 neuronal precursor cells, and 293T cells) deacylated, and promote its differentiation into T cells (Miyamoto et al., 2011).

Furthermore, the cell extracts can alter the expression of ribosomal genes. Recent studies have shown that oocyte extracts can mutate nuclear membranes, nuclear chromatin, and nucleoli of recipient cells within hours of treatment, inhibiting transcription of ribosomal genes. Ostrup et al. (2011) found that the remodeling of ribosomal genes involving nucleolar remodeling complex is the first event epigenetic reprogramming mediated by Xenopus egg extract.

However, the research on the mechanism of extracting somatic cell reprogramming is still not deep enough. The cell extract reprogramming technique mainly uses the nucleoplasm extract derived from the “target” cells to treat the recipient cells, and the stability of the results is easily affected by the selection of the “target” cells and the maintenance of genetic stability during the culture process. In addition, the functional stability of cells obtained by this technique still needs further research (Collas, 2003).

Problems and Prospects

At present, researchers have successfully improved the embryo survival rate of SCNT technology by using the cell extract method, and improved the gene transduction method of iPS technology (Kim et al., 2009; Rathbone et al., 2013). In addition, cell extract reprogramming techniques for tumor research has a fascinating prospect, owing to previous studies have shown that oocyte extracts can dedifferentiate tumor cells (Allegrucci et al., 2011).

The effect of cellular extract on the cloning efficiency is still controversial. The oocyte extracts from different species and different periods often produce different experimental results for the development of reconstructed embryos. From the current researches, after the extract induces reprogramming, the donor cells return to a lower differentiation state, which does not necessarily improve the cloning efficiency. Preparing suitable oocyte extracts and finding out the key factor should be the focus in the future research.

In addition, the active components of cell extracts are still unclear, and the molecular mechanism of reprogramming is still mysterious. The factors that play key roles in the regulation of differentiation in extracts are determined by certain methods, and the mechanism of its function is a hot spot. At present, our group is working on this aspect and collecting different components of the extract. Then observing the influence of the increase and decrease of the components on the induction effect further clarifies the composition of the extract and its mechanism of action, and adds specific inducers in the cell extract system to improve the efficiency of cell differentiation.

Footnotes

Acknowledgments

This study was supported by Sichuan Provincial Science and Technology Program (2017NZ0076) and Innovation Team Project for Conservation and Utilization of Yak Genetic Resources (13CXTD01).

Author Disclosure Statement

The authors or authors' institutions have no financial or other relationship with other people or organizations that may inappropriately influence the authors' work.

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