Elsevier

Journal of Proteomics

Volume 90, 2 September 2013, Pages 38-51
Journal of Proteomics

iTRAQ proteome analysis reflects a progressed differentiation state of epiblast derived versus inner cell mass derived murine embryonic stem cells

https://doi.org/10.1016/j.jprot.2013.03.015Get rights and content

Highlights

  • First proteomic comparison of entire embryonic versus epiblast stem cells

  • Marked differences in protein expression levels between mESC and mEpiSC

  • High concordance of abundance altered proteins in FT and NT stem cells

  • Intermediate filament protein levels reflect progressed differentiation state of mEpiSC.

Abstract

Mouse embryonic stem cells (mESC) and mouse epiblast stem cells (mEpiSC) share similar pluripotency factors like NANOG or POU5F1, however, their state of pluripotency differs significantly. mESC and mEpiSC can be derived from embryos generated by fertilization (FT) or by somatic cell nuclear transfer (NT). In this study we performed a 4-plex iTRAQ LC–MS/MS based approach, facilitating the multiplexed comparison of the four indicated types of stem cells. From four replicates of each cell type, 1650 proteins were quantified. 234 non redundant proteins with significant abundance alterations between FT/NT-mESC and FT/NT-mEpiSC, and 44 between FT and NT derived cells were detected. Bioinformatic analysis revealed that several pluripotency associated proteins, among them POU5F1, DNMT3L, TIF1B, and proteins involved in DNA repair like MSH2 and MSH6, are more abundant in mESC compared to mEpiSC. The abundance level of these proteins is not affected by the mode of embryo generation, whereas several cytoskeleton proteins show a higher abundance in NT-mESC compared to FT-mESC. In addition, a number of cytoskeletal proteins are enriched in mEpiSC, e.g., myosins, filamins and intermediate filament proteins, reflecting the progressed differentiation state of epiblast derived versus inner cell mass derived murine pluripotent stem cells.

Biological significance

This study aims to get new insights in the pluripotency state of stem cells and to deepen the knowledge of early cell differentiation. In an iTRAQ MS approach, we quantitatively compared proteomes of inner cell mass derived stem cells (mESC) with epiblast derived stem cells (mEpiSC). These stem cell types are derived from embryos of different developmental stages, and therefore vary considerably in their state of pluripotency and reflect different stages of early differentiation. The proteins which show significant abundance differences between the two stem cell lines represent (i) promising targets to further decipher molecular processes during early embryo development and (ii) useful molecular markers to monitor early differentiation events of stem cells by targeted approaches. This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations.

Introduction

Pluripotent embryonic stem cells (ESC) are able to differentiate into almost every cell type and are therefore promising tools for the development of new therapeutic approaches in regenerative medicine. The first embryonic stem cells were derived from mouse embryos (mESC) [1], [2] and although human ESC (hESC) were made available more than a decade later by Thomson et al. [3], mESC are still a highly important tool for a broad variety of applications in basic and clinical research. Furthermore mESC represent an important model to study early embryonic development at the molecular level since— in contrast to embryos— stem cells can be easily cultivated and engineered using latest methods of molecular biology. Besides mESC, which are derived from the inner cell mass (ICM) of blastocysts (around day 3.5 embryos), pluripotent stem cells can also be obtained from late epiblast cells of postimplantation embryos (around day 6.5 embryos) [4], [5]. These murine epiblast stem cells (mEpiSC) are capable of self-renewal and extensive in vitro and in vivo differentiation [6], but in contrast to mESC their capacity to contribute to the germ line remains to be demonstrated. Further, there are interesting differences in the expression of pluripotency factors between mEpiSC and mESC: while both cell types express NANOG, POU5F1 and SOX2, mEpiSC lack the expression of KLF4, DPPA3 (STELLA) and DAX1 which are important pluripotency factors for mESC. Thus mEpiSC and mESC differ fundamentally in the mechanisms maintaining pluripotency (reviewed in [7]). Moreover, mEpiSC express epiblast and early germ layer markers indicative of a more advanced, primed state of pluripotency [8]. Therefore, it is particularly interesting to quantitatively compare mESC with mEpiSC at the molecular level in order to i) get new insights into the different states of pluripotency, ii) learn how pluripotency is maintained and iii) decipher molecular mechanisms of the transition from mESC to the more differentiated mEpiSC. Studies addressing differences in the epigenetic status and transcriptome profiles of mESC and mEpiSC gave interesting insights in the biochemical networks defining the two states of pluripotency. However, since protein levels cannot be predicted from the epigenetic status or from transcriptome profiles, studies at the level of proteomes are indispensable to complement the knowledge of biochemical mechanisms regulating and maintaining these states of pluripotency. Recently, two comprehensive studies addressing subcellular proteomes of mESC and mEpiSC were published.

Rugg-Gunn et al. investigated in a comprehensive study the cell surface proteome of mESC, mEpiSC, trophoblast stem cells and extra-embryonic endoderm (XEN) stem cell lines [9]. The focus of this study was to find cell surface proteins serving as molecular markers for the different stem cell types. The comparison of mESC vs. mEpiSC proteomes revealed nine membrane proteins being specific for each cell type. Furthermore, Rugg-Gunn et al. demonstrated that several of these proteins can be used to quantify and isolate these cell types and are therefore useful markers for experiments addressing cell differentiation and cell reprogramming.

The second recently published study [10] investigated nuclear proteomes of mESC and mEpiSC. The dataset consists of 1597 proteins which were quantified and matched to existing databases of pluripotency networks (PluriNet [11] and PluriNetwork [12]). Interestingly, the dataset of Song et al. revealed that important proteins of the chromatin regulatory networks are differently abundant in nuclei of mESC and mEpiSC. Both studies represent remarkable datasets, helping to further characterize the two different states of pluripotency. However, no study of whole cell lysates containing all organelles and the cytoplasm has been published so far.

Beside the embryonic stage from which pluripotent embryonic stem cells are derived, the method of embryo generation can influence the molecular phenotype of stem cells. Both types of stem cells (mESC and mEpiSC) can be derived from embryos generated by fertilization (FT) or by somatic cell nuclear transfer (NT). Although it is known that NT-derived embryos frequently have abnormalities leading to losses of embryos at the peri-implantation stage, it has been demonstrated that NT-mESC are fully pluripotent, have a normal epigenetic status and are transcriptionally and functionally indistinguishable from FT-mESC [13], [14]. Ding et al. demonstrated further, that the miRNA composition, as well as the proteome profiles of NT-mESC and FT-mESC are highly similar and could not be distinguished by the techniques used in their study (array based miRNA profiling and 2D-DIGE analysis for proteome profiling) [15]. Therefore it was concluded, that the defects of NT-blastocysts did not affect corresponding mESC because abnormalities were compensated during the in vitro culture procedure used for mESC line establishment. In contrast, NT-derived mEpiSC can be transcriptionally and epigenetically different from their fertilization-derived counterparts [16]. Whether these differences are reflected at the proteome level has not been investigated so far.

In this manuscript, we describe a quantitative study analyzing whole cell lysates from mESC and mEpiSC to address the proteome in a manner unbiased from subcellular fractionation. In addition, to elaborate a possible impact of the mode of embryo generation (NT or FT) on the corresponding proteomes, stem cells derived from FT as well as NT embryos were analyzed using an iTRAQ LC–MS/MS approach.

Section snippets

ESC culture

For mESC samples two cell lines were used, one derived from a fertilized blastocyst (FT-mESC), the other from a blastocyst generated by nuclear transfer (NT) using a cumulus donor cell (NT-mESC). Both cell lines were of B6D2F1 (C57BL/6J × DBA/2J) genetic background. The derivation and culture of these cell lines have been described previously [17]. In detail, mESC were cultured on mitomycin-C inactivated primary mouse embryonic fibroblast (MEF) cell layer (ICR mouse strain derived) in

Generation and analysis of the proteome dataset

Our study aimed to quantitatively compare proteomes of mESC vs. mEpiSC and to check whether the mode of embryo generation (NT or FT), from which the stem cells are derived, has an impact at the proteome level. We performed a nano-LC–MS/MS analysis in combination with 4-plex iTRAQ [21], facilitating a multiplexed comparison of four types of stem cells, FT-mESC, NT-mESC, FT-mEpiSC and NT-mEpiSC. Four replicates of each cell type were generated and processed independently. The labeling scheme as

General remarks

Embryonic stem cells have the remarkable capability to renew themselves, and – when specific molecular differentiation signals are added – to differentiate into almost every cell type. This special feature, referred to as pluripotency, requires a well-balanced molecular network which has to be maintained in a failsafe manner but must show enough plasticity to facilitate the development to numerous states of differentiated cells. To enable a targeted manipulation of embryonic-derived stem cells

Conclusion

Quantitative analysis of mESC and mEpiSC revealed marked differences between the proteomes of these cell types. Compared to substantial proteomic differences originating from the developmental stage of the embryo used for stem cell generation, the mode of embryo generation (FT or NT) causes only minor alterations at the proteome level. Our results demonstrate, that the progressed differentiation state of epiblast derived versus inner cell mass derived pluripotent stem cells is reflected at the

Acknowledgments

We thank Florian Flenkenthaler, Kathrin Otte and Kaveh Mashayekhi for critical reading of the manuscript.

This work was supported by EU FP 7 (“Plurisys” HEALTH-F4-2009-223485; “Epihealth” HEALTH-2012-F2-278418 and “EpiHealthNet” PITN-GA-2012-317146 grants).

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