In Vitro Differentiation of Bone Marrow Mesenchymal Stem Cells Into Endometrial Epithelial Cells in Mouse: a Proteomic Analysis

Int J Clin Exp Pathol 2014;7(7):3662-3672 www.ijcep.com /ISSN:1936-2625/IJCEP0000322 Original Article In vitro differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mouse: a proteomic analysis Qing Cong1,2, Bin Li1,2, Yisheng Wang1,2, Wenbi Zhang1,2, Mingjun Cheng1,2, Zhiyong Wu1,2, Xiaoyan Zhang1,2, Wei Jiang1,2, Congjian Xu1,2,3,4 1Obstetrics and Gynecology Hospital of Fudan University, 2Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, 3Department of Obstetrics and Gynecology of Shanghai Medical School, 4Institute of Biomedical Sciences, Fudan University, Shanghai, P.R. China Received March 24, 2014; Accepted June 23, 2014; Epub June 15, 2014; Published July 1, 2014 Abstract: Objective: Mouse bone marrow mesenchymal stem cells (BMSCs) have been demonstrated to differentiate into female endometrial epithelial cells (EECs) in vivo. Our previous studies demonstrated that BMSCs can differentiate in the direction of EECs when co-cultured with endometrial stromal cells in vitro. Here, we obtain and analyse differential proteins and their relevant pathways in the process of BMSCs differentiating into EECs by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis. Methods: A 0.4-µm pore size indirect co- culture system was established with female mice endometrial stromal cells (EStCs) restricted in the upper Transwell chamber and BMSCs in the lower well plate. After indirect co-culture for several days, the BMSCs were revealed to progressively differentiate towards EECs in vitro. Then, four groups were divided according to different co-culture days with single culture groups of BMSCs as controls. Proteins were detected using iTRAQ based on 2DLC-ESI-MS/ MS and data were analysed by bioinformatics. Results: A total number of 311 proteins were detected, of which 210 proteins were identified with relative quantitation. Among them, 107 proteins were differentially expressed with a 1.2-fold change as the benchmark, with 61 up-regulated and 46 down-regulated proteins. Differential proteins CK19 and CK8 were epithelial markers and upregulated. Stromal marker vimentin were downregulated. Top canoni- cal pathways was “remodeling of epithelial adhesions junctions” and “actin cytoskeleton signaling”. Top networks was “cell-to-cell signaling and interaction, tissue development and cellular movement” regulated by ERK/MAPK and α-catenin. Conclusion: To the best of our knowledge, this is the first preliminary study of differential protein expression in the differentiation process of BMSCs into EECs in vitro. We further elucidated BMSCs differentiated in the direction of EECs. In addition, ERK/MAPK and α-catenin played important roles by regulating core differential proteins in the “cell-to-cell signaling and interaction, tissue development and cellular movement” network. Keywords: Bone marrow mesenchymal stem cells, endometrial epithelial cells; differentiation, proteomic analysis Introduction been identified in the endometrium of female bone marrow transplant recipients; these cells The human endometrium is a dynamic tissue appear to be histologically indistinguishable that undergoes more than 400 cycles of regen- from the endogenous endometrial cells and eration, differentiation, and shedding during a express markers of glandular and stromal dif- woman’s reproductive years [1]. Endometriosis ferentiation [8]. In addition, cells derived from is the development of endometrial tissue out- the bone marrow of male donor mice have been side of the uterus [2]. It affects 10%-15% of found to contribute to EECs and stroma de novo reproductive-age women and can cause pelvic in the eutopic and ectopic endometrium of pain and infertility [3]. Increasing evidence has female mice in vivo [4]. Stem cell markers oct-4 shown that stem cells can contribute to endo- and c-kit were found to be expressed in ectopic metrium and endometriosis [4-15]. Donor bone epithelial cells, which suggests the stem cell marrow mesenchymal stem cells (BMSCs) have origin [5]. Differentiation of MSCs into endometrial epithelial cells Figure 1. Isolation, characterization and co-culture of BMSCs and EStCs. A: Flow cytometric analysis of BMSCs cell surface markers expression from primary culture. Mesenchymal stem cell surface marker CD29 expression rate was 81.22% compared to the isotype and haemapoietic stem cell surface marker CD34 expression rate was 20.86% compared to the isotype. B: Primary culture of EStCs and immunocytochemistry characterisation. Negative control was stained with no primary antibody (up). EStCs were positive for stromal marker vimentin (down). Original magnifications × 400. C: Schematic diagram of co-culture system. Recently, our group demonstrated in mice that culture system than in the control group BMSCs could differentiate in the direction of (BMSCs cultured alone) (P < 0.05). Pan CK was EECs in vitro by indirect co-culture BMSCs with positive in the co-cultured BMSCs and negative endometrial stromal cells (EstCs) [9]. In this co- in the control group. culture system, cells cannot pass the Transwell membrane (cross-culture room, pore size 0.4 Isobaric tags for relative and absolute quantita- μm, Millipore, USA), while small molecular sub- tion (iTRAQ), is a shot-gun-based technique that stances can pass freely in the culture media. allows the concurrent identification and relative Thus, EStCs could induce BMSCs by paracrine quantification of hundreds of proteins in up to 8 signalling in the microenvironment, which is different biological samples in a single experi- somewhat similar to environments that exist in ment [16, 17]. The technology has many advan- vivo. After 7 days of indirect co-culture, expres- tages, such as having relatively high throughput sion of epithelial markers (cytokeratin 7, cyto- due to sample multiplexing, and it has been keratin 18, cytokeratin 19, and epithelial mem- shown to be suitable for the identification of brane antigen) was tested in BMSCs using low abundance proteins such as transcription real-time RT-PCR, and expression of pan cyto- factors [18]. keratin (CK) was tested using immunofluores- cence staining. The mRNA levels of CK7, CK18 To further illuminate BMSCs differentiate in the and CK19 were significantly higher in the co- direction of EECs and obtain information in this 3663 Int J Clin Exp Pathol 2014;7(7):3662-3672 Differentiation of MSCs into endometrial epithelial cells Figure 2. Experimental workflow of proteomic analysis of in vitro differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mouse using iTRAQ technique. process, we tend to acquire differential pro- Protein digestion, iTRAQ labelling and strong teins using iTRAQ and potential pathways by cation exchange fractionation (SCX) IPA (Ingenuity Pathway Analysis Software). All samples were centrifuged at 14,000 rpm for Materials and methods 60 s to collect cells in the Eppendorf microcen- trifuge tubes. Each sample was then lysed by Animals adding 50 μL of hypotonic buffer consisting of All wild-type female C57BL/6 mice were pur- 7 M urea, 2 M thiourea, 65 mM DTT chased from Shanghai Slac Laboratory Animal (DL-Dithiothreitol), 0.02% SDS cocktail and son- Corporation. They were maintained under spe- icated once. Total protein contents were deter- cific pathogen-free conditions with a light/dark mined using the commercial Bradford assay cycle of 12/12 h. All experiments were approved reagent (Bio-Rad, California, USA). A standard by the institutional experimental animals review curve for the Bradford assay was made using board of the Obstetrics and Gynaecology γ-globulin as a control. Hospital of Fudan University. From each sample, 100 µg was denatured, and Indirect co-culture system of BMSCs and EStCs the cysteines were blocked as described in the iTRAQ protocol (Applied Biosystems, Foster The protocol for the isolation and flow cytome- City, CA). Each sample was digested with 0.2 try characterization of BMSCs has been previ- mL of a 50 µg/mL trypsin (Promega, Wisconsin, ously reported [9]. Isolation and immunocyto- USA) solution at 37°C overnight and labelled chemistry characterization of EStCs was based with the iTRAQ tags as follows (Figure 2): on a previous report [19]. BMSCs and EStCs BMSCs cultured alone for 3 days, iTRAQ115; were co-cultured indirectly in transwell system BMSCs co-cultured for 3 days, iTRAQ116; (cross-culture room, pore size 0.4 μm, Millipore, BMSCs cultured alone for 7 days, iTRAQ117 USA). In brief, the BMSCs were seeded in the and BMSCs co-cultured for 7 days, iTRAQ118. bottom of 6-well plate (6 × 105 cells/well) and The labelled samples were combined, desalted the EStCs (6 × 104 cells/well) were seeded on with Sep-Pak Vac C18 cartridge 1 cm3/50 mg the transwell membrane to separate the cells (Waters, USA), and fractionated using a but allowing soluble factors to pass freely Shimazu UFLC system (Shimazu, Japan) con- (Figure 1A). The culture medium was changed nected to a strong cation exchange (SCX) col- every 2 days. Totally, 2.9 × 107 BMSCs of pas- umn (polysulfethyl column, 2.1 mm × 100 mm, sage 2 were seeded in 6-well plates co-culture 5 μm, 200 Å, The Nest Group, Inc. USA). SCX system to generate enough protein of BMSCs separation was performed using a linear binary co-cultured for 3 days and 7 days for proteomic gradient of 0-45% buffer B (350 mM KCl, 10 analysis and Western blot. mM KH2PO4 in 25% ACN, pH 2.6) in buffer A (10 3664 Int J Clin Exp Pathol 2014;7(7):3662-3672 Differentiation of

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