A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of Primary Cultured Cells and Subcultured Cells Using Mouse Adipose-Derived Mesenchymal Stem Cells

Adipose-derived mesenchymal stem cells (MSC-ATs) are representative cell sources for cell therapy. However, how cell stress resulting from passage influences the MSC-AT protein expression has been unclear. In this study, a protein expression analysis was performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using mouse primary cultured cells (P0) and cells passaged three times (P3) as samples. A total of 256 proteins were classified as cellular process-related proteins, while 179 were classified as metabolic process-related proteins in P0. These were considered to be adaptive responses of the cells to an in vitro environment. However, seven proteins of growth were identified (Csf1, App, Adam15, Alcam, Tbl1xr1, Ninj1, and Sbds) in P0. In addition, four proteins of antioxidant activity were also identified (Srxn1, Txndc17, Fam213b, and Apoe) in P0. We identified 1139 proteins expressed in both P0 and P3 cells that had their expression decreased to 69.4% in P3 cells compared with P0 cells, but 1139 proteins are very likely proteins that are derived from MSC-AT. The function of MSC-ATs was maintained after three passages. However, the LC-MS/MS analysis data showed that the protein expression was degraded after three passages. MSC-ATs retained about 70% of their protein expression ability in P3 cells.


Introduction
Mesenchymal stromal stem cells (MSCs) are considered to have the ability to differentiate into mesenchymal cells, such as osteoblasts, adipocytes, muscle cells, and chondrocytes [1,2]. These cells are also expected to have an immunosuppressive effect and are regarded as promising cellular therapeutic agents for immunological diseases resistant to treatment.
MSCs have been established from various tissues (umbilical cord blood, placenta, adipose tissue, etc.), among which adipose tissue contains a particularly large amount of cells.
Clinical research and treatment using adipose-derived mesenchymal stem cells (MSC-ATs) [3] is already underway in many medical institutions around the world [4]. The clinical practical application of islet cell transplantation therapy was reported in 2000 in the Edmonton protocol [5] and many subsequent papers [6][7][8][9][10]. The technology of islet transplantation is thought to be useful for the processing of therapeutic cells using MSC-ATs. We recently reported that the University of Wisconsin (UW) [11] organ preservation solution has a better cell survival/proliferation ability than Hank's balanced salt solution (HBSS) [12]. There is a possibility that the adipose tissue collected through the patient's skin may be infected with skin bacteria. One method of sterilizing tissues collected from a living body involves immersing and storing such tissue for 16 h using HBSS [13], which also contains antibiotics. After such storage, MSC-ATs can be isolated from adipose tissue. In addition, adipose tissue collected from a patient can be transported to a remote location.
It was recently reported that the stress of long-term culture of cells in vitro also occurs in stem cells, such as induced pluripotent stem (iPS) cells, causing DNA damage and cellular carcinogenesis [14]. Some researchers recommend reducing the number of passages of MSC-ATs to maintain the quality of primary cultured cells. However, MSC-ATs of primary cultured cells are reportedly contaminated with various types of cells, such as blood cells, through the process of cell isolation. This is because the stromal vascular fraction (SVF) [15] obtained when collecting MSC-ATs using centrifugation contains many kinds of cells (e.g., adipocytes, fibroblasts, smooth muscle cells, endothelial cells, blood cells, endothelial progenitor cells, preadipocytes, vascular progenitors, hematopoietic progenitors, and hematopoietic stem cells) [16,17]. For MSC-ATs isolated from adipocytes collected from patients, the number of cells can be increased by increasing the number of passages. Because this processing can be done outside the body, the patient can thus obtain many of her/his own cells after undergoing only one procedure of fat collection surgery. However, it is also important to maintain the quality of the cells. Therefore, researchers and clinicians have fervently discussed how many passaging operations of clinical MSC-ATs should be performed.
With liquid chromatography (or high-performance liquid chromatography (HPLC)) with tandem mass spectrometry (LC-MS/MS), the components to be analyzed are separated by a liquid chromatograph (LC) and ionized via a dedicated interface (ion source) and the generated ions are then separated by MS. LC-MS/MS is an analytical technique that dissociates and fragments mass ions and detects them with MS [18]. Recently, an online LC-MS/MS system for quantitative proteomics based on data-dependent protein IDs and shotgun-based quantitative proteomics methods was developed [19][20][21][22][23] by connecting the measuring equipment for a protein analysis to a computer and linking to an online protein database. In this way, a comprehensive protein expression analysis can be performed by checking the peptide sequence data of the protein contained in the sample.
A comprehensive expression analysis of the protein expressed by the cell is important for accurately understanding the mechanism underlying the effect of cell therapy accompanying the administration of the culture supernatant of cells and the cells themselves. The present study was performed to identify the functional protein components in mouse MSC-ATs of primary cultured cells (P0) and mMSC-ATs passaged three times (P3) using LC-MS/MS. The proteins specifically contained in primary cultured cells were identified as those expressed only in the SVF derived from AT. By examining the proteins expressed in both P0 and P3 cells, we can identify the proteins expressed by MSC-ATs. Determining the protein component of MSC-ATs that exerts a therapeutic effect is expected to be useful for cell therapy in the future.

Materials and Methods
2.1. Reagents. Fetal bovine serum (FBS) was obtained from Biowest (Nuaille, France). DMEM (high glucose) with Lglutamine, phenol red, and sodium pyruvate was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Plastic dishes were obtained from TPP (Trasadingen, Switzerland). All other materials used were of the highest commercial grade.

Animal
Care. All experimental protocols were in accordance with the guidelines for the care and use of laboratory animals set by Research Laboratory Center, Faculty of Medicine, and the Institute for Animal Experiments, Faculty of Medicine, University of the Ryukyus (Okinawa, Japan). The experimental protocol was approved by the Committee on Animal Experiments of University of the Ryukyus (permit number: A2017101). C57BL/6 male mice (8 weeks of age; Japan SLC, Shizuoka, Japan) were maintained under controlled temperature (23 ± 2°C) and light conditions (lights on from 08:30 to 20:30). Animals were fed standard rodent chow pellets with ad libitum access to water. All efforts were made to minimize the suffering of the animals.

Isolation of MSC-ATs from Mouse via the Inguinal Pad
Fat. AT was obtained from the inguinal pad fat of three 8-week-old mice. The method of isolating MSC-ATs from AT was in accordance with the AT-derived stem cell product standard document (RMRC-A 01: 2015) of Ryukyus Regenerative Medicine Research Center. In brief, these ATs were stored in cold HBSS and washed vigorously using HBSS three times before starting digestion. Next, the tissues were cut into small fragments with a scalpel for enzymatic digestion (2 mg collagenase type IV/ml; HBSS) in 50 ml tubes (rotation speed: 20 × 37°C × 60 min) using the shaker (BioShaker BR-42FM; TAITEC, Saitama, Japan). These tubes were then centrifuged (800g) for 5 minutes. The SVF [24] containing various kinds of cells, including MSC-ATs, was confirmed at the bottom of the tube after centrifugation. The MSC-ATS were collected as a cell pellet and then washed with fresh DMEM containing 10% FBS to remove the enzyme after the digestion. The digested tissue was then incubated in a T25 flask.
All of the mouse studies were approved by the Institutional Animal Care and Use Committee of University of the Ryukyus.

Preparation of mMSC-ATs.
Mouse MSC-ATs were cultured (37°C, 5% CO 2 ) in an uncoated T25 flask (TPP 90026). The passage of cells was performed every 3 to 4 days after reaching 80% confluence following sowing. The cells were then washed with PBS (calcium, magnesium-free), and mouse MSC-ATs were dissociated using a dissociation solution (Trypsin/EDTA (Lonza CC-3232)). Subculturing was carried out by plating on an uncoated T25 flask. DMEM containing 10% FBS was used for the culture medium.

Protein Identification by a Nano-LC-MS/MS Analysis.
We used an EzRIPA Lysis kit (ATTO Corporation, Tokyo, Japan) for cell lysis according to the manufacturer's instructions. A protein solution of 4493 μg/ml (P0) and 3105 μg/ml (P3) was obtained from mADSCs, and 6.0 μg protein was used for sample preparation. Finally, 0.4 μg of protein was used for nano-LC-MS/MS. The comprehensive expression analysis of proteins using LC-MS/MS and data analyses were performed according to the method reported previously [25].

Results and Discussion
The application of cell therapy in regenerative medicine is expected to be useful for the treatment of many kinds of diseases. For example, MSC-ATs, which can be collected from AT, have been applied to the treatment of a wide range of diseases in light of the low invasiveness compared with surgery. It is generally recognized that MSC-ATs are stable in quality from P0 cells to P5 cells [26]. As such, many manuals of commercially available MSC-ATs state that the quality is guaranteed for five passages. It was reported that multiple passaging processes reduce both the cell proliferative activity and the cell surface marker expression [27] and induce chromosome abnormalities [15]. In addition, increasing the number of passages also increases the risk of microbial infection of cells. Therefore, the general perception among clinical researchers dealing with therapeutic cells is that MSC-ATs are only useful as therapeutic cells within the first five passages [28].
However, it is easy to imagine that preparing cells collected from a living organism without subculturing may enable the production of therapeutic cells with new and special functions. Therefore, clinicians may try using MSC-ATs isolated from AT without any culturing or P0 MSC-ATs. Close attention must be paid in such cases, as immune rejection can be caused when using therapeutic cells not only for autologous transplantation but also other transplantations as well.
3.1. The Characteristics and Cell Quality of mMSC-ATs (P0). mMSC-ATs were cultured to 80% confluence using DMEM containing 10% FBS after isolation from AT. The whole medium was exchanged every two days. Microscopy was performed to confirm the absence of abnormalities with regard to the mMSC-AT (P0) size and shape and the culture state ( Figure 1(a), left panel). Flow cytometry was performed using markers of mMSC-ATs (CD44, CD90.2), hematopoietic stem cells (CD34), and leukocytes (CD45). CD44 and CD90.2 were expressed in mMSC-ATs, while CD34 and CD45 were not detected (Figure 1(a), right panels). We induced differentiation into adipocytes (Figure 1(b), left panel) and osteoblasts (Figure 1(b), right panel) using mMSC-ATs. Mature adipocytes were stained with Oil Red O, and mature osteoblasts were stained with Alizarin Red S.
3.2. The Characteristics and Cell Quality of mMSC-ATs (P3). mMSC-ATs were cultured to 80% confluence using DMEM containing 10% FBS after isolation from adipose tissue. The whole medium was exchanged every two days. The passage of cells was performed every 3 to 4 days after reaching 80% confluence. Microscopy was performed to confirm the absence of abnormalities with regard to the mMSC-AT (P3) size and shape and the culture state (Figure 1 A proteome analysis using LC-MS/MS provided evidence supporting the safe application of cell therapy with MSCs and supplied information on the potential application of MSCs in various treatments. A protein analysis indicates the protein components present in the cell component. In this study, mMSC-ATs were used, but when human MSC-ATs are used, this analysis will show the protein components that should be administered to patients.

A Comprehensive Protein Expression
Analysis of mMSC-ATs (P0 and P3). We performed mMSC-AT isolation according to a protocol similar to that used in clinical studies at the University of the Ryukyus, and isolated mMSC-ATs were subjected to LC-MS/MS after 0 or 3 passages. The presence of a large amount of albumin in the medium reduces the accuracy in protein analyses. Therefore, the protein extracts obtained from the cells after washing with phosphatebuffered saline (PBS) were used in this study.
There were 1785 types of proteins identified from the mMSC-AT (P0) samples (Table 1) and 1825 types of proteins identified from the mMSC-AT (P3) samples (Table 2). Among the 1785 types of proteins in mouse P0 cells, there were 336 types of proteins unique to the primary cultured cells (group P0). A total of 1449 types of proteins in mouse P0 cells were also identified in mouse P3 cells (group P0&P3). Among the 1825 types of proteins in mouse P3 cells, there were 376 types of proteins unique to the cells passaged 3 times (group P3) ( Figure 2). Therefore, the 336 types of proteins whose expression was eliminated by passage were deemed likely to have been derived from the different types of cells contained in the SVF.
The amount of protein quantified in this paper is a theoretical value estimated based on the emPAI function of the Scaffold software program. The ratio of the number of measured peptides to the number of theoretical peptides is linearly related to the logarithm of the protein concentration, and the number obtained by subtracting 1 from the index of the peptide number ratio was defined as the emPAI. The larger the emPAI value, the greater the amount of protein.
We recently published a paper on the correlation between different emPAI values (>0, >1, >2, >3, >5, and >10) and the results of protein expression analyses. The results showed that, for an emPAI value > 10, the presence of protein can be                                                               When multiple spectra are matched to the same peptide, the way of counting into one is called "peptide number". #2 When multiple spectra are matched to the same peptide, the method of counting is called "spectrum number". #3 "Unique" is a peptide that is not matched to other proteins and has been assigned only to the relevant protein.                                                                     When multiple spectra are matched to the same peptide, the way of counting into one is called "peptide number". #2 When multiple spectra are matched to the same peptide, the method of counting is called "spectrum number". #3 "Unique" is a peptide that is not matched to other proteins and has been assigned only to the relevant protein.
detected with a high probability, even if the number of samples is n = 1 [25]. Proteins quantified using emPAI were listed from the top in the tables showing the GO analysis results (Tables 1 and 2) in descending order of concentration. Since the data of this study were derived from a single protein expression analysis obtained from mMSC-ATs of three mice, the data reliability must be carefully considered.  Figure 3). Stml2 (CD4-positive, alpha-beta T cell activation), CD36, CD180 (B cell proliferation), CD47 (opsonization), and CD166 (adaptive immune response) were detected as factors related to the CD cell surface markers related to the immune system. Cats, Ha1b, Erap1, Mdr1a, Psb8, and Ha11 were detected as factors related to the MHC class antigen and thereby related to the immune system. Csf1 (osteoclast differentiation), CD109 (osteoclast fusion), and Rab35 (antigen processing and presentation) were detected as factors related to the bone immune system. Isg15, Tri25, Ifm2, Bst2, Oas1a, and Asc were detected as factors related to the antiviral with a scatter plot (y-axis = P3, x-axis = P0). The average quantitative value of P3-expressed proteins decreased to 69.4% compared to P0-expressed proteins (Atp5f1, B2m, Hprt1, Rplp1, Ppia, Rps18, Pgk1, Tfrc, Ywhaz, and Gapdh; Supplementary Figure 1 Table 3 are listed by their detailed molecular functions.      Protein S100-A13 RAGE receptor binding, calcium ion binding, copper ion binding, fibroblast growth factor binding, lipid binding, metal ion binding, protein homodimerization activity, zinc ion binding  clinical culture medium. Therefore, the increased expression of Tln1, Flnc, and Ass1 protein at P3 compared with P0 is not caused by FBS. Regarding the change in the expression of Ass1, the activation level of Hif1α was considered to be higher at P0 than at P3, because the oxygen concentration is lower in vivo than in vitro [29,30]. Therefore, the results of this study reflect not only the effect of cell division frequency but also the influence of oxygen concentration. When cultured cells are planted in a living body in a low-oxygen environment, the hypoxic response may be activated. Therefore, in order to interpret the results of this study more accurately, we must obtain data on the protein expression of MSCs cultured under hypoxic and high-oxygen conditions.

Conclusions
The functions of proteins classified by the GO analysis were quantified using the LC-MS/MS measurement system for the amount of proteins and components contained in primary cultured cells of mMSC-ATs and cells passaged three times. The ability of mMSC-ATs to differentiate into expression markers of cells, fat, and osteoblasts did not change, even after three passages. However, the protein expression decreased to 69.4%. The proteins whose expression levels decreased after three passages included Ass1 among the Hif-related proteins. Furthermore, it was revealed that 336 kinds of proteins are specifically expressed in primary cultured MSC-ATs. In conclusion, MSC-ATs used as therapeutic cells retained their cell properties after three passages but showed a decreased protein expression on LC-MS/MS.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.