Informed consent was obtained from all participants in the present study and the study was approved by the Tianjin Medical University General Hospital Institutional Review Board and Ethics Committee (Tianjin, China). Patients with NMO were recruited between January 2016 and December 2017 from Tianjin Medical University General Hospital. The five patients (all female; patient characteristics are presented in Table SI) were enrolled on the basis of the following criteria: i) They conformed with the diagnostic criteria proposed by Wingerchuk et al (3) in 2007; ii) exclusion criteria included a history of diabetes, cardio- or cerebrovascular diseases and inflammatory, and autoimmune diseases other than NMO; iii) none of the patients had received corticosteroid or other immunosuppressant therapy within the last 4 weeks; and iv) all patients exhibited no other autoimmune diseases and all patients with NMO were monitored over a one year period (detailed information are described in Table SI). A total of five age-matched healthy female volunteers were enrolled as health controls.

Aliquots of 5-10 ml adult donor bone marrow aspirates were provided by the Department of Hematology of Tianjin Medical University General Hospital. Bone marrow-derived mononuclear cells (BM-MNCs) were isolated and cultured in Gibco^® Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (D-MEM/F-12 medium; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Chicago, IL, USA), 100 U/ml penicillin/streptomycin, 2 mM L-glutamine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 2 ng/ml human basic fibroblast growth factor and 10 ng/ml human epidermal growth factor (PeproTech, Inc., Rocky Hill, NJ, USA). Platelet-derived growth factor-BB (PDGF-BB) was also purchased from PeproTech, Inc. Following culture at 37°C in a humid atmosphere with 5% CO₂ for 3 days, the culture medium was completely replaced and non-adherent cells were removed. When the cells had reached ~80-85% confluence, the adherent cells were detached by treatment with 0.125% trypsin and 0.1% EDTA (Sigma-Aldrich; Merck KGaA), and put back into the medium at a 1:2 dilution under the same culture conditions.

At passage 3, adherent cells were identified by surface markers with monoclonal antibodies cluster of differentiation (CD)29, CD166, CD44, CD73, CD34 and CD90 (BioLegend, Inc., San Diego, CA, USA), CD45 (BD Pharmingen; Becton, Dickinson and Company, Franklin Lakes, NJ, USA), CD105 (Abcam, Cambridge, MA, USA) using a FACS flow cytometer (BD Biosciences, Becton, Dickinson and Company; the details of all the antibodies are described in Table I).

BM-MSCs were cultured in DMEM/F-12 medium and observed under an inverted light microscope. At the same time, BM-MSCs were stained for β-tubulin for the morphology examination. BM-MSCs were grown on glass chamber slides at 90% confluence and fixed with 4% paraformaldehyde at room temperature for 30 min. Subsequently, the BM-MSCs were first permeated with cold acetone for 10 min and washed with ice-cold PBS. The BM-MSCs were then blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich, Merck KGaA) in PBS at room temperature for 30 min, followed by incubation with rabbit antihuman β-tubulin antibody (Thermo Fisher Scientific, Inc.) overnight at 4°C. After three washes with ice-cold PBS, the BM-MSCs were incubated with goat tetramethylrhodamine-conjugated goat anti-rabbit immunoglobulin G secondary antibody (Thermo Fisher Scientific, Inc.; details of the antibodies are described in the Table II) at room temperature for 30 min and their nuclei were stained with 1 µg/µl 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min. The cells were then washed twice with ice-cold PBS and the images were visualized using a fluorescence microscope (Nikon Corporation, Tokyo, Japan).

To assess the proliferation state of the BM-MSCs cells following the various treatments an MTT (Sigma-Aldrich; Merck KGaA) proliferation assay was performed according to the manufacturer's protocol. In brief, BM-MSCs cells were seeded into 96-well plates at a density of 2×10⁴ cells/ml for 1-4 days. An aliquot of 20 µl MTT labeling reagent (5 mg/ml) was added daily to each well and the plates were incubated at 37°C for 4 h. The resulting formazan crystals were solubilized by adding 100 µl DMSO per well and the plates were incubated at room temperature for 10 min. The absorbance of formazan was measured at 575 nm and this was taken as the measure for the proliferation state of the cells. Trypan blue cell exclusion was also used at room temperature for 5 min to assess cell viability and the cell number with a light microscope.

The apoptotic rate of the BM-MSCs was evaluated using a commercial fluorescein thiocyanate (FITC)-Annexin V apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), as recommended by the manufacturer. BM-MSCs (5×10⁵ cells) were harvested, washed and incubated in the Annexin V binding buffer. Subsequently, the BM-MSCs were stained with Annexin V-FITC and propidium iodide (PI), and flow cytometric analysis was then performed using a flow cytometer (BD FACSAria™ III) and the data were analyzed with FlowJo 7.6 software (FlowJo LLC, Ashland, OR, USA). Apoptotic cells were identified in the early (FITC-Annexin V positive, PI negative) and the late (FITC-Annexin V and PI double-positive) stages.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA, following the manufacturer's protocol and the concentration of total RNA was measured using a spectrophotometer following treatment with DNase I (Invitrogen; Thermo Fisher Scientific, Inc.). A sample (2 µg) of RNA was used for reverse transcription in a 20 µl reaction containing EasyScript^® reverse transcriptase kit (TransGen Biotech Co., Ltd., Beijing, China.). The thermal program consisted of 42°C for 30 min, and then 85°C for 5 sec to inactivate enzymes. RT-qPCR was performed using a Bio-Rad RT-PCR system instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with an SYBR^®-Green PCR kit (Roche Diagnostics, Indianapolis, IN, USA). Genes involved in the cell cycle, proliferation and cell apoptosis were selected for investigation. Details of all the primers are described in Table III. The thermal cycling program consisted of 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 60 sec at 60°C. Relative quantities of target genes were determined for unknown samples using the comparative threshold cycle (ΔΔCq) method (16) and normalized against GAPDH as the control. Each test sample was amplified in three different wells with a 20 µl reaction volume, following the manufacturer's protocol for each specific experiment and each test was repeated at least in triplicate.

BM-MSCs were induced to differentiate into osteoblasts and adipocytes using the following procedure. The induction medium for osteogenesis was Gibco^® Iscove's modified Dulbecco's medium (IMDM; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 0.1 µM dexamethasone, 0.2 mM ascorbic acid 2-phosphate and 10 mM glycerol 2-phosphate (Sigma-Aldrich; Merck KGaA). The induction medium for adipogenesis was IMDM supplemented with 10% FBS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 µg/ml insulin and 60 µM indomethacin (Sigma-Aldrich; Merck KGaA). After 3 days, the culture medium was completely replaced and the medium was subsequently changed twice weekly thereafter. After the predetermined culture time had elapsed, adipocytes were stained with Oil Red O at room temperature for 30 min; the osteoblasts were identified using alkaline phosphatase (ALP) and Alizarin Red S assays were performed at room temperature in the dark for 50 min (Sigma-Aldrich; Merck KGaA).

The total proteins of the BM-MSCs were extracted with Pierce™ IP Lysis buffer (Thermo Fisher Scientific, Inc.) and the concentration was determined with bicinchoninic acid (BCA) methods with a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Lt., Beijing, China). The proteins were separated using 10% SDS-PAGE and 15 µg protein were loaded in each lane. Subsequently, the proteins were transferred electrophoretically to a polyvinylidene difluoride membranes (Merck KGaA). The membranes were blocked with 5% defatted milk at room temperature for 1 h and then incubated with the primary antibodies overnight. The next day, the membranes were incubated with the HRP-conjugated secondary antibodies for 1 h. Protein bands were detected using an Amersham ECL Western blotting detection kit (GE Healthcare Life Sciences, Little Chalfont, UK). The protein bands were statistically analyzed with ImageJ 1.4 software (National Institutes of Health, Bethesda, MD, USA). For western blotting analysis, the β-actin antibody (used as the loading control) was purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA); the antibodies against cyclin A2 (CCNA2), cyclin-dependent kinase inhibitor 2A (CDKN2A), amphiregulin (AREG) and Bcl-xl were from Cell Signaling Technology, Inc., (Danvers, MA, USA); and the anti-Fas antibody came from R&D Systems, Inc., (Minneapolis, MN, USA). All antibodies are described in further detail in Table II.

Each experiment was repeated at least three times. All data are presented as the mean ± standard deviation. The difference between means was statistically analyzed using a t-test. All statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Aliquots of 5-10 ml adult donor bone marrow aspirates (five from patients with NMO and five from age- and sex-matched healthy subjects) were provided by the Department of Hematology of Tianjin Medical University General Hospital. After isolation, BM-MSCs at passage 3 were harvested to analyze their immunophenotype using flow cytometric analysis. BM-MSCs from patients with NMO and healthy controls were revealed to express CD105, CD73, CD90, CD29, CD44 and CD166, although they lacked expression of CD34 and CD45 (Table IV). All the BM-MSCs formed a monolayer of bipolar spindle-like cells, with a 'whirlpool-like' array. However, certain BM-MSCs from the patients with NMO were observed to be aberrant, with an irregular and ragged appearance following staining with β-tubulin under a fluorescence confocal microscope. Compared with the elongated morphology of the BM-MSCs from the healthy controls, certain BM-MSCs from patients with NMO also exhibited a 'cobblestone'-like' appearance (Fig. 1A).

The differentiation capabilities of BM-MSCs were subsequently investigated following induction with the differently conditioned media. BM-MSCs were able to differentiate into osteoblasts and adipocytes, as tested by positive staining of ALP and Alizarin Red S (for osteoblasts), and Oil Red O (for adipocytes). The results revealed that the BM-MSCs from the healthy controls and the patients with NMO were easily induced to differentiate into osteoblasts and the adipocyte lineage (Fig. 1B). There appeared to be no obvious differences in the differentiation capability between BM-MSCs from either the healthy controls or the patients with NMO. These data indicated that there was little discrepancy in morphology between BM-MSCs from healthy control subjects and the patients with NMO, since all the BM-MSCs had very similar differentiation capabilities.

The proliferative capability of the BM-MSCs was measured using a non-radioactive MTT assay method. The extent of cell proliferation was measured following culture for 0, 1, 2, 3 and 4 days, in a regular MSC culture medium. As presented in Fig. 2A, the proliferation rate of BM-MSCs from the patients with NMO was decreased compared with the healthy controls from day 3. However, following 4 days of culture, the proliferation of the BM-MSCs from the patients with NMO was significantly decreased (P<0.05). In addition, along with the proliferation assay, the mRNA expression levels of an array of cell cycle- and proliferation-associated genes in the BM-MSCs from the patients with NMO and healthy controls were detected by RT-qPCR (Fig. 2B and C). These experiments revealed that the expression levels of AREG and CCNA2 were significantly decreased in BM-MSCs from the patients with NMO compared with the healthy controls (P<0.05), whereas the expression of CDKN2A significantly increased in the patient group (P<0.05). Changes in the protein levels of the above genes revealed a similar tendency. Three experimental subjects were randomly selected from the NMO patient group and the healthy subject group, respectively, and these exhibited the same tendency (Fig. 3A and B). These data suggested that the proliferative capabilities of BM-MSCs from the patients with NMO were lower compared with those of BM-MSCs from the healthy controls.

The viability of BM-MSCs was subsequently compared between the patients with NMO and the healthy controls, as indicated by the apoptotic rate of BM-MSCs under normal culture conditions. As presented in Fig. 4, the apoptotic rate of BM-MSCs from the patients with NMO was three times higher compared with the healthy controls. In addition, the expression levels of an array of cell apoptosis and cell death-associated genes were compared between the two groups. Of the genes assessed, the expression of Fas was revealed to be significantly increased (P<0.05), whereas that of the pro-survival gene Bcl-xl was significantly decreased in BM-MSCs from the patients with NMO at the mRNA and the protein levels (P<0.01; Fig. 4B-D). These data suggested that senescence may be increased in the BM-MSCs from patients with NMO compared with the healthy controls. Furthermore, the BM-MSCs from patients with NMO were at a disadvantage in terms of their self-renewal capability.

Several growth factors and cytokines have been considered to be critical factors regulating MSC proliferation, migration and differentiation, among which PDGF is regarded as the most potent mitotic stimulatory factor (17,18). PDGFs are disulfide-linked homodimers or heterodimers, consisting of several 12.0-13.5 kDa polypeptide chains designated as the PDGF-A, -B, -C and -D chains and the five naturally occurring PDGFs are PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD (18). Considering that PDGF-BB is the major growth factor in bone matrix and its safety and effectiveness have been verified in a number of clinical trials (19,20), it seemed logical during these experiments to supplement the NMO BM-MSC culture medium with PDGF-BB in order to optimize the BM-MSCs' biological properties. The MTT assay experiment revealed that 20 ng/ml PDGF-BB was able to significantly promote the proliferation rate of BM-MSCs from patients with NMO on days 3 and 4 (P<0.05; Fig. 5A). Furthermore, the cytometric images indicated that the apoptotic rate of the NMO patient group was decreased from 9.745±0.245% to 3.672±0.128% (P<0.01; Fig. 5B). Additionally, improvements at the molecular level were also confirmed by RT-qPCR. As presented in Fig. 5C, the expression levels of AREG, CCNA2 and Bcl-xl were significantly increased (P<0.01); conversely, the expression levels of CDKN2A and Fas were significantly decreased (all P<0.05). The protein expression levels following PDGF-BB treatment in MSC cells from the identical patients were also examined. Three patients were selected and the protein expression levels of AREG, CCNA2 and Bcl-xl were revealed to be significantly increased in the patients with NMO MSC with PDGF (P<0.05), whereas the expression levels of Fas and CDKN2A were significantly decreased (P<0.01; Fig. 6A and B).