Human bone marrow samples were collected from normal donors by bone marrow aspiration at The First Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, China) and mononuclear cells were isolated by density gradient centrifugation at 400 × g at 20°C for 25 min. The current study was approved by the First Affiliated Hospital, College of Medicine, Zhejiang University. The cells were cultured in low-glucose Dulbecco's modified Eagle's medium (10-014-CVR; Corning Incorporated, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; 10099-141; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C and 5% CO₂ in a humidified incubator. The medium was replaced after the initial 48 h. Subsequently the medium was changed every 3 days. The adherent cells were passaged when 90% confluence was reached. BM-MSCs at passage 2–6 were used in the subsequent assays.

BM-MSCs of passages 3–5 were harvested using 0.25% trypsin-ethylenediaminetetraacetic acid solution (EDTA; 25200-056; Gibco; Thermo Fisher Scientific, Inc.). Cells were then incubated with the following anti-human monoclonal antibodies: CD90-fluorescein isothiocyanate (FITC; 1:20; 11-0909), CD105-phycoerythrin (PE; 1:20; 12-1057), CD73-APC (allophycocyanin; 1:20; 17-0739), CD45-FITC (1:20; 11-9459), CD34-PE (1:20; 12-0349), CD19-APC (1:20; 17-0199) and CD11b-PE (1:20; 12-0113) antibodies (eBioscience, Inc., San Diego, CA, USA) at 4°C for 30 min. Following fixing with 75% ethanol (Sinopharm Chemical Reagent Co., Ltd.), permeabilisation with permeabilisation solution [CCS012A; Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China] and blocking with Human BD Fc Block (564219; BD Pharmingen, San Diego, CA, USA), cells were detected using an FC 500 MCL flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) with mouse IgG1 K isotype control FITC (11-4714), mouse IgG1 K isotype control PE (12-4714), mouse IgG1 K isotype control APC (17-4714) isotype-matched antibodies (eBioscience, Inc.) applied as controls. BM-MSCs at passages 3–5 were cultured in osteogenic or adipogenic differentiation medium (HUXMA-90021 and -90031, respectively; Cyagen Biosciences, Inc., Santa Clara, CA, USA) for the differentiation assays. Cells were stained with Alizarin Red or Oil red O solution (HUXMA-90031; Cyagen Biosciences, Inc.) to evaluate osteogenic or adipogenic differentiation, respectively. The cells were analyzed using CXP Analysis Software 5 (Beckman Coulter, Inc.).

The human ALL Reh cells were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and were divided into cells cultured either alone or co-cultured with with BM-MSCs. Reh cells were cultured in Iscove's modified Dulbecco's medium (SH30228; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% FBS at 37°C and 5% CO₂ in a humidified incubator. For co-culture experiments, BM-MSCs were initially seeded on 6- or 12-well plates at 5×10⁴ cells/ml density. After 4–6 h of culture, Reh cells were added to the adherent MSCs layer at 5×10⁵ cells/ml density. After 48 h of co-culture, Reh cells were collected by directly separating suspending cells as described by previous studies (13,14).

Forkhead Box P3/Transcription Factor Staining Buffer Set (eBioscience, Inc.) was used for intracellular staining with the anti-human monoclonal Ki-67-PE antibody (12-5699; 1:20; eBioscience, Inc.). Reh cells were collected and washed with phosphate-buffered saline (PBS) 2 times. Following incubation with fixation/permeabilization solution at 4°C for 30 min, Reh cells were washed and incubated with 5 µl Ki-67-PE antibody at 4°C for 30 min. Cells were resuspended in 500 µl PBS for flow cytometric detection using an FC 500 MCL flow cytometer. Ki-67 was detectable during the active phases of cell cycle (G₁, S, G₂ and M) and not during the resting phase (G₀), therefore, the positive cells were considered to be proliferating cells (15).

Reh cells were harvested and washed twice with cold PBS. Cells were resuspended in 100 µl 1X binding buffer provided in the Apoptosis Detection kit (559763; BD Pharmingen, San Diego, CA, USA). Following incubation with the Annexin V-PE antibody and 7-aminoactinomycin D from the Apoptosis Detection kit (559763; BD Pharmingen) at room temperature for 15 min, Reh cells were analyzed using the FC 500 MCL flow cytometer. Cells that were Annexin V-positive were considered to be apoptotic.

Reh cells were fixed with chilled 75% ethanol for 2 h at −20°C. Following rehydration in PBS for 15 min, cells were washed twice and resuspended in 1 ml propidium iodide (CCS012A; Multi Sciences Biotech Co., Ltd., Hangzhou, China), then incubated at room temperature for 30 min. Samples were analyzed using the FC 500 MCL flow cytometer. Data were analyzed using MultiCycle software, version 3.2 (Beckman Coulter, Inc.).

A previously reported (16), NR2F2-targeting shRNA (5′-AGGTAACGTGATTGATTCAGTATCTTA-3′) was cloned into the pGLV3 plasmid (Genepharm, Inc., Sunnyvale, CA, USA). A scrambled sequence (5′-TTCTCCGAACGTGTCACGT-3′) was also cloned into the pGLV3 as a negative control. shRNAs were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Highly concentrated lentivirus was produced by co-transfecting 293T cells (Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) with pMD2.G (Thermo Fisher Scientific, Inc.), psPAX2 (Thermo Fisher Scientific, Inc.) and shRNA plasmids with Lipofectin (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The viral suspension was collected, filtered and used to transfect BM-MSCs directly following removal of medium. After 10 h, virus-containing medium was replaced with fresh medium. The positive expression of green fluorescent protein was assessed by fluorescent microscopy (Eclipse-Ti K11301; Nikon Corporation, Tokyo, Japan) and used to evaluate transfection efficiency.

Transfected BM-MSCs were seeded onto a 96-well plate at a concentration of 5×10³ cells/well. Five parallel wells of cells were seeded for each group (knockdown BM-MSCs group and negative control BM-MSCs group). Following incubation for 48 h, 10 µl 3-(4,5-dimethyl-2-thiazolyl) 2,5-diphe-nyltetrazolium bromide (MTT; LK-MTT001; Hangzhou Lianke Biology Technology Ltd., Co., Hangzhou, China) was added to all wells. Medium was discarded after 4 h. The optical density (OD) values were evaluated by the SpectraMax M5 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at 570 nm following addition of 100 µl dimethyl sulfoxide (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China).

The concentration of VEGFA in the co-culture medium was assessed using an ELISA kit (BMS277/2; eBioscience, Inc.). Briefly, co-culture medium supernatant was collected at 24 or 48 h following separation of Reh cells by concentration using centrifugation at 300 × g at 20°C for 10 min. An adequate volume of sample was incubated with pre-coated plates according to the manufacturer's protocol. The OD value was detected at 450 nm using the SpectraMax M5 microplate reader. Standard curves were produced using the standards of known concentration supplied in the kit and used to calculate protein concentrations.

Co-cultured BM-MSCs were isolated by thoroughly washing with PBS until all Reh cells were removed. Total cellular RNA was extracted from co-cultured BM-MSCs using TRIzol reagent (15596-026; Thermo Fisher Scientific, Inc.) PrimeScript RT reagent kit with gDNA Eraser (RR047A; Takara Biotechnology Co., Ltd., Dalian, China) was used for RT. The RT protocol was as follows: 37°C for 15 min; 85°C for 5 sec; stored at 4°C. qPCR was performed using SYBR Premix Ex Taq II (RR420A; Takara Biotechnology Co., Ltd.) according to manufacturer's instructions in a LightCycler system (Roche Diagnostics, Basel, Switzerland). Each reaction was performed in triplicate. The cycling conditions were as follows: Initial denaturation at 95°C for 5 min, 45 cycles of 95°C for 5 sec and 60°C for 20 sec, melting curve analysis at 95°C for 15 sec, 65°C for 15 sec and then maintained at 95°C. The expression of glyceraldehyde 3-phophate dehydrogenase (GAPDH) was used as a reference gene. The primers used were as follows: NR2F2, forward 5′-CAACCAGCCGACGAGATT-3′ and reverse 5′-ATTGCTCTATGACTGAGGAGGAGAC-3′; VEGFA, forward 5′-ACAGGTACAGGGATGAGGACAC-3′ and reverse 5′-AAGCAGGTGAGAGTAAGCGAAG-3′; GAPDH, forward 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse 5′-AGGGGCCATCCACAGTCTTC-3′.

The transfected BM-MSCs were harvested by digesting using 0.25% trypsin-EDTA solution. Cells were lysed on ice for 1 h in radioimmunoprecipitation assay lysis buffer (AR0105; Wuhan Boster Biological Technology, Ltd., Wuhan, China) with phenylmethylsulfonyl fluoride solution (AR1179; Wuhan Boster Biological Technology, Ltd.). Cell lysates were centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was incubated at 100°C for 10 min following addition of an appropriate volume of loading buffer (5-fold dilution; Hangzhou Fu De Biological Technology, Co., Ltd., Hangzhou, China). Samples were loaded onto a 10% polyacrylamide gels containing sodium dodecyl sulfate (Hangzhou Fu De Biological Technology, Co., Ltd.), electrophoretically separated (110 V for 100 min) and transferred onto a nitrocellulose membrane. Membranes were blocked at 20°C for 2 h with 5% non-fat milk then incubated with the primary antibodies at 4°C overnight, and then incubated with fluorescent IgG IRDye^® 650 goat anti-mouse (1:10,000; 926-65010) and goat anti-rabbit (1:10,000; 926-65020) secondary antibodies (LI-COR, Inc., Lincoln, NE, USA) at room temperature for 1 h. Rabbit monoclonal NR2F2 (1:1,000; #6434) and mouse monoclonal GAPDH (1:1,000; #5079) primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and Multi Sciences (Lianke) Biotech Co., Ltd., respectively. Immunoreactive bands were visualized using an Odyssey Infrared Imaging System (LI-COR, Inc.). GAPDH was used as a loading control.

All independent experiments were repeated ≥3 times. Statistical analysis was performed using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). Differences between mean values were evaluated using Student's two-tailed t-test. Values are presented as the mean ± standard deviation and P<0.05 was considered to indicate a statistically significant difference.

BM-MSCs exhibited a spindle-like shape and fibroblast-like morphology. BM-MSCs were CD90- and CD105-positive, and predominantly CD73-positive. However, the cells were negative for CD19 and CD11b, and hematopoietic markers, including CD34 and CD45. The characteristics of BM-MSCs have been described in a previous report (17).

In order to assess whether BM-MSCs promote Reh cell proliferation, Reh cells were isolated and the expression of the Ki-67 antigen was analyzed. As demonstrated in Fig. 1A and B, Reh cells that were co-cultured with BM-MSCs for 48 h exhibited a significantly increased percentage of Ki-67-positive cells compared with Reh cells cultured alone (P<0.05). The present study also assessed whether BM-MSCs affected the apoptosis of Reh cells. Co-culture with BM-MSCs for 48 h had no observable effect on the apoptosis of Reh cells (Fig. 1C and D). Additionally, the cell cycle pattern of Reh cells was analyzed in the co-culture system. When co-cultured with BM-MSCs, the percentage of Reh cells in the G₂/M phase was markedly increased compared with Reh cells cultured alone. Whereas the percentages of cells in G₀/G₁ or S phases were not significantly different (Fig. 2).

To analyze the expression levels of NR2F2 in BM-MSCs, BM-MSCs were separated from Reh cells following co-culture for 48 h. The mRNA and protein levels of NR2F2 in BM-MSCs were observed to be upregulated when co-cultured with Reh cells compared with culture alone (Fig. 3A and B). In order to further elucidate whether NR2F2 is involved in mediating the BM-MSC-induced proliferation of Reh cells, NR2F2 expression was measured in BM-MSCs, and was identified to be downregulated by transfection of an NR2F2 antisense shRNA. Western blot analysis demonstrated that the expression of NR2F2 was significantly knocked down by the shRNA (Fig. 3C). As demonstrated in Fig. 4A and B, the percentage of Ki-67-positive Reh cells in the NR2F2 knockdown BM-MSC co-culture group was significantly reduced compared with the negative control group (P<0.05). Furthermore, the percentage of Ki-67-positive Reh cells in the NR2F2 knockdown BM-MSC co-culture group was similar to the percentage observed when Reh cells were cultured alone. Additionally, the cell viabilities of BM-MSCs in the knockdown and control groups were compared to exclude the possibility that silencing NR2F2 affected the cell viability. As demonstrated by the MTT assay, no significant difference in cell viability was observed between the two groups (Fig. 4C). These data suggested that NR2F2 is involved in the regulation of BM-MSC-induced proliferation of Reh cells.

As it has been previously reported that VEGFA provides important proliferative cues for ALL cells in a BM-MSC-conditioned system (6), the present study attempted to evaluate whether the effects of NR2F2 on the co-culture system were mediated by VEGFA. BM-MSCs were isolated from the co-culture after 48 h and the mRNA expression levels of VEGFA were measured in NR2F2 knockdown and negative control cells. Following NR2F2 knockdown, VEGFA was significantly downregulated (P<0.05; Fig. 5A). To further investigate the association between NR2F2 and VEGFA, the concentration of VEGFA in the co-culture supernatant was measured by ELISA at 24 and 48 h. A relatively high concentration of VEGFA was detected in the supernatant of BM-MSCs (Fig. 5B). However, the concentration of VEGFA was barely detectable in the supernatant of Reh cells cultured alone (data not shown). The concentration of VEGFA was significantly reduced in the NR2F2-knockdown group compared with the negative control at 24 and 48 h (P<0.05; Fig. 5C). Thus, these results demonstrated that VEGFA is involved in the mediation of the effects of NR2F2 on BM-MSC-induced proliferation of Reh cells.