Under the approval of the Ethics Committee of the First Affiliated Hospital of Soochow University, human placentas and peripheral blood samples were obtained after healthy donors gave written informed consent.
The animal studies were performed following the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH), and licensed by the Animal Research Committee of the First Affiliated Hospital of Soochow University.
Preparation of MSCs, Ob-MSCs, De-MSCs and Re-MSCs
Normal full-term human placentas (≥37 weeks gestational age) were collected from healthy donors. Placenta MSCs were isolated and cultured as described previously . The tissue was digested with 0.01 % collagenase type II solution, and then filtered through a 100-μm cell strainer. The cells were centrifuged and resuspended in Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG, Hyclone, Logan, UT, USA) supplemented with 10 % fetal bovine serum (FBS, Hyclone, USA), 100U/mL penicillin G, 100 μg/mL streptomycin, and then seeded in 6-well plates. Cells were incubated at 37 °C in a 5 % CO2 atmosphere. Nonadherent cells were removed after 3 days, and the medium was replaced every 3 days. When cells reached approximately 70–80 % confluence, the adherent cells were trypsinized by 0.05 % trypsin/EDTA and expanded.
To obtain De-MSCs, MSCs were cultured in osteogenic induction medium for 7 days and then subjected to complete medium (DMEM-LG with 10 % FBS) without inducible factors for 2 weeks. The osteogenic induction medium contains 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerolphosphate (Sigma-Aldrich, USA) and 0.25 mM ascorbate (Sigma-Aldrich, USA) in DMEM-HG (Hyclone, USA) with 10 % FBS. To gain MSC-derived osteoblasts (Ob-MSCs), MSCs were subjected to osteogenic induction medium for 7 days. Similarly, after osteogenic induction of De-MSCs for 7 days, osteoblasts derived from De-MSCs (Re-MSCs) were obtained.
Multilineage differentiation of MSCs and De-MSCs
To induce osteoblasts, both MSCs and De-MSCs were subjected to osteogenic medium for 21 days. Then, cells were incubated for 30 mins with Alizarin Red S (Sigma-Aldrich, USA) at pH 4.1 at room temperature to evaluate calcium accumulation.
For adipogenesis, cells were seeded in 6-well plates and treated in DMEM-HG (Hyclone, USA) with 1 μM dexamethasone (Sigma-Aldrich, USA), 10 μg/mL insulin (Sigma-Aldrich, USA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, USA), 0.2 mM indomethacin (Sigma-Aldrich, USA) and 10 % FBS. The adipogenic differentiation medium was changed every 3 days for 2 weeks. Oil Red O (Sigma-Aldrich, USA) was used to visualize lipid-rich vacuoles.
For chondrogenic differentiation, approximately 2–3 × 106 cells in 0.5 mL medium were centrifuged at 500 rpm/min for 10mins in a 15 mL polypropylene tube to form a pellet. Without disturbing the pellet, cells were cultured for 21 days in DMEM-HG supplemented with 10 % FBS, 10 ng/mL transforming growth factor beta 1 (TGF-β1) (Peprotech, Rocky Hill, NJ, USA), 0.5 μg/mL of insulin (Sigma-Aldrich, USA), 50 μM ascorbic acid (Sigma-Aldrich, USA). On day 21, cells were kept in 10 % formalin for 1 h at room temperature, dehydrated in serial ethanol dilutions, and embedded in paraffin blocks. Paraffin sections were stained histologically with Toluidine Blue solution (Sigma-Aldrich, USA) to demonstrate the presence of intracellular matrix mucopolysaccharides.
Cell proliferation evaluation and alkaline phosphatase (ALP) assay of MSCs and De-MSCs osteogenesis in vitro
The viability and proliferation of MSCs and De-MSCs were measured in osteogenic medium with a Cell-Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) for 7 days. At the desired time points, cells were incubated in CCK-8 solution for 2 h. The absorbance was read at 450 nm by microplate reader (BioTek, Winooski, VT, USA) and the live cell number was correlated to optical density (OD).
MSCs and De-MSCs (104 cells/cm2) were seeded in 6-well plate and replaced with osteogenic induction medium. ALP activity was respectively assayed before (0 days) and 7, 14, and 21 days after induction, by a BCIP/NBT alkaline phosphatase color development kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions, and ALP-positive cells were stained in blue.
Osteogenesis of MSCs and De-MSCs in vivo
Collagen scaffolds were prepared according to the previous method  and cut into 3 mm × 3 mm × 3 mm collagen bundles. A total of 50 μL suspension of the MSCs or De-MSCs cells (1 × 106 cells/bundle) was seeded on the collagen scaffolds and then implanted subcutaneously in the back of nonobese diabetic/severe combined immunodeficient mouse recipients (NOD/SCID). Seven days after transplantation, the scaffolds were collected and digested by collagenase to harvest the loaded cells. To investigate the osteogenic capability of MSCs and De-MSCs in vivo, ALP activity assay was performed on some cells, the others were evaluated for expression of human osteogenesis-related genes by quantitative real-time PCR assays (qRT-PCR). Thirty days later, the implants were gathered, fixed and sliced for hematoxylin and eosin (H&E) staining and immunohistochemical staining for collagen II expression (rabbit anti-human collagen II, Abcam, Cambridge, MA, USA).
qRT-PCR for gene expression
To evaluate the osteogenic differentiation potential of MSCs and De-MSCs, the gene expression at the mRNA level was examined. Seven days after induction in vitro or implantation in vivo, total RNA of different cells were extracted using Trizol reagent (Invitrogen, Waltham, MA, USA) and reverse-transcribed into complementary DNA (cDNA) with the M-MLV reverse transcriptase kit (TaKaRa, Shiga, Japan). By using a Brilliant SYBR Green QPCR Master Mix (TakaRa, Japan), the cDNA templates were subjected to qRT-PCR to semi-quantify the gene expressions of human bone morphogenetic protein 2 (BMP2), human Runt-related transcription factor 2 (Runx2) and human Osterix (Osx), normalized to the expression of human glyceraldehyde-3-phosphate dehydrogenase (GADPH). BMP2: forward primer 5′-GCACCAAGATGAACACAG-3′, reverse primer 5′-AGGGCATTCTCCGTGGCAGT-3′; Runx2: forward primer, 5′-TCTTCACAAATCCTCCCC-3′, reverse primer, 5′-TGGATTAAAAGGACTTGG-3′; Osx: forward primer, 5′-CAACTGGCTCTTCTGCGGCAAGAG-3′, reverse primer 5′-GCTGGTGTTTGCTCAGGTGGTC-3′; GAPDH: forward primer, 5′-AGAAGGCTGGGGCTCATTTG-3′, reverse primer 5′-AGGGGCCATCCACAGTCTTC-3′. The relative expression level of target gene was calculated by the 2−ΔΔCt method. Fold was used to show the change times of mRNA level normalized by the undifferentiated MSCs in vitro and differentiated MSCs in vivo.
Flow cytometry (FCM) analysis for surface markers on cells in vitro
MSCs, Ob-MSCs, De-MSCs and Re-MSCs were collected and FCM analysis was performed with following mouse anti-human monoclonal antibodies (eBioscience, San Diego, CA, USA), FITC-, PE- or APC-conjugated CD29, CD34, CD45, CD90, CD166, CD105, CD28, CD80, CD83, CD86, HLA-DR, MHC-II, programmed cell death 1 ligand 1 (PD-L1), Programmed cell death 2 ligand 1 (PD-L2), B7-homolog 3 (B7-H3, CD276). Conjugated mouse IgG k-chain served as isotype control. Samples were detected by FCM (FC 500FCL, Beckman Coulter, Indianapolis, IN, USA) and analyzed by Flowjo 7.6.1 software (TreeStar, Ashland, OR, USA).
Mixed lymphocyte reaction (MLR) assay in vitro
Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and cultured in RPMI 1640 with 10 % FBS, 100U/mL penicillin G and 100 μg/mL streptomycin. CD3+ T cells were isolated from PBMCs using CD3+ T cell isolation kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. MSCs, Ob-MSCs, De-MSCs or Re-MSCs were treated by mitomycin C (MMC, 10 μg/mL, Sigma-Aldrich, USA) for 2 h at 37 °C, then washed three times with PBS containing 1 % FBS.
For MLR assay, T cells (1 × 105 cells per well) with anti-human CD3 and CD28 antibodies (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) (30 ng/mL, respectively) were cultured in the presence of the MMC-treated MSCs, Ob-MSCs, De-MSCs or Re-MSCs (2 × 104 cells/well). All the T cells were further cultured for 3 days, 1 μCi [3H] thymidine was added into the wells 18 hours before the experiment stopped and counts per minute (cpm) was measured by β-scintillation counter (Shimadzu Corporation, Kyoto, Japan).
Detection of primed lymphocytes by FCM in vivo
Six-week-old female BALB/c mice were immunized with MMC-treated MSCs, Ob-MSCs, De-MSCs and Re-MSCs in normal saline (N.S.), respectively. The mice treated with vehicle were served as control group. 1 × 106 cells in 200 μL N.S. were subcutaneously injected into mice. The PBMCs from mice were harvested 5 days and suspend splenocytes were harvested 7 days after priming. The different cell populations were assayed by FCM. The antibodies specific to mouse (eBioscience, USA) conjugated with PE, FITC, APC were applied to mark mouse CD25, CD4, CD80, CD11b, CD11c, and CD45R (B220).
Values were presented as mean ± standard deviation (SD) and analyzed statistically by GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA, USA). Two-tailed unpaired Student t test was applied between two groups, while one-way ANOVA followed by Tukey’s multiple comparison test was used among more than two groups. Probability values were considered statistically significant at P < 0.05.