Cell culture and characterization

The C57BL/6J pESC and J1 mouse ESC lines were cultured on dishes coated with 0.1 % (w/v) gelatin (Sigma-Aldrich, St. Louis, MO, USA) and expanded in Serum-Free Clonal Grade Medium (Millipore, Billerica, MA, USA). Cells were passaged every 5 days by 1 % accutase (Millipore), and observed by phase-contrast microscope (Nikon, Japan) during the process of culture.

Immunofluorescence staining was performed to detect stemness marker expression. pESCs and ESCs were plated on gelatin-coated (Sigma-Aldrich) glass cover lips and fixed by cold 4 % paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, followed by washes three times with PBS and permeabilization with 0.25 % Triton X-100 (Sigma-Aldrich) for 10 min. Cells were then blocked with 10 % bovine serum albumin (BSA; Sigma-Aldrich) for 45 min and incubated overnight at 4 °C with 1:200 diluted primary antibodies, including goat anti-OCT3/4, rabbit anti-NANOG; and mouse anti-SSEA-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three washes with PBS, the cells were incubated for 30 min at room temperature with fluorescein isothiocyanate (FITC)-labeled secondary antibodies (Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained with 4,6′-diamidino-2-phenylindole (DAPI; Invitrogen). Images were obtained with a laser confocal microscope (FV1000; Olympus, Japan).

To confirm whether pESCs and ESCs possessed pluripotent differentiation capacity in vivo, ESCs and pESCs were dispersed using 1 % accutase, and 1 × 106 cells were resuspended in 100 μl Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) and injected subcutaneously into nude mice. After 4 weeks, the specimens were harvested and fixed in 4 % paraformaldehyde, dehydrated through a graded ethanol, embedded in paraffin, sectioned at 7 μm, deparaffinized and stained with hematoxylin and eosin (H&E).

Cell differentiation in embryoid bodies

pESCs and ESCs were dispersed and resuspended in DMEM supplemented with 20 % fetal bovine serum (FBS, Gibco), 50 U-μg/ml penicillin–streptomycin (Invitrogen). Subsequently, 1 × 105 cells were transferred into ultra-low attachment dishes (Fisher Scientific, Pittsburgh, PA, USA) to form embryoid bodies (EBs). The medium was changed every 2 days. pESCs and ESCs were able to form EBs when cultured for 3 days, and were continuously cultured in suspension before surface antigen expression detection. EBs in suspension culture for 5 days were fixed in 4 % paraformaldehyde, dehydrated and embedded in paraffin, sectioned at 5 μm, and incubated with primary antibodies at 4 °C overnight. The primary antibodies comprised mouse anti-SSEA-1, rabbit anti-CD151 and cytokeratin (Santa Cruz Biotechnology), and rat anti-CD73 (eBioscience, San Diego, CA, USA). After removal of primary antibodies with three washes with PBS, FITC-labeled secondary antibodies (Invitrogen) were added and incubated for 1 hour at room temperature. The cells were washed three times with PBS and counterstained with DAPI, observed under a laser confocal microscope.

Cell differentiation during adherent culture

EBs cultured in suspension for 5 days were plated onto 0.1 % (w/v) gelatin-coated dishes and cultured with DMEM supplemented with 20 % FBS, 50 U-μg/ml penicillin–streptomycin, 2 mM 


-glutamine (Invitrogen), 1 % nonessential amino acids (Hyclone), 1 % β-mercaptoethanol (Sigma-Aldrich). To measure the expression of three germ layer markers, total RNA of adherently cultured EBs at different time points (5, 10, and 15 day) was isolated using TRIzol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of the normalized RNA samples using a RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocols. Relative levels of mRNA were determined from cDNA by quantitative real-time PCR with a SYBR Green PCR kit (Takara, Japan) in a total sample volume of 20 μl, and the samples were run in triplicate on a Bio-Rad CFX96 Real-Time PCR Detection System in accordance with the manufacturer’s instructions. The primer sequences and the fragment sizes are presented in Table 


. All primers were obtained from Takara.


was used as the reference gene. Single-peak melting profiles were obtained for the amplifications, and the size of the PCR product was confirmed by agarose gel electrophoresis. Each experiment was repeated three times. The ΔΔCT method [


] was used to calculate relative amounts of transcripts.

Table 1

Primers of three germ layer genes and the reference gene for quantitative real-time PCR





























Enrichment of MSCs from EB outgrowths

To enrich mesenchymal stem cells (MSCs), EBs cultured in suspension were adherently cultured as already described for 10–15 days. Cells were then cultured and expanded with MSC medium (MSCM; Lonza, Basel, Switzerland) for 5–6 passages to enrich spindle-shaped cells. Cells were passaged at a high ratio of 1:2 during expansion.

MSCs derived from pESCs and ESCs were named parthenogenetic MSCs (pMSCs) and embryonic MSCs (eMSCs), respectively. Cells were detached from culture dishes by Accutase, collected and washed three times with ice-cold PBS, and resuspended in PBS. FITC-conjugated primary antibodies (CD29, CD44, CD73; all from eBioscience) were added and incubated overnight, followed by two washes with ice-cold PBS. MSC surface antigen expressions were then tested by flow cytometry using FACS Calibur (BD Biosciences) analysis. Isotype-specific antibodies served as controls. Cells were analyzed using CellQuest software (BD Biosciences). At least 1 × 105 cells were analyzed, and three independent tests were performed for each experiment.

For osteogenic differentiation, pMSCs and eMSCs were cultured in osteogenic differentiation medium (DMEM supplemented with 20 % FBS, 50 U-μg/ml penicillin–streptomycin, 50 μM ascorbic acid (Sigma-Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich), and 50 nM dexamethasone (Sigma-Aldrich)) for 21 days. The medium was changed every 3 days. After 21 days, cells were fixed in 4 % PBS-buffered paraformaldehyde and processed for Alizarin red S, Von Kossa staining, and reverse transcription-PCR (RT-PCR) assays for alkaline phosphates (Alp) and osteocalcin (Ocn), to test the osteogenic differentiation.

For chondrogenic differentiation, cells were cultured in chondrogenic medium (DMEM supplemented with 20 % FBS, 50 U-μg/ml penicillin–streptomycin, 50 μM ascorbic acid, 10 ng/ml transforming growth factor-β1 (TGF-β1; R&D Systems, Minneapolis, MN, USA), and 500 ng/ml insulin-like growth factor (IGF; R&D Systems)). The medium was changed every 3 days. After 21 days, cells were processed for Safranin O staining and PCR assays for aggrecan and type II collagen (Col-II).

For adipogenic differentiation, cells were exposed to adipogenic induction medium (DMEM supplemented with 20 % FBS, 50 U-μg/ml penicillin–streptomycin, 200 μM indomethacin (Sigma-Aldrich), and 10 μg/ml insulin (Sigma-Aldrich)) for 14 days. The medium was changed every 3 days. Adipogenic differentiation was indicated by oil-red O staining of lipid droplet formation in cytoplasm and RT-PCR assays for peroxisome proliferator-activated receptor gamma (Pparγ) and CCAAT/enhancer-binding protein alpha (C/ebpα).

For fibroblastic differentiation, cells were treated with DMEM supplemented with 20 % FBS, 50 U-μg/ml penicillin–streptomycin, 50 ng/ml recombinant human connective tissue growth factors (CTGF; BioVendor, Brno, Czech Republic) and 50 μM ascorbic acid, with medium changed every 3 days (pMSCs and eMSCs after CTGF induction were named pFs and ESC-derived fibroblasts (eFs), respectively). Growth factors in pFs and eFs were further measured every 5 days by enzyme-linked immunosorbent assays (ELISA) in triplicate. After the medium was removed by gentle aspiration using a vacuum manifold, the cells were then washed with PBS, and lysed with the addition of 1 ml RIPA buffer for total protein extraction. The cells were removed by scraping, and transferred into 1.5 ml conical tubes. The mixture was incubated on ice for 30 min, with occasional vortexing. The cell extracts were then assayed using mouse EGF, FGF, IGF1, VEGF, PDGFα, PDGFβ, TGFα, and TGFβ1 ELISA™ kits (Lian Shuo Biological, Shanghai, China) in accordance with the manufacturer’s instructions. Subsequently, the OD450 nm value was measured with an enzyme-labeled instrument (Thermo Fisher Scientific). The expression levels of mouse fibroblasts (Fs) were used as controls.




, tenacin-C (


), matrix metalloproteinase-1 (


), Vimentin, and fibroblast-specific protein-1 (


) expression was screened by quantitative real-time PCR 20 days after induction. The primer sequences and the fragment sizes are presented in Table 


. To further detect the marker of derived fibroblasts, immunofluorescent staining was performed for rat anti-Vimentin (BOSTER), mouse anti-cytokeratin, goat anti-FSP1, and COL-I (all from Santa Cruz Biotechnology). Fibroblasts derived from pESCs and ESCs were named pFs and eFs, respectively.

Table 2

Primers of fibroblast phenotypic hallmark genes for quantitative real-time PCR

























Preparation of TESE

To prepare TESE, 150 μl of 10 × DMEM was added into 1 ml ice-cold collagen solution (4 mg/ml type I collagen from rat tail dissolved in 0.1 % acetic acid). After neutralization with 500 μl 0.1 % NaOH solution, 100 μl of cell suspension (5 × 106 cells/ml) was added into the solution and mixed immediately. The whole operation was carried out on ice. The mixture was transferred into dishes (d = 35 mm; Fisher Scientific, Pittsburgh, PA, USA) and then incubated at 37 °C and 5 % CO2 for gelling. Twenty-four hours later, 2 ml DMEM was added, and the TESE was cultured for another 2 days before use [18]. We prepared TESE from pFs, eFs, and Fs, and collagen gel without cells acted as control.

TESE grafting onto mouse skin defects

Twenty BALB/c mice were purchased from the Experimental Animal Center of The Fourth Military Medical University and treated in accordance with the guidelines provided by the Institutional Ethics Committee of Northwest University. Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (20 mg/kg body weight). Then 8 % sodium sulfate was used to depilate the wounding area of animals (instead of shaving) 24 hours before wounding, to ensure synchronization of hair growth. A circular full-thickness 1.5-cm-diameter skin defect was created on the back of each mouse using a biopsy punch (15 mm; purchased from Shanghai LZQ Precision Tool Technology Co., Ltd, Shanghai, China). TESE grafts derived from pFs, eFs, Fs, and control (


 = 5 for each group) were implanted onto the skin defects. Grafts were covered with vaseline gauze and adhesive bandages for 3 days. Animal behavior and wounds were monitored throughout the experiment. Images were recorded at days 0, 3, 6, 9, 12, 15, and 18 post operation with a digital camera (Canon, Japan) to visualize the wound. The wound area was measured by tracing the wound margin and calculated using Image-Pro Plus Software (Media Cybernetics LP, Silver Spring, MD, USA). Investigators measuring samples were blind to groups and treatment. The wound closure percentage was calculated as follows:

$$ left(mathrm{Original} mathrm{defect} mathrm{area} hbox{–} mathrm{actual} mathrm{defect} mathrm{area}right) / mathrm{original} mathrm{defect} mathrm{area} times 100 % $$

Animals were sacrificed at day 18, and the regenerated skin specimens (including dermis and epidermis) were fixed in 10 % buffered formalin for paraffin embedding. Sections were cut at 7 μm, deparaffinized, and stained with H&E. Sections were also stained with a mouse Cytokeratin-14 antibody (1:200; Santa Cruz Biotechnology), and a secondary Alexa-Fluor 594-labeled goat anti-mouse IgG (1:500; Invitrogen) was used.

Statistical analysis

Data are expressed as mean ± SD of at least three independent samples. Statistical comparisons between groups were performed with one-way ANOVA and two-way ANOVA analysis. p < 0.05 and p < 0.01 were considered significant.