Blood sample and ethics statement
The use of human peripheral blood was approved by the Institutional Review Board (IRB). The IRB or Ethics Committee of Beijing Chao-Yang Hospital approved this study. The participants provided their written informed consent to participate in the study. The ethics committees or IRBs of Beijing Chao-Yang Hospital approved this consent procedure. This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the National Institute of Biological Sciences and with the Guide for the Care and Use of Laboratory Animals. PBCs were either obtained from a blood bank or were freshly collected from volunteers. Peripheral blood mononuclear cells (PBMNCs; lymphocytes and monocytes) were obtained by density gradient centrifugation with Ficoll-paque plus (GE) at room temperature. The age of the healthy donors (male or female) ranged from 20–40 years. The human mesenchymal stem cells (hMSCs) were derived and donated by Dr. Xia [12].
Generating integration-free hiPSCs and hiPSC culture
The human episomal vectors pEV SFFV-OS (OCT4-2a-SOX2), pEV SFFV-MK (MYC-2a-KLF4), and pEV SFFV-B (BCL-XL) were kindly donated by Dr. Xiao-Bing Zhang. PBMNCs were cultured for 5 days before nucleofection. To generate integration-free iPSCs, cells were nucleofected with 20 μg EV plasmid DNA (10 μg OS + 5 μg MK + 5 μg BCL-XL). PBMNCs (1.5 × 106) were nucleofected by Amaxa Nucleofector® Program U-008 (Lonza) and then seeded into a 35-mm dish preseeded with feeder. The first colonies appeared at days 10–14 after coculture of PBMNCs with feeder. The number continued to increase. About 25 iPSC colonies were selected for further culture. The hiPSC medium was composed of knockout Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 15 % knockout serum replacement (KSR; Invitrogen), 5 % fetal bovine serum (FBS; Hyclone), 1 × nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 1 mM L-glutamine (Invitrogen), and 8 ng/ml basic fibroblast growth factor (bFGF; Peprotech). iPSCs were passaged every 4–6 days by treatment with dispase (Invitrogen). After 10 passages, iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability (embryoid body (EB) and teratoma formation) examination.
Karyotype analysis
One day after human iPSCs were subcultured, the cells were exposed to 0.25 μg/ml colcemid for 3.5 hours, digested, collected, and exposed to a hypotonic solution (0.4 % sodium citrate:0.4 % KCl = 1:1) for 16 minutes. The cells were fixed twice with methanol/acetic acid (3:1) for 1 hour in total. Then the cells were dropped onto cold, wet and clean glass slides. The slides were incubated for 4 hours at 70 °C. Then the slides were treated with 0.01 % trypsin at 37 °C for 10–12 seconds and washed with 0.9 % NaCl and then stained with Giemsa solution (Giemsa:phosphate buffer = 1.5 ml:40 ml, pH 7.4) at 37 °C for 2.5 minutes. The karyotype was determined by microscopic examination. More than twenty chromosomal spreads were counted per population.
Immunocytochemical analysis
Colonies were fixed for 2 hours at room temperature with 4 % paraformaldehyde and then incubated at room temperature for 15 minutes with 1 % Triton X-100/phosphate-buffered saline (PBS). Cells were washed three times in PBS and blocked at 37 °C for over 3 hours with 4 % normal goat serum (Chemicon). Subsequently, cells were incubated at 4 °C overnight with primary antibody to Oct4 (1:500, Santa Cruz), SSEA4 (1:500, Life Technology), Nanog (1:500, Bethyl laboratories), TRA-1-60 (1:250, Life Technology), and TRA-1-81 (1:250, Life Technology). Cells were washed three times in PBS and incubated at 37 °C for 2 hours with goat anti-rabbit Alexa-Flour 594-conjugated (Life technologies) and goat anti-mouse Alexa-Fluor 488-conjugated (Life Technology) secondary antibodies (1:500 in 1 % normal goat serum in PBS). Unbound secondary antibodies were removed in three washes with PBS. Nuclei were identified by 1 μg/ml DAPI (Invitrogen) staining at room temperature for 5 minutes. Images were acquired using a confocal laser scanning microscope (LSM 510 META; Carl Zeiss).
In vitro and in vivo differentiation
In vitro differentiation was performed by the EB formation method. The undifferentiated hiPSC colonies were manually dissected into smaller pieces using a fire-drawn glass needle. They were cultured in nonadherent petri dishes containing hiPSC medium without bFGF. EBs were characterized by the pieces of colonies taking on a round appearance, with smooth borders. Some irregularly shaped but smooth-bordered EBs may be several EBs clustered together.
In vivo differentiation was established by formation of teratomas and examined by hematoxylin and eosin staining. The undifferentiated hiPSC colonies from a confluent 60-mm dish were manual dissected into smaller pieces. Cells were suspended in 200 μl PBS containing 2 % KSR and were injected under the inguinal skin of SCID mice. After 7–8 weeks the teratomas were excised and fixed in 10 % neutral buffered formalin for 24 hours. Dehydration was performed with graded ethanol series followed by three consecutive steps of clarification in xylene and paraffin-embedment during tissue processing. Approximately 5 μm adjacent sections were made using a microtome. The slides were allowed to dry overnight at 42 °C. Deparaffinization was performed in two cycles using xylene. Decreasing alcohol series were used for rehydration, followed by a final rinse with deionized water for 5 minutes. Hematoxylin solution was added to the tissue for 1 minute. The sections were stained blue in NaOH until the nuclei stained bluish-purple and detected under microscopy. Sections were placed in deionized water for 3 minutes after being washed in distilled water. Dehydration was performed with graded ethanol series. Eosin solution was added to the tissue for 1 minute. Dehydration was performed with 100 % EtOH followed by clarification with xylene. The slides were mounted with resinous mounting medium and visualized under a microscope.
Cell pellet formation and chondrocyte differentiation
The undifferentiated hiPSC colonies were manually dissected into smaller pieces and cultured for 10 days in nonadherent petri dishes containing hiPSC medium without bFGF for EB formation. Approximately 25 % of the initial media was replaced with an equal amount of the differentiation media (DMEM, 20 % FBS, 1 × non-essential amino acids, 0.1 mM β-mercaptoethanol, 1 mM L-glutamine) every 2 days. EBs were then seeded onto 10 cm gelatin-coated dishes. Within 10 days of cellular confluence, the cells were incubated with 0.25 % trypsin/EDTA at 37 °C for 5 minutes, and reseeded on new gelatin-coated dishes. At 90–100 % confluence of cells, which is often observed in 5 to 7 days, the cells were harvested with 0.25 % trypsin/EDTA. The cells were sorted by CD73 and CD105 double positivity. Then they (3 × 105) were placed in a 15-ml polypropylene tube, centrifuged at 1200 rpm for 3 minutes at room temperature, and resuspended in chondrogenic differentiation medium (DMEM-HG supplemented with 10 % ITS (Invitrogen), 10−7 M dexamethasone, 1 mM ascorbate-2-phosphate (Invitrogen), 1 % sodium pyruvate (Invitrogen), and 10 ng/ml transforming growth factor-beta 1 (Peprotech)). The cells were recentrifuged and maintained in small pellet form for 21 days. The culture medium was replaced every 3 days. The hMSCs were also collected and cultured in this chondrogenic differentiation medium for 21 days.
Flow cytometry analysis
Before cells were placed in a 15 ml polypropylene tube, the cell populations acquired a homogenous, fibroblast-like morphology. The iPSC–MSC-like cells were grown to attain confluence, harvested by 0.25 % trypsin/EDTA, washed with PBS, and resuspended in staining buffer consisting of 2 % FBS in PBS. Cell suspensions were mixed with PE mouse anti-human CD73, APC mouse anti-human CD105, APC mouse anti-human CD45 and PE mouse anti-human CD34. The isotype controls used were mouse IgG1 K isotype control APC and mouse IgG1 K isotype control PE, which were appropriate isotypes related to antibodies (eBioscience) in our experiments. Samples were run on a LSR II Flow Cytometer (BD Biosciences) instrument. For each analysis, a minimum of 10,000 cells were assayed. hMSCs were also used as a positive control and detected.
Histological analysis of chondrogenic differentiation
Chondrogenic differentiation was assessed by alcian blue and toluidine blue staining of pellet sections. The aggregates were fixed in 10 % neutral-buffered formalin for 24 hours. Dehydration was performed with graded ethanol series followed by three consecutive steps of clarification in xylene and paraffin-embedment during tissue processing. Approximately 4-μm adjacent sections were made using a microtome. The slides were allowed to dry for 2 hours at a temperature of 60 °C. Deparaffinization was performed over three cycles using xylene. Decreasing alcohol series were used for rehydration, followed by a final rinse with deionized water for 5 minutes. The sections were then stained with 0.1 % alcian blue reagent or 1 % toluidine blue staining for 4–5 hours and were rinsed with distilled water. Dehydration was performed with graded ethanol series followed by three consecutive steps of clarification in xylene and the slides were mounted in Permount® (Fisher) and visualized under a microscope. Additional chondrogenic differentiation sections were further assessed by immunohistochemistry.
Immunohistochemistry
Pellet sections for immunohistochemical staining were performed with a sequence of treatments as for the histological analysis. The sections were transfered onto glass slides suitable for immunohistochemistry examination. After deparaffinization and rehydration, we brought the slides to a boil in 1 mM EDTA pH 8.0 followed by 8 minutes at a sub-boiling temperature and then allowed the slides to cool at room temperature. The slides were rinsed with deionized water three times. Endogenous peroxidase activity was blocked by incubating sections in 3 % H2O2 solution in methanol at room temperature for 15 minutes. The slides were rinsed with deionized water and immersed with PBS for 5 minutes. Appropriately diluted primary antibody (50–100 μl) was applied to the sections on the slides and then they were incubated in a humidified chamber at room temperature for 1 hour. The following primary antibodies were applied: rabbit polyclonal antibodies against collagen II (1:50, Abcam) or mouse monoclonal antibodies to collagen X (1:50, Abcam). The slides were washed three times with PBS, 5 minutes each time. The samples were then incubated with the corresponding secondary antibodies (anti-rabbit and mouse; Roche) for 15 minutes at room temperature. The slides were further washed three times with PBS, 5 minutes each time, followed by DAB (Roche) detection under microscopy. The slides were washed three times with PBS, 2 minutes each time. The cell nuclei were counterstained by immersing the slides in hematoxylin for about 1–2 minutes. Dehydration was performed with graded ethanol series followed by three consecutive steps of clarification with xylene and the slides were finally mounted in Permount® (Fisher) and visualized under a microscope. Negative controls were processed following the same procedure omitting the primary antibodies to exclude the level of background staining.
Sulfated glycosaminoglycan content
Sulfated glycosaminoglycan (sGAG) content of chondrogenic pellets was analyzed using the dimethylmethylene blue (DMMB; Sigma) spectrophotometric method. Cells or chondrogenic pellets were digested in 50 μl papain solution (10 U/mL in PBS with 0.1 M sodium acetate, 2.4 mM EDTA, 5 mM L-cysteine; Sigma) at 60 °C for 2 hours. sGAG content in sample digest (50 μl) was measured by mixing with DMMB dye solution (1 ml of 16 mg/l 1,9-dimethylmethylene blue, 40 mM glycine, 400 mM NaCl, pH 3.0) and measuring absorbance (525 nm). The concentration of sGAG in each sample was calculated against a standard curve of shark chondroitin sulfate (Sigma). DNA content was determined using the Qubit dsDNA HS assay kit and Qubit Fluorometer system (Invitrogen).
In vivo chondrogenesis study
Six-week-old SCID mice were intraperitoneally anesthetized using avertin (1.25 %, Sigma). hiPSC–MSC-like cells, which were chondrogenically induced for 10 days in vitro, were implanted into the kidney capsule of the mice. The specimens were harvested 6 weeks after implantation. These specimens were fixed in 10 % neutral-buffered formalin, dehydrated, clarified, and embedded in paraffin. Sections were subjected to standard procedures with alcian blue staining, toluidine blue staining and collagen II and X immunochemical staining.
Real-time polymerase chain reaction analysis
Total RNA was extracted by TRIzol reagent (Invitrogen). RNA was then converted into cDNA using the Reverse Transcriptase System (Promega). cDNA samples were subjected to real-time polymerase chain reaction (PCR) with KAPA SYBR FAST qPCR kit Master Mix (2×) using a real-time PCR System. The primer sequences were: hAGGRECAN-F:TCGAGGACAGCGAGGCC, hAGGRECAN-R: TCGAGGGTGTAGCGTGTAGAGA; hβ-ACTIN-F: TTTGAATGATGAGCCTTCGTCCCC, hβ-ACTIN-R: GGTCTCAAGTCAGTGTACAGGTAAGC; hCOL2-F:TGGACGATCAGGCGAAACC, hCOL2-R:GCTGCGGATGCTCTCAATCT; hSOX9-F:AGCGAACGCACATCAAGAC, hSOX9-R:CTGTAGGCGATCTGTTGGGG; hCOL10-F: ATGCTGCCACAAATACCCTTT, hCOL10-R: GGTAGTGGGCCTTTTATGCCT. The 20 μl PCR systems contained 1 μl cDNA template, SYBR Green qPCR Master Mix, 0.25 mM forward primer and reverse primer. The reactions were incubated in a 96-well plate at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. All reactions were repeated three times. β-actin was used as an internal control.
Statistical analysis
All quantitative data were collected independently at least three times and yielded reproducible results. Data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance. Differences were considered statistically significant at P < 0.05.