Animals
The umbilical cord was obtained using pregnant cats (n = 2, 4 ~ 5 years, mixed breed) who visited Kyungpook National University Animal Hospital. In the cesarean-section delivery, the cats were pre-medicated with 0.1 mg/kg acepromazine maleate (Samwoo medical, Korea) and 5 mg/kg propofol (Myeongmun pharmaceutical, Korea) was injected intravenously. Isoflurane (Hana pharmaceutical, Korea) was used to maintain anesthesia. Under sterile conditions, umbilical cords were collected from cesarean section deliveries. Cats were hospitalized and discharged for two weeks after surgery.
Cell Isolation
Cell isolation was performed through some modifications as previously described (1, 2, 25). The collected wharton’s jelly was placed in a sterile culture dish and washed with 0.9% physiological saline and vessel was removed. Tissue was then finely chopped and resuspended in 2 mg/ml collagenase type I solution (US Sigma-Aldrich) at 37 °C for about 20 minutes. After enzymatic digestion, it was centrifuged at 3,000 rpm for 5 minutes and washed in phosphate buffered saline (PBS) (Gibco, USA). Cell pellets were resuspended in culture medium in hypoglycemic Dulbecco's Modified Eagle's Medium (LG-DMEM; Gibco, USA) containing 10% FBS (fetal bovine serum; Gibco, USA) and 1% Penicillin-Streptomycin solutions (Gibco, USA). fWJ-MSCs were seeded in T-75 flasks (Corning, USA) and incubated in a humidified atmosphere with 5% CO2. The culture medium was changed three times a week and passed when the cells reached 80–90% confluence.
Reverse Transcriptase Polymerase Chain Reaction
Total RNA was extracted from the cultured cells with the RNeasy minikit (Qiagen, Germany) according to the manufacturer’s protocol. RNA concentrations were measured by Nanodrop 2000 (ThermoScientific, USA). cDNA was prepared by 1 mg of total RNA for reverse transcription using Superscript II reverse transcriptase (Invitrogen, USA) and oligo dT primers (Invitrogen, USA). The cDNA was amplified using T100™ Thermal Cycler (Biorad, USA). Primers are shown in the Table 1.
Table 1
Genes | Forward primer (5’-3’) | Reverse primer (5’-3’) | Product size |
OCT4 | GCCCGAAAGAGAAAGCGAAC | CGACGATTGCAGAACCACAC | 161 bp |
SOX2 | GCCCTGCAGTACAACTCCAT | TGGAGTGGGAGGAAGAGGTA | 175 bp |
KLF4 | ACCAAGAGCTCATGCCACCT | AAGGCTTCTCACCTGTGTGG | 183 bp |
MYC | AGGAGAAACGAGCTGAAACG | GTTCTCGTCGCTTCCTCAAC | 181 bp |
GAPDH | GTGGAGGGACTCATGACCAC | GTGAGCTTCCCATTCAGCTC | 176 bp |
Cumulative Population Doubling Level Analysis
Proliferation and growth efficiency of fWJ-MSC was determined by CPDL using the formula CPDL = ln (Nf/Ni)ln2, where Ni is the initial seeding cell number, Nf is the final harvest cell number and ln is the natural logarithm. fWJ-MSCs (5 × 104 cells) were plated three times in 6-well culture plates (Corning, USA) and passaged 4 days later. Final cell count was counted and 5 × 104 cells were plated again. To calculate the cumulative doubling level, we calculate the doubling population for each pass and then multiply by the doubling of the previous passages.
Scratch test, Colony Forming Unit test
In scratch test, fWJ-MSCs were incubated in 6-well plates until 90% confluence was reached. Cell scratches were made with a 100 µl pipette tip. DMEM medium containing 5% FBS was used for 24 hours. After 24 hours, inverted microscope was used to observe the healing state of the cells.
In CFU test, Passage 5 fWJ-MSCs were seeded at 1 × 103 cells in 6-well plates and incubated DMEM culture medium containing 10% FBS. After 2 weeks, the plastic adherent colonies were stained with 1% toluidine blue (Scytek, USA).
Karyotype Analysis
In order to detect any chromosomal abnormalities in the fWJ-MSC, karyotyping was performed by the conventinal methods at passage 5. fWJ-MSCs were arrested with 500 ul colcemid (Gibco, USA) in the incubator (37 °C, 5% CO2) for 1 hours. The cells were suspended in a hypotonic solution (0.075 M KCl; Sigma-Aldrich, USA) and incubated for 20 min at 37 °C. And the cells were fixed by washing in Canoy’s fixative (methanol : glacial acetic acid = 3 : 1; Sigma-Aldrich, USA). Chromosome diffusion was obtained by pipetting the suspension into a clean glass and air drying. fWJ-MSCs undergoing metaphase were pictured with a CCD camera (Olympus, Tokyo, Japan), chromosomes were counted, and pattern was analyzed by software (ChlPS-Karyo; GenDix Inc, Korea).
Flow Cytometry
To establish the immunophenotype of fWJ-MSCs, cells were stained with 6 antibodies for FACS analysis, following the protocol provided by the manufacturer (Beckman Coulter, USA). fWJ-MSCs were trypsinized and washed several times with PBS at passage 5. The suspended cells were aliquoted (approximately 1 × 106 cells) for specific antibody staining. The cells were immunostained with the following antibodies shown in the Table 2. The antibodies were conjugated with Fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Analysis was determined by the use of FACS (Gallios Flow Cytometer; Beckman Coulter, USA) and software (Kaluza for Gallios; Beckman Coulter, USA).
Table 2
Marker | Antibody | Company / Catalog# |
CD105 | MOUSE ANTI HUMAN CD105 | BIO-RAD / MCA1557 |
CD90 | PE Mouse Anti-Human CD90 | BD Pharmingen / 555596 |
CD45 | FITC Mouse Anti-Human CD45 | BD Pharmingen / 555482 |
CD44 | PE anti-mouse/human CD44 Antibody | BioLegend / 103024 |
CD34 | FITC Mouse Anti-Human CD34 | BD Pharmingen / 555821 |
CD14 | MOUSE ANTI HUMAN CD14 | BIO-RAD / MCA1568 |
Osteogenic Differentiation
Osteogenic differentiation medium (StemPro Osteogenesis Differentiation Kit; Gibco, USA) was used to prove osteogenic differentiation capability. At passage 5 when cells reached confluency of 80 to 90%, medium was changed to the osteogenic induction medium, and incubated for 3 weeks, changing once every 3 days. After 3 weeks, calcium deposition was found by staining with Alizarin Red S and Von Kossa. For Alizarin Red S staining, cells were washed with PBS and fixed with 70% ethanol for 1 hour at 4° C. The cells were then washed several times with distilled water. Cells were stained with Alizarin Red S (IHCworld, USA) for 10 minutes at room temperature. Cells were washed with nonspecific dye with 5 distilled water. For quantitative measurements, Alizarin Red S stain was solubilized for 1 hour using 100 mM cetyl pyridinium chloride (Sigma-Aldrich, USA). Solubilized Alizarin Red S absorbance was measured at 570 nm using a spectrophotometer. For Von Kossa staining, stain cells with 5% silver nitrate (Scytek, USA) for 30 to 60 minutes while exposing the cells to UV light, then stain with 5% sodium thiosulfate (Scytek, USA) for 2–3 minutes, then counterstaining for 5 minutes with nuclear red stain (Scytek, USA).
Adipogenic Differentiation
To determine if fWJ-MSCs can be differentiated into adipocytes, cells were treated with adipose production differentiation medium (StemPro Adipogenesis Differentiation Kit; Gibco, USA). At passage 5, when the cells reached 80–90% confluence, the medium was changed to adipose production differentiation medium and incubated for three weeks, changing once every three days. After 3 weeks, oil red O staining (Scytek, USA) was performed to detect lipid fibrosis. Cells were fixed with 10% neutral buffered formalin to fix for at least 1 hour and rinsed with 60% isopropanol before incubating for 10 minutes in freshly diluted Oil Red O. Oil red O stain was solubilized using isopropanol and absorbance was measured using a spectrophotometer at 500 nm.
Chondrogenic Differentiation
To confirm that fWJ-MSCs are differentiated into chondrocytes, cartilage formation differentiation medium (StemPro Chondrogenesis Differentiation Kit; Gibco, USA) was used. First, cells (5 × 105 cells) were seeded in 15 ml polypropylene tubes and centrifuged with pellets at passage 5. The pellet was incubated in 1 ml of cartilage forming differentiation medium and incubated for 4 weeks. Differentiation medium was replaced 3 times a week. After differentiation, pellets were embedded in paraffin and 4 um sections were cut. For histological evaluation, sections were stained with toluidine blue (Scytek, USA). For quantitative measurements, toluidine blue staining was solubilized with 100 mM cetylpyridinium chloride (Sigma-Aldrich, USA) for 1 hour. Solubilized toluidine blue absorbance was measured at 600 nm using a spectrophotometer.
Quantitative Real-Time Polymerase Chain Reaction
Quantitative Real-time PCR (qRT-PCR) was performed by mixing cDNA with primers and LightCycler® 480 SYBR Green I Master (Roche Diagnostics, Germany). qRT-PCR was performed using an LightCycler 480 II with supplied software (Roche applied science, Germany) according to the manufacturer’s instructions. RNA expression levels were compared after normalization to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used in this study are listed in Table 3.
Table 3
List of Real-time PCR Primer
Genes | Forward primer (5’-3’) | Reverse primer (5’-3’) | Product size |
MSX2 | GCCTCCAAGACACATGAGC | CCTGGGTCTCTGTGAGGTTC | 185 bp |
LPL | TGGCGGAGGAATTTCACTAT | AGGAGAAAGGCGACTTGGAG | 176 bp |
LEPTIN | AGCAGCTTGGCTGACAATTT | CCAGCAATCACTCCTGGTCT | 178 bp |
FABP4 | CATCAGTGTGAATGGGGATG | CCACTTCTGCACCTGTACCA | 169 bp |
COL2A1 | CCCTAGAGGTCCTCCTGGTC | CAAAGGCAGACATGTCGATG | 188 bp |
GAPDH | GTGGAGGGACTCATGACCAC | GTGAGCTTCCCATTCAGCTC | 176 bp |
Statistical Analysis
The data were analyzed by Student’s t-test using Excel software (Microsoft, USA) and expressed as the mean ± standard error. Statistical significant data are indicated by asterisks (***p < 0.001, **p < 0.01, *p < 0.05).