Preparation of MSCs
Human umbilical cords were obtained from full-term Caesarean section deliveries and then stored in Dulbecco’s modified Eagle medium (DMEM)/F12 (1:1) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO, Invitrogen Inc. Carlsbad, CA, USA). To isolate stem cells, the cord was cut into 4–5 cm long pieces, and the vessels were pulled away to leave only Wharton’s Jelly (WJ). WJ was cut into 1–2-mm3 pieces and digested with 1 mg/ml of collagenase type 1 (Millipore Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS) at 37 °C for 30 min. The digested mixture was then passed through a 100-μm filter (BD Biosciences, Franklin Lakes, NJ, USA) to obtain cell suspensions. The cells were washed with PBS solution and then cultured in DMEM/F12 medium containing 10 % fetal bovine serum, 2 mmol/L glutamine, 1 % nonessential amino acids, and 1 % penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) at 37 °C and 5 % CO2. Non adherent cells were removed when the medium was changed on day 3. The multipotent MSCs, which were adhering to the plastic bottom were then collected. The multipotent MSCs phenotype is defined as the co-expression of antigens (CD105, CD73, and CD90 [≥95 % positive]) and the absence of hematopoietic lineage markers (CD45 and CD34 [≤2 % positive]). In summary, multipotent MSCs phenotype was confirmed by positive expression markers (CD105 CD73 and CD90-FITC) and negative expression markers (CD45 and CD34-FITC).
Induction of Adipogenic and Chondrogenic Differentiation in MSCs
To induce chondrogenic differentiation, MSCs were treated with chondrogenic medium. The medium was changed twice a week. The chondrogenic medium consisted of DMEM-F12 supplemented with 0.1 μM dexamethasone, 50 μg/mL AsA, 100 μg/mL sodium pyruvate (Sigma-Aldrich), 40 μg/mL proline (Sigma-Aldrich), 10 ng/mL TGF-β1, and 50 mg/mL ITS+ premix (Becton Dickinson; 6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 ng/mL selenius acid, 1.25 mg/mL bovine serum albumin and 5.35 mg/mL linoleic acid).
To induce adipogenic differentiation, the MSCs were incubated in adipogenic medium which was changed twice a week. The adipogenic medium consisted of DMEM-F12 supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma- Aldrich), 1 μM hydrocortisone (Sigma-Aldrich), 0.1 mM indomethacin (Sigma-Aldrich), and 10% FBS.
Preparation of PSL Cells from MSCs
We used small molecular compounds (STC-F002), extracted from sea algae (Eklonia cava). The MSCs was cultured in DMEM-F12 medium containing 10% FBS. When 90% of the dish bottom was covered with these cells, approximately day 5, 10ug/mL of STC-F002 was added to the culture medium (U.S. Patent No. 10,131,881(2018)).
Approximately 10 days later, distinct types of colonies appeared which looked similar to human Embryonic Stem cell (hES) colonies. The hES cell like colonies were carefully picked and mechanically disaggregated into small clumps.
Expression of Pluripotent Stem Cell Markers in PSL
The PSL cells strongly expressed all the characteristic pluripotent stem cell markers. These included SSEA4, a cell-surface glycosphingolipid used for detecting human ES cells; Oct3/4, a protein involved in the self-renewal of human ES cells, Sox2, a transcription factor that controls genes involved in embryonic development and AP, ubiquitous membrane-bound glycoprotein for detecting pluripotent stem cell40. In comparison MSCs were either negative or weakly positive for the pluripotent stem cell markers.
Primary antibodies used for IH of SSEA4 (1:100, Developmental Studies Hybridoma Bank), OCT4 (1:200, R&D Systems), SOX2 (1:200, R&D Systems). The secondary antibodies were Alexa488-conjugated goat anti-rabbit IgG (1:500, Invitrogen). The nuclei were stained with 1 μg/ml Hoechst 33342 (Invitrogen).
Induction of Osteogenic, Chondrogenic, Hepatogenic and Neurogenic Differentiation in PSL
To induce osteogenic differentiation, fifth- to seventh-passage umbilical cord derived stem cells were treated with small molecules for 10 days. The PSL were then cultured in osteogenic medium for 1 week with medium changes twice a week. Osteogenic medium consists of DMEM-F12 supplemented with 0.1 μM dexamethasone (Sigma-Aldrich), 10 mM β- glycerol phosphate (Sigma-Aldrich), and 0.2 mM ascorbic acid (AsA; Sigma-Aldrich). Von kossa staining was used for osteogenic differentiation
To induce chondrogenic differentiation, PSL cells were cultured in chondrogenic medium for 2 weeks. Medium changes were carried out twice a week. Chondrogenic medium consisted of DMEM-F12 supplemented with 0.1 μM dexamethasone, 50 μg/mL AsA, 100 μg/mL sodium pyruvate (Sigma-Aldrich), 40 μg/mL proline (Sigma-Aldrich), 10 ng/mL TGF-β1, and 50 mg/mL ITS+ premix (Becton Dickinson; 6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 ng/mL selenous acid, 1.25 mg/mL bovine serum albumin (BSA), and 5.35 mg/mL linoleic acid). Alcian blue was used to stain for chondrogenic differentiation
To induce hepatogenic differentiation, PSL cells were cultured in hepatogenic medium, which was changed twice a week. Hepatogenic medium consisted of IMDM supplemented with 10nM dexamethasone, 100ng/ml HGF, 50ng/ml FGF and 5.5 ug/mL ITS+ premix. Hepatogenesis was assessed by fluorescence imaging (alpha-fetroprotein)
To induce neurogenic differentiation, a multistep protocol was used. PSL cells were cultured in step 1 medium for 7 days, and then step 2 medium for 7 days. Step 1 medium consisted of DMEM-F12 supplemented with 2% B27 supplement, 2mM L- glutamin, 30ng/ml EGF, and 25ng/ml bFGF. Step 2 medium consisted of DMEM-F12 supplemented with 2% FBS and 25ng/ml BDNF. Neurogenesis was assessed on day 6 and day 10, and imaging for nestin was performed on day 12.
Injection of MSCs and PSL
BALB/c mice (6–8 weeks old) were provided by Oriental Bio Inc. (Seongnam, Korea) and were allowed to adapt to the new environment for 7 days. Experiments were performed in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals in Research and Use Committee of Bundang Hospital of Seoul National University, Korea. This study was approved by the Seoul National University Bundang Hospital Institutional Animal Care and Use Committee (BA1109-091/064-01)37.
The mice were divided into an MSCs (n=9) and an PSL (n=9) group. MSCs and PSL cells were injected into the tail veins of the mice in each group. 5x105 MSCs and 5x105 PSL cells were administered per mouse. In addition, a control group was used for the base line.
Preparation of the miRNA-221 molecular beacon
A miRNA- 221 molecular beacon was used to bio-image the MSCs and PSL cells.
To construct the miRNA-221 MB, NH2-modified molecular beacon, miRNA-221 oligos (5′- ATGAACAGACGCCCCAAACCGACCCAAAG-BHQ2-3′) were synthesized by Bioneer Inc. (Daejeon, Korea). MBs with complementary sequences (miRNA-221) were also synthesized. The Cy5.5-COOH fluorophore were purchased from molecular probe (Thermo Fisher, Waltham, USA). The miRNA-221-linked MB contains cy5.5 (excitation/emission wavelength: 595/625 nm) and BHQ2.
Synthesized miR-221 MB probe was confirmed with selectivity assay by fluorescent spectrometry37.
The miR-221 MB probe was incubated with the MSCs and PSL cells check the expression of intracellular miR-221 in a tube for 5 min at room temperature. miR-221 MB was also able to bind to miR-221 in the human MSCs and PSL cells from different pathological origins.
In vivo imaging
MSCs and PSL cells were traced in vivo with the miRNA conjugated fluorescent dye. The miRNA conjugated fluorescent dye system was conducted and transferred to MSCs and PSL cells. Mice were anesthetized before MSCs and PSL cells were injected. Cell transplantation was conducted as described above. miRNA-221 was tracked from approximately 30 min after miRNA221-labeled MSCs and PSL cell injection. Near-infrared (NIR) fluorescence images (595/625 nm) of the mice were taken using an IVIS Spectrum 2D in vivo imaging system (Perkin Elmer, Connecticut, USA) to monitor miRNA-22137. cell tracing was performed at about 10 min after substrate injection.
Imaging analysis were normalized mice imaging with MB probe injection to eliminate signal interference using mice imaging without cell injection
Immunohistochemical analysis of animal tissues
Samples from each organ were snap-frozen in OCT compounds (Sakura). Cryosections (5–15 μm) were air-dried and then fixed in 4% paraformaldehyde (Seongnam, Bioseasang, Korea) for 10 min.
After blocking with 5% (v/v) goat serum (Life Technologies) for 1 h at room temperature, the samples were incubated with the primary SSEA4 antibody (Santacruz, USA) for 12h at 4 °C. After washing in PBS, they were stained with the secondary antibody and H33342 (Life Technology, Eugene, USA) for 30 min at room temperature. Immune staining was observed and recorded using a Zeiss LSM 800 confocal laser microscope with a 20x NA 0.6 objective (Carl Zeiss Microscopy GmbH). The Emission range of the lasers was 405 nm for H33342 and 625 nm for miRNA-221-MB. The microscopy image data was reconstructed using a maximum intensity projection algorithm implemented in the Zeiss ZEN lite blue 2.5 software (Carl Zeiss Microscopy GmbH).