6.1. Cell culture and fibroblastic differentiation
- Telomerase transformed iMSCs (Materials and reagents #2.1a) were cultured in complete MEM-α (Media recipes #1) in a humidified atmosphere of 5% CO2 at 37°C (Equipments #4.10) in T-75 tissue culture flasks (Materials and reagents #2.1j).
- Cells were harvested at 80% confluency by adding 500ul of 0.25% trypsin-EDTA solution (Materials and reagents#2.1k) and incubating for 1min at 37°C (Equipments #4.10).
- iMSCs were then plated in 6 well plates (Materials and reagents #2.1b) at a density of 250× cells/well.
- When the cells reached 70-80% confluency, a fibroblastic differentiation medium (Media recipes #2) was added.
- The medium was changed every day for 21 days.
- iMSCs (Materials and reagents #2.1a) cultured in MEM-α were used as a negative control, and primary human fibroblasts [F180] (Materials and reagents #2.1b) cultured in DMEM were used as a positive control in the absence of CTGF and LAA.
- The differentiated PDGFRα-positive cells were subsequently harvested on the 21st day for storage through cryopreservation for future studies.
6.2 Cryopreservation of the differentiated PDGFRα-positive cells
- On day 21, the differentiated PDGFRα-positive cells were washed twice with PBS (Materials and reagents #2.2c) to remove the old media.
- Subsequently, 100µL of trypsin-EDTA (Materials and reagents #2.2b) was added and distributed over the entire surface area of the wells, ensuring complete coverage of the adherent cell layer and incubated at 37°C for 2 mins (Equipments #4.10).
- The differentiated cells were observed under the microscope (Equipments #4.5) to confirm complete detachment. Fresh complete media (MEM-α, Media recipes #1) was added to the cells, dispersed by gentle repeated pipetting to avoid clumping and achieve single-cell suspension. Similarly, for the controls, iMSCs were resuspended in MEM-α, and fibroblasts were resuspended in DMEM (Media recipes #1).
- The cell suspension was then collected in 15ml falcon tubes and centrifuged (Equipments #6) at 1500 rpm (g-101) at 24°C for 5 mins.
- The supernatant was discarded, and the pellets were resuspended in 90% of fresh complete media (Media recipes #1), to which 10% of cryoprotectant (DMSO; Materials and reagents #2.2c) was added dropwise.
- The above cell suspension was distributed into cryovials with approximately 1x106 cells per vial (Materials and reagents #2.2a)
- The cryovials were then stored in a -80°C freezer (Equipments #11) for 24 hours up to 1-week; they were transferred to a liquid nitrogen tank (Equipments #4.12) until the future use.
6.3 Thawing and sub-culturing of the differentiated PDGFRα-positive cells
- The frozen cell suspension in cryovial stored at -80°C/liquid nitrogen (Equipments #4.11) was thawed in a 37°C water bath (Equipments #4.13) for less than a minute with gentle swirling after which it was transferred into a 15 mL sterile tube. An additional 4-6mL of complete media (Media recipes #1) was added to this tube.
- The above tube with cells was centrifuged (Equipments #4.6) at 1500rpm for 5 mins at 24°C to get rid of DMSO.
- The supernatant was discarded, and the pellet was resuspended in 4-6 mL of complete media, after which the cell suspension was transferred into a T-25 culture flask (Materials and reagents #2.1j) and incubated at 37°C with 5% CO2 (Equipments #4.10).
- The cells were observed over the following few days, and media was changed every 2 days until the cells were healthy and reached 60-80% confluency. They were sub-cultured and used for further experimentation.
6.4 Measuring cell viability using MTT
1) Two sets of PDGFRα-positive cells were used for MTT analysis: pre-cryopreservation on day 21 (Group 1: before freezing) and post thawing after 3-4 weeks (Group 2: after freezing).
2) Approximately 1 × 104 PDGFRα-positive cells were seeded in triplicates in 96-well plates (Materials and methods #2.3b) and maintained in 200μL complete MEM-α medium (Media recipes #1) at 37 °C with 5% CO2 (Equipments #4.10).
2) After 72 hours, the old culture medium was discarded, and 20μL of MTT (5 mg/mL; Materials and methods #2.3a) dissolved in 100μL of PBS (Materials and methods #2.3d) was added to each well.
3) Plates were incubated for 2h at 37 °C (Equipments #4.10).
4) The MTT+PBS solution was discarded carefully, following which 100μL of DMSO; (Materials and methods #2.3f) was added per well.
5) Absorbance was recorded at 570 nm on a microplate reader (Equipments #4.9). Figure 1 shows absorbance values for Group 1 and 2 with a minor (2.3%), a non-significant difference.
6.5 Confirming the presence of cell surface markers of differentiated PDGFRα-positive cells using flow cytometry analysis
- PDGFRα-positive cells and the positive and negative control cells were harvested using 0.5% trypsin-EDTA (Materials and methods #2.4h).
- 5× cells were counted using a hemocytometer (Materials and reagents #2.4d).
- The cells were centrifuged (Equipments #6) at 6000 RPM for 15 min at 4°C to obtain a pellet.
- The pellet was washed twice with Dulbecco’s Phosphate Buffered Saline (PBS; Materials and reagents #2.4e).
- The cells were resuspended in 100µL of FACS buffer containing 2% FBS and 1mM EDTA (Materials and reagents #2.4f).
- Recommended volumes (as per the manufacturer) of specific cell surface markers were added. The markers used were as follows: CD-34-PE (Materials and reagents #2.4a), CD-44-APC (Materials and reagents #2.4b) and CD-140α-BB515 (Materials and reagents #2.4c).
- Cells were incubated at room temperature for 15 to 20 mins with respective antibodies in separate flow cytometry tubes before detection.
- A minimum of 15000 gated events with forward and side light scatter characteristics were collected for analysis using BD FACS Aria™ III sorter (Equipments #4.4). Figure 2 illustrates maintenance of differentiation in PDGFRα-positive cells post thawing after 3-4 weeks of cryopreservation (Group 2) as obtained from flow cytometry analysis. Figure 3 shows functional validation of freshly differentiated PDGFRα-positive cells before cryopreservation (Group 1) and is published in our previous study .
6.6 Gene expression analysis for validation of the cryopreserved and differentiated PDGFRα-positive cells
RNA isolation and cDNA synthesis
- RNA was isolated from PDGFRα-positive cells along with iMSCs (negative control; Materials and reagents #2.1a) and fibroblasts (positive control; Materials and reagents #2.1b) and using PureLink® RNA Mini Kit (Materials and reagents #2.4a) using the manufacturer’s protocol.
- Due to many cells, lysis was done by sonication using a rotor-stator cell homogenizer (Equipments #4.8) for 30 to 45 seconds.
RNA was eluted in 50µL of RNase-free water and quantified using Nanodrop 2000 spectrophotometer (Equipments #4.1)
- Purity was determined using the ratio A260/A280.
- 5µg of the purified RNA was reverse transcribed to cDNA in a final reaction volume of 20µL using the High-capacity cDNA synthesis kit (Materials and reagents #2.4b) in a Veriti thermal cycler (Equipments #4.2) as per manufacturer’s instructions.
Relative fold change analysis by quantitative real-time PCR
- Quantitative real-time PCR (qPCR) was performed using the Qiagen Rotor-Gene QPCR system (Equipments #4.3) with GoTaq®SYBR green master mix (Materials and reagents #2.4c) in a total reaction volume of 20
- Cycling parameters used were as follows: initialization at 95°C for 2 mins; denaturation at 95°C for 15 seconds; annealing/extension at 60°C for 1 min; total cycles = 40.
- Purity confirmation of PCR product was done by melt curve analysis using sequential heating at 95°C for 15 seconds, 60°C for 1 min, and 95°C for 15 seconds.
- A list of primer sequences used for specific gene amplification of extracellular matrix and stem cell differentiation markers is given in Table 1 (Materials and reagents #2.4e).
- The relative fold change was obtained using the 2^-ddCt method using the housekeeping gene glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (Materials and Reagents #2.4e). Figures 3 and 4 show the gene expression analysis results for extracellular matrix proteins and stem cell differentiation markers, respectively, adapted from our published study .
6.7 Protein expression analysis of differentiated PDGFRα-positive cells using western blotting
- Approximately 1× PDGFRα-positive cells along with iMSCs (negative control; Materials and reagents #2.1a) and fibroblasts (positive control; Materials and reagents #2.1b) were used for protein lysis.
- Protein lysis of the cells was carried out using the M-PER™ mammalian protein extraction reagent with the HALT™ protease inhibitor cocktail (Materials and reagents #2.5b) added to it in a 1:1000 dilution.
- Protein lysates were prepared as per the manufacturer’s instructions and quantified using Bradford reagent (Materials and reagents #2.5c) using a microplate ELISA reader (Equipments #4.9).
- 50µg of protein was loaded onto 10% SDS-PAGE gels for western blotting.
- The gels were transferred onto nitrocellulose membranes (Materials and reagents #2.5a).
- The membranes were incubated with blocking buffer (Media recipes #2) for 1 hour at room temperature.
- Primary antibodies for the genes were added as mentioned in Materials and Reagents #2.5d and e.
- The above antibodies were incubated overnight at 4°C (cold room).
- The next day, membranes were incubated for 1 hour at room temperature with the appropriate secondary antibodies (Materials and reagents #2.5f).
- The membranes were visualized using Clarity Western ECL substrate reagent (Materials and reagents #2.5g) in a Chemi-Doc visualizer (Equipments #4.7).
- The proteins were quantified and analyzed using ImageJ (Softwares #5b). Figure 5 shows the results of the protein expression analysis for stem cell differentiation markers and is adapted from our previous study .