Isolation and culture of CDCs:
1- Harvest intact hearts from euthanized pigs.
2- Collect 1-2 g of auricular explants. Wash the sample with PBS and disrupt the tissue mechanically into 1–2 mm3 fragments. Wash again these fragments to eliminate residual blood and cellular debris.
3- The tissue is then subjected to three successive enzymatic digestions with a solution of 0.2 % trypsin and 0.2 % collagenase IV in PBS at 37 °C for 5 min each.
4- Digested tissue is washed with Complete Explant Medium (CEM). Finally, explants are cultured in 90 mm Petri plates with CEM at 37 °C and 5 % CO2.
5- After three weeks, discard tissue fragments. Trypsinize fibroblasts-like cells migrating from tissue explants and seed onto 30 mm poly-D-lysine coated plates with Cardiosphere Growing Medium (CGM). Under these conditions, suspended cells clusters called cardiospheres are formed, and cells migrating from them are the CDCs.
6- These cells are seeded again into culture flasks at 3 500 cell/cm2 with CGM and expanded at 37 °C and 5 % CO2.
1- Porcine CDCs are detached from culture flasks with 0.25 % trypsin solution and suspended in PBS containing 2 % FBS.
2- Incubate 2 × 105 cells for 30 min at 4 °C with appropriate concentrations of the monoclonal antibodies: CD90, CD29, CD31, CD44, CD45, CD61, CD105, CD117, Sca-1, SLA-I (Swine Leukocyte Antigen class I) and SLA-II (Swine Leukocyte Antigen class II) from Serotec.
3- Wash cells and resuspend in PBS.
4- Flow cytometric analysis is performed on a FACScalibur cytometer (BD Biosciences) after acquisition of 105 events. Cells is primarily selected using forward and side scatter characteristics and fluorescence is analysed using CellQuest software (BD Biosciences).
5- Isotype-matched negative control antibodies should be used in all the experiments.
6- Calculate the mean relative fluorescence intensity by dividing the mean fluorescent intensity (MFI) by the MFI of its negative control.
1- Culture cell monolayer at 80 % of confluence in adipogenic, chondrogenic and, osteogenic differentiation media during 21 days.
2- After 21 days, fix cells with a paraformaldehyde 4 % solution for 20 minutes.
3- Wash cells 3 times with PBS and stain differentiated cell:
- Adipogenic staining: incubate cells with isopropanol 60 % for 5 min, dry, stain with 3.5 mg/ml de Oil Red O in isopropanol 60 % for 5 min and wash with distilled water.
- Chondrogenic staining: wash cells twice with HCl 0.1 M, stain with 10 ng/ml Alcian Blue 8GX (diluted in HCl 0.1 M) for 2 h and wash with HCl 0.1 M and distilled water.
- Osteogenic staining: stain with 20 mg/ml Alizarin Red S (pH = 4.1‐4.2) for 30 min and wash with distilled water.
4- Observe by optical microscopy.
1- For the isolation of total RNA, detach porcine CDCs from culture flasks with 0.25 % trypsin solution and remove the supernatant. Add 1 mL of Trizol (ThermoFisher) to the cell pellet. Cells are transferred to an Eppendorf tube, 200 μL of chloroform are added and samples are incubated for 5–10 min at room temperature (RT). After a centrifugation for 15 min at 12000 × g, the aqueous phase is mixed with 500 μL of isopropanol and incubated at -80 °C for 20 min to precipitate the RNA. Consecutive centrifugations and ethanol washing are made. Finally, the pellet is resuspended in DEPC-treated water.
2- Measure RNA quality and concentration using an UV spectrophotometer, as Synergy™ Mx Microplate Reader (Biotek, Winooski, VT, USA). Only the RNA samples with a 260/280 nm absorbance ratio between 1.8 and 2.1 and with a 260/230 mm absorbance ratio greater than 2.0 should be retrotranscribed to complementary DNA (cDNA) and amplified by PCR.
3- Synthesis of cDNA from 1 μg of RNA in reverse transcription reaction for 1 h at 37°C using Superscript III reverse transcriptase (Invitrogen).
4- Primers for selected genes can be designed by using the NCBI Primer-BLAST tool (www.ncbi.nlm.nih.gov/tools/primerblast/). Example primer sequences are detailed in Figure 1.
5- Prepare amplification reactions with adequate concentrations of reverse and forward primers, cDNA, DEPC water and Taq DNA Polymerase Recombinant kit (Invitrogen), according to manufacturer’s instructions. Amplify in a PXE 0.2 thermocycler (Thermo) following the protocol described in Figure 2.
6- Run the PCR products in an agarose gel (2%) and use adequate volumes of SYBR Safe DNA Gel Stain (Thermo Fisher Scientific) to visualize amplified cDNAs with UV excitation.
7- Gene expression levels are analysed and normalized with the Gene Tools software (Synoptics Limited) using beta-actin (ACTB) as reference gene. The relative quantification is made by measuring the brightness intensity of each band using the GeneSnap software.