**4. Materials and Methods**

### *4.1. Isolation of Human DPSCs*

The DPSCs were isolated from the dental pulp of human permanent healthy teeth, which were extracted based on the orthodontic indications in the Department of Oral Surgery, Faculty of Medicine, Jagiellonian University Medical College in Krakow and then donated for scientific research following approval by the Bioethics Committee at the Jagiellonian University in Krakow (approval number: 1072.6120.41.2017).

In the first step of isolation of DPSCs, the pulp chamber was exposed using the pulp drill (size: D Ø: 0.25 mm and 0.30 mm) to extract the dental pulp. Subsequently, the pulp chamber was gently rinsed with phosphate-buffered saline (PBS; GE Healthcare Life Sciences HyClone Laboratories, Malborough, MA, USA) containing 100 IU/mL penicillin and 100 μg/mL streptomycin solutions (Gibco, ThermoFisher Scientific, Waltham, MA, USA) to wash out the remaining pulp tissue. Following the mechanical disruption, the isolated pulp tissue was subjected to further enzymatic digestion using a mixture of collagenase I (3 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) and dispase (4 mg/mL, Sigma-Aldrich) for 30 min at 37 ◦C. The enzymes were inactivated by adding a complete cell culture medium (DMEM/F12 supplemented with 10% FBS, Sigma-Aldrich; and 100 IU/mL penicillin, 100 μg/mL streptomycin, Gibco, ThermoFisher Scientific). Released cells as well as the larger pieces of dental pulp were washed and seeded on 24-well culture plates favouring primary cell adhesion (Corning Primaria Culture Plate; Falcon Coring, Tewksbury, MA, USA) and were further cultured under standard conditions (37 ◦C, 5% CO2, 95% humidity). Fresh culture medium was added after 24 h post-seeding. The tissue pieces were removed after 10 days and the adherent cells were washed with PBS and cultured in the complete cell culture medium. The scheme of isolation of DPSCs is presented in Figure 1a. DPSCs were passaged with 0.25% trypsin/EDTA (Gibco, ThermoFisher Scientific) when the confluence of cells reached close to 80–90%. The cells were isolated from three separate teeth derived from three individual donors and were further cultivated individually as separate isolates of DPSCs. DPSCs collected from a single human donor were used in every experiment. Thus, three independent DPSC lines derived from three teeth that were harvested from three individual human donors were used to replicate the experiments.

## *4.2. Isolation of Human UC-MSCs*

The UC-MSCs were isolated from human umbilical cords obtained from The Polish Stem Cell Bank, with permissions from the Polish Ministry of Health (MZ-PZ-TSZ-025-15906-36/AB/14). The umbilical cord was washed with PBS to remove the remaining blood. Subsequently, the vein and arteries were dissected, the Wharton's jelly tissue was cut into 1–2 mm pieces and placed on tissue culture dishes (Falcon) containing complete cell culture medium (DMEM/F12 supplemented with 10% FBS, Sigma-Aldrich,; and 100 IU/mL penicillin, 100 μg/mL streptomycin, Gibco, ThermoFisher Scientific). The tissue pieces were removed after 5 days, the adherent cells were washed with PBS and cultured in the complete cell culture medium. The scheme of isolation of UC-MSCs is presented in Figure 1b. The cells were cultured under standard cell culture conditions. UC-MSCs were passaged with 0.25% trypsin/EDTA (Gibco, ThermoFisher Scientific) when the confluence of cells reached close to 80–90%. The cells isolated from the Wharton's Jelly tissue from the UC of every donor were cultivated separately. In the three replicated experiments, two independent UC-MSC lines derived from the two Wharton's jelly tissues obtained from UCs collected from two separate human donors were used.

#### *4.3. Cell Counting and Viability Assessment*

To assess the number of cells and examine their viability, the cell suspension was mixed with 0.2% Trypan Blue Stain (ThermoFisher Scientific), placed in cell counting slides, and analysed by Countess Automated Cell Counter (ThermoFisher Scientific).

#### *4.4. Antigenic Phenotyping by Flow Cytometry*

To compare the phenotype of DPSCs and UC-MSCs, the cells of both fractions were resuspended in standard staining medium (DMEM/F12 supplemented with 2% FBS; both from Sigma-Aldrich, and were further immunolabelled with the following monoclonal antibodies against human antigens: anti-CD45 (FITC, clone: HI30, Biolegend, San Diego, CA, USA), anti-CD14 (FITC, clone: M ϕP9, BD Biosciences, Franklin Lakes, NJ, USA), anti-CD34 (FITC, clone: 581, BD Biosciences, Franklin Lakes NJ, USA), anti-CD29 (PE/Cy5, clone: TS2/16, Biolegend), anti-CD44 (PE, clone: BJ18, Biolegend), anti-CD73 (PE, clone: AD2, Biolegend), anti-CD90 (PE, clone: 5E10, Biolegend), anti-CD105 (PE, clone: 43A3, Biolegend), anti-Stro-1 (Alx647, clone: STRO-1, Biolegend,) and anti-HLA-DR (PE, clone: L243, Biolegend). For each analysed antigen, appropriate isotype control was used as following: mouse IgG1 (FITC, clone: MOPC-21, BD Biosciences, mouse IgG2 (Alx488, clone: 133303, R&D Systems, Minneapolis, MN, USA), mouse IgG1 (PE/Cy5, clone: MOPC-21, BD Biosciences,), mouse IgG1 (PE, clone: MOPC-21, BD Biosciences,), mouse IgG2 (PE, clone: IS6-11E5.11, Miltenyi Biotec, Bergisch Gladbach, Germany) and mouse IgG1 (APC, clone: IS5-21F5, Miltenyi BiotecStaining was performed for 30 min at 4 ◦C according to the manufacturer's protocols. Cells were further washed and analysed using LSR Fortessa flow cytometer and FACS Diva software (Becton Dickinson, Franklin Lakes, NJ, USA).

#### *4.5. Di*ff*erentiation of DPSCs and UC-MSCs*

#### 4.5.1. Osteogenic, Chondrogenic, and Adipogenic Di fferentiation

Plates with 12-wells were coated with 0.1% gelatin (Sigma-Aldrich). In the case of osteogenic and adipogenic di fferentiation, 2.0 × 10<sup>4</sup> cells were seeded per well in the complete cell culture medium (DMEM/F12 supplemented with 10% FBS, Sigma-Aldrich; and 100 IU/mL penicillin, 100 μg/mL streptomycin, Gibco, ThermoFisher Scientificto reach 60% confluence. Subsequently, the medium was replaced with complete StemPro Osteogenesis Di fferentiation Kit or StemPro Adipogenesis Di fferentiation Kit (Gibco, ThermoFisher Scientific), stimulating osteogenic or adipogenic di fferentiation, respectively. The cultures were refed every 3–4 days. In the case of chondrogenic di fferentiation, micro mass cultures were generated by seeding 5 μL droplets of cell solution (1.6 × 10<sup>7</sup> viable cells/mL) and were incubated for 2 h under high humidity conditions. Next, the micro masses were flooded with the complete cell culture medium. After 24 h, the medium was changed to StemPro chondrogenesis di fferentiation medium (Gibco, ThermoFisher Scientific). The cultures were re-fed every 2–3 days.

Cells were examined for osteogenic, chondrogenic, and adipogenic di fferentiation on days 7, 14 and 21 of culture, following histochemical staining performed for identifying the cell phenotype.

## 4.5.2. Cardiomyogenic Di fferentiation

12-well plates were coated with 50 μg/mL collagen type I (Sigma-Aldrich), and 2.0 × 10<sup>4</sup> cells were seeded per well in complete cell culture medium (DMEM/F12 supplemented with 10% FBS, Sigma-Aldrich; and 100 IU/mL penicillin, 100 μg/mL streptomycin, Gibco, ThermoFisher Scientific) for 24 h. The following day, the culture medium was changed with a cardiomyogenesis-stimulating medium containing DMEM/F12 supplemented with 2% FBS (both from Sigma-Aldrich) and 10 ng/mL bFGF, 10 ng/mL VEGF and 10 ng/mL TGF-β1 (all growth factors were from Peprotech, London, UK). The growth factors were supplemented daily and the whole medium was replaced every two days. Cells were examined for cardiac di fferentiation on days 7, 14 and 21 of culture, following the staining for cardiac-specific markers.

## 4.5.3. Endothelial Di fferentiation

Twelve-well plates were coated with a solution containing 50 μg/mL fibronectin (BD Bioscience) and 0.1% gelatin (Sigma-Aldrich). Cells were seeded with the density of 2.0 × 10<sup>4</sup> cells per well in the complete cell culture medium (DMEM/F12 supplemented with 10% FBS, Sigma-Aldrich; and 100 IU/mL penicillin, 100 μg/mL streptomycin, Gibco, ThermoFisher Scientific) for 24 h. The following day, the culture medium was changed with EGM-2MV Endothelial Cell Growth Medium (Lonza, Basel, Switzerland). EGM-2MV was replaced every two days. Cells were examined for endothelial di fferentiation on days 7, 14 and 21 days of culture, following staining for endothelial markers.

The expression of selected osteogenic, chondrogenic, adipogenic, cardiomyogenic and angiogenic-specific markers was evaluated by measuring the levels of corresponding mRNAs as well as by histochemical or immunocytochemical staining.

#### *4.6. Gene Expression Analysis by Real-Time RT-PCR*

Total RNA was isolated using the GeneMATRIX Universal RNA Purification Kit (Eurx, Gdansk, Poland) according to the manufacturer's protocol for RNA isolation from cell cultures. Briefly, to lyse the cells, lysis bu ffer (Eurx) with 1% Bond-Breaker reagen<sup>t</sup> (ThermoFisher Scientificwas used. RNAs were treated with Turbo DNase (Ambion, ThermoFisher Scientificto remove DNA contamination. RNA concentration was determined by Nano Photometer ® device (Implen, Munich, Germany). Purified RNAs were stored at −80 ◦C.

cDNA synthesis was performed using NG dART RT kit (EURx, Gdansk, Poland) according to the manufacturer's protocol with the following conditions: one cycle at 25 ◦C for 10 min, one cycle at 50 ◦C for 40 min, and one cycle at 85 ◦C for 5 min by employing C1000 Touch ™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). The samples of cDNAs were stored at −20 ◦C for further analysis. Expression of selected human genes associated with osteogenic (Osteocalcin, Osteopontin, *Runx2*), chondrogenic (*Acan, Col10A1, Col1A1, Sox9*), adipogenic (*CEBP*<sup>α</sup>*, PPAR*γ), cardiomyogenic (*Gata-4, Myl2c, Nkx2.5*) or endothelial (*Gata-2, Tie-2*, VE-cadherin) di fferentiation were examined by real-time PCR using an ABI PRISM 7000 sequence detection system (Applied Biosystems, ThermoFisher Scientific, Waltham MA, USA). β2-microglobulin was used as a control housekeeping gene.

Real-time PCR was performed using SYBR Green qPCR Master Mix (EURx), cDNA template (10 ng), forward primer (1 μM) and reverse primer (1 μM; both from Genomed, Warsaw, Poland). The sequences of primers used are included in Table 1. Reactions were performed under the following conditions: one cycle at 50 ◦C for 2 min, one cycle at 95 ◦C for 10 min, followed by 40 cycles at 94 ◦C for 15 s, 60 ◦C for 30 s and 72 ◦C for 30 s. Relative quantification of genes expression was calculated using the comparative ddCt method.


**Table 1.** List of primers employed in real-time RT-PCR.
