*4.8. Immunocytochemistry*

Immunocytochemistry staining was performed to evaluate (i) Gata-4 and troponin T-C expression in DPSCs and UC-MSCs di fferentiated into cardiomyocytes as well as (ii) Gata-2 and VE-cadherin expression in angiogenic di fferentiation. For this purpose, on days 7, 14 and 21 of the di fferentiation culture, the medium was removed, cells were washed with PBS (GE Healthcare Life Sciences HyClone Laboratories, Malborough, MA, USA), and fixed with 4% paraformaldehyde (POCH, Avantor Performance Materials Poland S.A.) for 20 min at RT. Cells were subsequently washed thrice with PBS, permeabilized with 0.1% Triton X-100 solution (Sigma-Aldrich) for 8 min at RT, and washed again thrice with PBS. Subsequently, cells were stained against: (i) cardiac-specific proteins–with primary anti-Gata-4 antibody (mouse monoclonal IgG2a, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-troponin T-C antibody (goat polyclonal IgG, 1:20; Santa Cruz Biotechnology) (ii) endothelial-specific proteins–with primary anti-Gata-2 (rabbit polyclonal IgG, 1:50; Santa Cruz Biotechnology) and anti-VE-cadherin (mouse monoclonal IgG1, 1:20; Santa Cruz Biotechnology) for 16 h at 4 ◦C. For (i) cardiac and (ii) endothelial markers detection, the following secondary antibodies were subsequently added, respectively: (i) donkey anti-mouse IgG antibody conjugated with Alexa Fluor 488 (1:250; Jackson ImmunoResearch, Cambridgeshire, UK) and donkey anti-goat antibody conjugated with Alexa Fluor 546 (1:250; ThermoFisher Scientific) (ii) goa<sup>t</sup> anti-mouse IgG antibody conjugated with Alexa Fluor 546 (1:250; ThermoFisher Scientific) and goa<sup>t</sup> anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:250; ThermoFisher Scientific). The concentrations of the used primary and secondary antibodies were selected based on the optimization experiments conducted previously. Moreover, appropriate IgG controls were used to confirm the specificity of antibodies prior to the current study. Staining with secondary antibodies was performed for 2 h in 37 ◦C protected from light. Cells were further washed with PBS and nuclei were stained with DAPI (2 μM, ThermoFisher Scientific) for 15 min in 37 ◦C protected from light. VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) was used to mount coverslips. The preparations were analysed with Leica DMI6000B ver. AF7000 fluorescent microscope (Leica Microsystems GmbH, Welzlar, Germany). The control (undi fferentiated) DPSCs and UC-MSCs were also stained with primary and secondary antibodies according to the protocol described above, and the results are presented in Figure S1.

#### *4.9. Mechanical Characterization of the Hydrogel Matrix*

Mechanical properties of BD PuraMatrix Peptide Hydrogel (Corning, Tewskbury, MA, USA) were investigated using atomic force microscopy (AFM, CellHesion head, JPK Instruments, Berlin, Germany) in force mapping mode. To probe hydrogel samples, commercially-available silicon nitride cantilevers (MLCT-C, Bruker, Billerica, MA, USA) with a nominal spring constant of 0.01 N/m were applied. Force curves, i.e., dependencies between cantilever deflection and relative sample position, were acquired over a grid of 8 × 8 pixels within a scan area of 50 × 50 μm. The maximum load force (F) was 5 nN and load speed of 8 μm/s was maintained. Young's modulus was determined using Hertz contact mechanics as described previously [77]. Briefly, the following relation between the load force (F) and resulting indentation ( Δz) for the paraboloidal assumption of the probing tip was applied:

$$F = \frac{4 \cdot \sqrt{\overline{R} \cdot E}}{3 \cdot (1 - v^2)} \cdot \Delta z^{\frac{3}{2}} \tag{1}$$

**Theorem 1.** *Maximum load force (F). In this theorem, R is the radius of tip curvature, E is Young's modulus and v is the Poisson ratio of the material (here assumed to be 0.5 treating hydrogels as incompressible material).*

JPK Data Processing software was used to apply this equation to the experimental data to obtain the value of Young's modulus for each force curves. The final Young's modulus was obtained by averaging all force curves and was expressed as a mean and standard deviation.

#### *4.10. 3D Encapsulation of DPSCs within the Hydrogel Matrix*

BD PuraMatrix Peptide Hydrogel (Corning) with a concentration of 1% (*w*/*v*) after decreasing the viscosity by vortexing was diluted with a cell culture-grade water (PAA,) to the concentration of 0.3%. The cells re-suspended in a 20% sucrose solution (Sigma-Aldrich) were centrifuged at 320× *g* for 7 min at RT to wash out residual salts from the culture medium (to prevent early hydrogel gelation). After centrifugation, the cells were re-suspended in a 20% sucrose solution, and the cell number and viability were assessed by Countess Automated Cell Counter as described in Section 4.3. To obtain a final hydrogel concentration of 0.15%, 0.3% hydrogel was diluted with the same volume of cell suspension in 20% sucrose solution (at 2× the final desired cell concentration). Thus, the final concentrations of hydrogel and sucrose were equal to 0.15% and 10%, respectively. The whole volume was carefully mixed and added to the centre of each well without introducing bubbles. The following volumes of the mixtures were used: 50 μL/well in a 96-well plate, 250 μL/well in a 24-well plate, and 400 μL/well in a 12-well plate (Corning) and are presented in Table 2. The gelation of the BD PuraMatrix hydrogel was initiated by the addition of 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) and by gently running culture media down the side of the well on top of the hydrogel. Within 1 h post 3D cell encapsulation in hydrogel and following their gelation, 70% of the medium was changed twice to stabilize pH. The DPSCs were cultured for two days under standard culture conditions.


**Table 2.** Preparing of 3D encapsulation of DPSCs within the hydrogel matrix.

#### *4.11. 3D and 2D Culture of DPSCs in Vitro*

To establish a 2D culture of the DPSCs, cell culture plates (Corning) were coated with 0.1% gelatin (Sigma-Aldrich). The cell suspension was prepared in 20% sucrose solution and seeded at a concentration of 2 × 10<sup>4</sup>/well in a 12-well plate 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, Waltham, MA, USA). DPSCs were cultured for two days under standard culture conditions. DPSCs encapsulated in the 3D hydrogel or seeded on 2D gelatin-coated cell culture plates were further cultured at 37 ◦C in a humidified atmosphere containing 5% CO2 and the following oxygen concentrations were used: (i) 2% of O2 (hypoxia) or (ii) about 18% of O2 (normoxia) for 7, 14 and 21 days.

#### *4.12. Assessment of DPSC Proliferation and Metabolic Activity In Vitro*

The proliferation and metabolic activity of DPSCs in 3D or 2D culture in the environment containing 2% (hypoxia) or about 18% of O2 (normoxia) were measured by MTS assay or by analysing ATP content, respectively. The tests were performed every 24 h until seven days post cell encapsulation/seeding.

For MTS assay, DPSCs were encapsulated in the hydrogel or seeded onto gelatin-coated surfaces of 96-well transparent plates (Corning, Tewskbury MA, USA) at a density of 10<sup>3</sup> cells/well for 2D culture conditions and 2 × 10<sup>3</sup> cells/well for 3D culture conditions. The analysis was performed using the Cell Counting Kit-8 (Sigma-Aldrich, St. Louis MO, USA). For this purpose, 50 μL of WST-8 reagen<sup>t</sup> was added into each well and incubated for 4 h. The absorbance was measured at 450 nm wavelength using the Multiskan FC Microplate Photometer (ThermoFisher Scientific, Waltham MA, USA). For the measurement of ATP concentrations, DPSCs were encapsulated in the hydrogel or seeded onto gelatin-coated surfaces of 96-well white plates (Perkin Elmer, Waltham, MA, USA). Subsequently, the assay was conducted using the ATP Lite Luminescence assay kit according to the manufacturer's instructions (Perkin ElmerLuminescence was measured using the Infinite ® M200PRO plate reader (Tecan, Mannedorf, Zurich, Switzerland).

#### *4.13. Osteogenic Di*ff*erentiation of DPSCs in 3D or 2D in Vitro Culture*

Osteogenic di fferentiation of DPSCs in 3D or 2D cultures was initiated 1–2 days after the encapsulation of DPSCs in hydrogel (3D culture) or seeding of DPSCs into cell culture plates coated with 0.1% gelatin (2D culture). For this purpose, a StemPro Osteogenesis Di fferentiation Kit (ThermoFisher Scientific) was used. The di fferentiation medium was changed every 3–4 days. On days 7, 14 and 21 of osteogenic di fferentiation, DPSCs from 3D and 2D culture were recovered for the analysis of expression of osteogenesis-associated genes by Real-Time RT-PCR. Moreover, the presence of calcium phosphate deposits was confirmed by the staining of cells in the hydrogel or those seeded on the 2D surface by Alizarin Red S solution as described in Section 4.7. For 3D culture, a larger number of washes (approx. 4–6) were performed to reduce the red background staining of the hydrogel.

#### *4.14. Recovery of DPSCs from the Hydrogel Matrix*

The DPSCs were isolated from the hydrogel according to the Puramatrix Peptide Hydrogel manufacturer's protocol titled "Cell recovery for sub-culturing or biochemical analyses" (Corning). Briefly, the hydrogel with the culture medium was mechanically disrupted by pipetting. The suspension was then transferred to a 15 mL centrifuge tube. The wells were washed with PBS (GE Healthcare Life Sciences HyClone Laboratories) to collect the remaining hydrogel fragments, transferred into a centrifuge tube, and centrifuged at 320× *g* for 7 min at RT. The hydrogel pellet was re-suspended in PBS (GE Healthcare Life Sciences HyClone Laboratories) and centrifuged at 320× *g* for 7 min at RT. Subsequently, the hydrogel pellet was re-suspended in 0.25% Trypsin/EDTA (Gibco; ThermoFisher Scientific) following digestion for approx. 10 min at 37 ◦C. The trypsin was inactivated with complete cell culture medium and the suspension was centrifuged at 320× *g* for 7 min at RT.
