Pluripotent stem cells (PSCs) have the self-renewal ability that proliferates limitlessly, and the pluripotency that develops potentially into every cell type of the body (Romito and Cobellis, 2016). In general, PSCs include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) that are derived from the particular cell mass (inner cell mass or epiblast) of pre-implantation embryos, and induced pluripotent stem cells (iPSCs) that are generated from differentiated cells by forcing the expression of transcription factors with the rejuvenating reprogramming technology (Takahashi et al., 2007; Yu et al., 2007). Of note, iPSCs have certain advantages in models of regenerative medicine because they are derived from patient customized somatic cells, and not embryos. Therefore, iPSCs are able to avert the ethical controversy surrounding the use of human embryo and may enable the development of personalized therapies for each patient (Hockemeyer and Jaenisch, 2016).

Endothelial cells (ECs), which lined the luminal surface of blood vessels, play critical roles in forming new blood vessels and restoring damaged blood vessels (Michiels, 2003). Cardiovascular diseases, which are mainly caused by endothelial dysfunction, represent one of the most common causes of mortality throughout the world. However, the pathological mechanism of how endogenous ECs become dysfunctional during development of cardiovascular diseases is not completely understood yet. Additionally, transplantation of functionally normal autologous ECs for the treatment of cardiovascular diseases is hindered by the lack of availability of ECs in the quantity required. Therefore, the use of PSCs has been proposed due to their ability to generate large numbers of autologous ECs (Mahla, 2016; Olmer et al., 2018; Shin et al., 2022).

Platelet/endothelial cell adhesion molecule-1 (PECAM-1 or CD-31) is a cell adhesion and signaling receptor widely distributed on the surface of platelets, leukocytes and ECs, and maintains the integrity of the blood vessel (Jackson, 2003). CD-31, which is widely recognized as an endothelial cell marker, plays a variety of roles in modulation of platelet function (Cicmil et al., 2002; Falati et al., 2006), angiogenesis (Alcalde et al., 1997), vasculogenesis (Breier and Risau, 1996) and mediation of leukocyte migration across the ECs (Duncan et al., 1999). Additionally, vascular endothelial growth factor (VEGF) as a signaling protein plays critical roles in promoting angiogenesis and vasculogenesis, and maintaining the integrity of ECs (Coultas et al., 2005; Liang et al., 2017). Therefore, PSCs were mainly cultured in endothelial cell growth medium containing VEGF in order to in vitro differentiation of ECs (Olsson et al., 2006). In our previous studies, porcine EpiSCs (pEpiSCs) cultured in endothelial cell growth medium supplemented with 50 ng/mL of VEGF for 8 days were efficiently differentiated into ECs (Jeon et al., 2021; Shin et al., 2022). Typical vascular functions of pEpiSCs-derived ECs confirm through capillary-like structure formation assay, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake and three-dimensional spheroid sprouting.

Although PSCs-derived ECs to develop cell therapeutics for cardiovascular disease are promising, clinical trials to be used for stem cell transplantation for cardiovascular disease patients requires further pre-clinical exploration and validation in animal models (Song et al., 2015). In addition, the medical community hopes to alleviate the worldwide human-organ critical shortage with genetically compatible animal parts. So the pig is an excellent model to investigate xenotransplantation as an alternative potential source of organs, due to physiological and immunological similarities with humans (Klymiuk et al., 2010; Nowak-Imialek et al., 2011).

In the present study, piPSCs were used to differentiate into ECs for testing pre-clinical exploration and validation in animal models. To prove in vitro differentiation of piPSCs into ECs, they were cultured in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days and then expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) and endothelial-associated marker (CD-31) in porcine differentiated cells derived from piPSCs in four differentiation media were examined.

All chemicals and reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) unless otherwise mentioned.

Induction of porcine induced pluripotent stem cells (piPSCs)

Porcine fetal fibroblasts (PFFs) production and culture were performed accordingly, as previously described by Baek et al. (2021). Lenti-viral transduction was carried out using the viPS^TM Vector Kit (Thermo Fisher Scientific^TM, USA). PFFs were transduced with lenti-viral vectors encoding six human transcription factors (POU5F1, NANOG, SOX2, C-MYC, KLF4 and LIN28) to initiate reprogramming via ectopic expression. The transduction to target cells was carried out under multiplicity of infection (MOI) 25 condition. After 24 h of the transduction, these cells were harvested by trypsinization and seeded onto mitomycin C inactivated mouse embryonic fibroblasts (iMEFs) in stem cell medium [Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; GIBCO, Grand Island, NY, USA) containing 1% MEM nonessential amino acids, 1% penicillin/streptomycin, 2 mM L-glutamine (GIBCO, Grand Island, NY, USA), 0.1 mM β-mercaptoethanol, 10% FBS, 10% knock-Out^TM serum replacer (KSR; Thermo Fisher Scientific^TM, Grand Island, NY, USA) and 20 ng/mL leukemia inhibitor factor (LIF)]. Porcine induced pluripotent stem cells (piPSCs: OSKMNL) were passaged using the manual method and medium was changed each day.

Isolation and culture of swine umbilical vein endothelial cells (SUVECs)

Umbilical cords were obtained from piglets at farrowing. Before cutting, both ends of the umbilical cord (periplacental and perifetal) were clamped to keep blood inside the vessel. Dissected umbilical cords were then transported to the laboratory in phosphate buffered saline (PBS) with 10 mM glucose, 100 U/mL penicillin and 100 mg/mL streptomycin. The umbilical veins were cannulated and washed with 0.1% (w/v) collagenase (GIBCO, Grand Island, NY, USA) in Medium199 and incubated for 15 min at 37℃. After digestion, cell suspension was centrifuged. The cell pellet was resuspended in Medium199 with 20% FBS, 100 mg/mL endothelial cells growth supplement (ECGS; CORNING, Corning, NY, USA), 100 mg/mL heparin, 100 IU/mL penicillin and 100 mg/mL streptomycin and then cultured at 37℃, 95% air and 5% CO².

In vitro differentiation of piPSCs into endothelial cells

For in vitro differentiation of piPSCs into endothelial cells, piPSCs (OSKMNL) were passaged by manually picking the colonies and then seeded onto 0.5% gelatin-coated culture plates in four differentiation media [Endothelial Cell Basal Medium 2 (EBM-2; Lonza, Muenchensteinerstrasse, Basel, Switzerland), Endothelial Cell Basal Medium 2 supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (EBM-2 + VEGF; R&D Systems, Minneapolis, MN, USA), StemDiff APEL^TM 2 medium (APEL-2; STEMCELL Technologies, Vancouver, BC, Canada), StemDiff APEL^TM 2 medium supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (APEL-2 + VEGF)] on cultured plates coated with matrigel^® (CORNING, NY, USA) (1:40 dilution with DMEM/F-12 medium) for 8 days at 39℃. These media were changed every 2-3 days for 8 days.

Alkaline phosphatase (AP) activity

AP staining was carried out using an Alkaline Phosphatase Detection Kit (Chemicon/Milipore, Darmstadt, Germany). piPSCs (OSKMNL) were fixed with 4% paraformaldehyde (PFA) for 1-2 min and then washed several times with PBS-T (20 mM Trics-HCl, pH 7.4, 0.15 M NaCl, 0.05% Tween-20). These cells were stained with stain solution (the ratio of Fast Red Violet: Naphthol AS-BI phosphate solution: water = 1:2:1) for 15 min in dark at room temperature. After the staining, they were washed with PBS-T and then covered with Dulbecco’s phosphate buffered saline (D-PBS) to prevent drying. Images were observed with LEICA microscope (LEICA, Wetzlar, Germany, type 090-135 001) and captured by NIS Elements microscope imaging software (Nikon, Minato-ku, Tokyo, Japan, version 3.0).

Immunocytochemistry

piPSCs (OSKMNL) cultured in four differentiation media were fixed with 4% PFA for 20 min at 4℃ and then washed with PBS-T three times for 5 min. After being washed in PBS-T, the cells were treated with the blocking solution (5% BSA in PBS-T) for 1 hour at room temperature. And the cover slips were incubated with primary antibodies in blocking solution at 4℃ for overnight; OCT-3/4 (1:100; Santacruz, Dallas, TX, USA), NANOG (1:100; Abcam, Cambridge, UK), SOX2 (10 ng/mL; R&D System, Minneapolis, MN, USA) and CD-31 (1:100; Novusbio, Centennial, CO, USA). After overnight, the cells were washed with PBS-T three times for 5 min and then incubated with secondary antibodies in blocking solution at room temperature for 1 hour; Alexa fluor^® 568 Donkey Anti-Goat IgG (1:150; Invitrogen, Waltham, MA, USA), Alexa Fluor^® 546 Goat Anti-Rabbit IgG (1:150; Invitrogen, Waltham, MA, USA) and Alexa Fluor^® 555 Donkey Anti-Mouse IgG (1:150; Invitrogen, Waltham, MA, USA). To indicate the nuclei in cells, the cells were treated with 5 µg/mL of Hoechst 33342 in PBS at room temperature for 15 min. All images were explored using the LEICA fluorescence microscope (LEICA, Wetzlar, Germany, DM 2500) and performed with the Leica Application Suite (LAS) (LEICA, Wetzlar, Germany, version 2.7).

Quantitative real-time PCR (qRT-PCR)

Total RNAs of cells differentiated from piPSCs (OSKMNL) were extracted using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using the Revoscript^TM RT Premix (iNtRON Biotechnology Inc., Seongnam-Si, Korea). Total RNAs and cDNA were measured by MaestroNano^® Spectrophotometer (MAESTROGEN, Hsinchu City, Taiwan). Quantitative real-time PCR (q-PCR) was executed using the THUNDERBIRD^® SYBR^® qPCR Mix (TOYOBO, Pudong, Shanghai, China) on the Rotor-Gene Q - Pure Detection system (QIAGEN, Hilden, Germany). The conditions were followed: pre-denaturation for 60 sec at 95℃, denaturation for 15 sec at 95℃ and extinction for 60 sec at 60℃ for 40 cycles. Data analysis was used to ΔΔCt method and gene expression was standardized relative to reference gene (18S). The primer list used for qRT-PCR represents in Table 1.

Table 1. Quantitative real-time polymerase chain reaction primer lists used in this study.

Gene	Forward	Reverse	Target size (bp)	References
18S	TCG GAA CYG AGG CCA TGA TT	GAA TTT CAC CTC TAG CGG CG	69	NR_046261.1
OCT-3/4	GGA TAT ACC CAG GCC GAT GT	GTC GTT TGG CTG AAC ACC TT	68	NM_001113060.1
SOX2	CAT GTC CCA GCA CTA CCA GA	GAG AGA GGC AGT GTA CCG TT	66	NM_001123197.1
NANOG	CCC GAA GCA TCC ATT TCC AG	GAT GAC ATC TGC AAG GAG GC	86	DQ_447201.1
CD-31	GGG GCC ACG ATG TGG CTT GG	CGC GAA GCA CTG CAG GGT CA	156	NM_000442.3

Western blotting

Proteins in whole cells were reacquired using Lysis buffer containing with protease/phosphatase inhibitor (Cell signaling technology, Danvers, MA, USA). After the collected cells were incubated in ice for 30 min, they were centrifuged to 15,000 rpm for 15 min at 4℃. After the centrifuge, only supernatant of the tube was collected. The harvested proteins were quantified using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Proteins harvested from cells were separated on SDS-PAGE and transferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA). After being blocked with 5% skim milk (Bioshop, Burlington, Ontario, Canada) for 1 hour, the membrane was washed three times with PBS-T for 15 min. The membranes were incubated with specific primary antibodies overnight at 4℃ in the following condition; CD-31 (1:1000; Novusbio, Centennial, CO, USA) and β-actin (1:4000; Santacruz, Dallas, TX, USA). The membrane was washed three times with PBS-T for 15 min and then incubated with the secondary antibody in 5% skim milk at 4℃ for 3-5 hrs in the following condition; Goat anti-Rabbit IgG (H&L) (1:1000; Tonbo Biosciences, San Diego, CA, USA) and Goat anti-Mouse IgG (H&L) (1:2000; Tonbo Biosciences, San Diego, CA, USA). HRP conjugated secondary antibody was used. After the membrane was washed three times with PBS-T for 15 min, Immunocomplexes were detected by ECL (Bio-Rad, Hercules, CA, USA). ChemiDoc^TM XRS⁺ System (Bio-Rad, Hercules, CA, USA) to detect protein by being exposed for 1 hour was used. Bio RAD The Discovery Series Quantity One 10D Analysis software (Bio-Rad, Hercules, CA, USA) was used to analyze.

Flow cytometry (FACS) analysis

Swine Umbilical Cord Vein Endothelial Cells (SUVEC), piPSCs (OSKMNL) and cells differentiated from piPSCs (OSKMNL) cultured in four differentiation media were washed with PBS (without Ca²⁺ or Mg²⁺) at room temperature. These cells were treated with 0.05% Trypsin EDTA at 39℃ for 5 min. The trypsin-treated cells were collected on the tube using PBS and then centrifuged at 300 g for 5 min. The cells were resuspended in stain buffer (1× PBS, 2% BSA, 0.1% NaN3, pH 7.1-7.4). And then the single cells were stained with fluorescently conjugated antibody [CD31-PE (1:100; BD Pharmingen^TM, San Diego, CA, USA)] for flow cytometry and incubated for 20-45 min on ice in dark. After the incubation, the cell pellet was washed twice with stain buffer and then resuspend in stain buffer. The stained cell samples were analyzed by FACS verse^TM (BD Biosciences, Franklin Lakes, NJ, USA) and Flow Jo.

Statistical analysis

At least three replicates were measured for each group. Two-way ANOVA with Bonferroni’s post hoc test and t-test using Graph Pad Prism software v7.00 (GraphPad) were used to test the significance of the data.

Morphology of piPSCs cultured for differentiation of endothelial cells

Since porcine induced pluripotent stem cells (piPSCs) were cultured in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days, morphological appearances in piPSCs (OSKMNL) were examined on days 2, 5 and 8 (Fig. 1A). piPSCs (OSKMNL) cultured in stem cell medium formed colonies from the day 2 and the colonies were continuously sustained until the day 8. piPSCs (OSKMNL) on passage 3 and 20 were positively expressed the activity of alkaline phosphate (AP) (Fig. 1B). However, all of piPSCs (OSKMNL) cultured in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 +50 ng/mL of VEGF) did not establish the colonies on days 2, 5 and 8 (Fig. 1A).

Figure 1.Morphology of porcine induced pluripotent stem cells (piPSCs) cultured in differentiation media. (A) Morphologies of porcine induced pluripotent stem cells (piPSCs: OSKMNL) cultured in stem cell medium and four differentiation media [StemDiff APELTM 2 medium (APEL), StemDiff APELTM 2 medium supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (APEL + VEGF), Endothelial Cell Basal Medium-2 (EBM-2) or Endothelial Cell Basal Medium-2 supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (EBM-2 + VEGF)] on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) on days 2, 5 and 8. (B) AP activity of piPSCs (OSKMNL) cultured on iMEF at passage 3 and 20. Scale bar = 200 µm.

Expressions of pluripotency-associated genes and endothelial-associated gene in differentiated cells derived from piPSCs in differentiation media

We evaluated expressions of pluripotency-associated genes (OCT-3/4, SOX2 and NANOG) and endothelial-associated gene (CD-31) in porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days (Fig. 2). The expressions of OCT-3/4, SOX2 and NANOG were significantly higher in piPSCs (OSKMNL) cultured in APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF than those of piPSCs (OSKMNL) cultured in stem cell medium (p < 0.05). Also, endothelial-associated gene (CD-31) was highly expressed in piPSCs (OSKMNL) cultured in EBM-2 and EBM-2 + 50 ng/mL of VEGF (p < 0.05). Based on this results, reprogramming factors (OSKMNL) integrated earlier in piPSCs were not removed and continuously expressed in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with cor^® (1:40 dilution with DMEM/F-12 medium) for 8 days.

Figure 2.Expression of pluripotency-associated genes and endothelial cell marker in differentied cells derived from piPSCs cultured in diffrentiation media. Relative mRNA level of pluripotency-associated genes (OCT-3/4, SOX2 and NANOG) and endothelial cell marker (CD-31) in porcine differentied cells derived from piPSCs (OSKMNL) cultured in four differentiation media [StemDiff APELTM 2 medium (APEL), StemDiff APELTM 2 medium supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (APEL + VEGF), Endothelial Cell Basal Medium-2 (EBM-2) or Endothelial Cell Basal Medium-2 supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (EBM-2 + VEGF)] on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) for 8 days. Values presented as mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus differentiaion media. Control: piPSCs (OSKMNL).

Immunocytochemistry of pluripotency-associated markers in differentiated cells derived from piPSCs in differentiation media

Expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) in porcine differentiated cells derived from piPSCs (OSKMNL) in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days were examined using immunostaining (Fig. 3). OCT-3/4, SOX2 and NANOG were highly expressed in piPSC (OSKMNL) cultured in stem cell medium as the control. However, differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days expressed pluripotency-associated markers. Generally, differentiated cells derived from PSCs should not express pluripotency-associated markers. However, in our result, pluripotency-associated markers continuously expressed in the differentiated cells derived from piPSCs (OSKMNL) in four differentiation media.

Figure 3.Immunocytochemistry of pluripotency-associated markers in differentiated cells derived from piPSCs cultured in differentiation media. Immunostaining images of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) in porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media [StemDiff APELTM 2 medium (APEL), StemDiff APELTM 2 medium supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (APEL + VEGF), Endothelial Cell Basal Medium-2 (EBM-2) or Endothelial Cell Basal Medium-2 supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (EBM-2 + VEGF)] on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) for 8 days. Blue: Staining of Hoechst 33342, Red: Staining of OCT-3/4, SOX2 and NANOG protein. Merge: Hoechst 33342 signal and OCT-3/4, SOX2 and NANOG protein. Scale bar = 50 µm.

Expression of endothelial-associated marker (CD-31) in differentiated cells derived from piPSCs in differentiation media

Expression of vascular endothelial-associated marker (CD-31) in porcine differentiated cells derived from piPSCs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days was examined using immunostaining and western blotting (Fig. 4A and 4B). As shown in Fig. 4, expression of CD-31 in differentiated cells derived from of piPSCs (OSKMNL) cultured in four differentiation media was not observed at all.

Figure 4.Expression of endothelial cell marker (CD-31) protein in differentiated cells derived from piPSCs cultured in differentiation media. (A) Immunocytochemistry of endothelial cell marker (CD-31) protein in porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media [StemDiff APELTM 2 medium (APEL), StemDiff APELTM 2 medium supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (APEL + VEGF), Endothelial Cell Basal Medium-2 (EBM-2) or Endothelial Cell Basal Medium-2 supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (EBM-2 + VEGF)] on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) for 8 days. Blue: Hoechst 33342 signal for nuclei. Red: Staining of vascular endothelial cells marker (CD-31) protein. Merge: Hoechst 33342 signal and CD-31 protein. Scale bar = 50 µm. (B) Western blotting of endothelial cell marker (CD-31) protein in porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media (AEPL-2, APEL-2 + VEGF, EBM-2 and EBM-2 + VEGF) for 8 days.

Flow cytometry of endothelial-associated marker (CD-31) in differentiated cells derived from piPSCs in differentiation media

Flow cytometry of vascular endothelial-associated marker (CD-31) in porcine differentiated cells derived from piPSCs (OSKMNL) in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 +50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days was analyzed (Fig. 5). Swine umbilical cord vein endothelial cell (SUVEC) was used as the control to confirm that PE-conjugated CD-31 was working. In the result, it can be seen that the vascular endothelial-associated marker (CD-31) in SUVEC, which is an endothelial cell, was working with PE-conjugated CD-31. However, PE-conjugated CD-31 in porcine differentiated cells derived from piPSCs (OSKMNL) in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) did not work. This indicates that porcine differentiated cells derived from piPSCs (OSKMNL) in differentiation media for a certain period of time did not differentiate into ECs.

Figure 5.Flow cytometry of endothelial cell marker (CD-31) in differentiated cells derived from piPSCs cultured in differentiation media. Flow cytometry of endothelial cell marker (CD-31) expression in porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media [StemDiff APELTM 2 medium (APEL), StemDiff APELTM 2 medium supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (APEL + VEGF), Endothelial Cell Basal Medium-2 (EBM-2) and Endothelial Cell Basal Medium-2 supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (EBM-2 + VEGF)] on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) for 8 days was analyzed. Swine umbilical cord vein endothelial cells (SUVEC) were used as the positive control. Cells in blue were treated with PE-conjugated CD-31 and cells in red were not treated with PE-conjugated CD-31.

Endothelial cells (ECs) differentiated from pluripotent stem cells (PSCs) are subjected to the stem cell-based therapy where cell transplantation would reflect promptly to cardiovascular diseases with fully functional ECs. To be classified according to origins, PSCs are derived from embryonic stem cells (ESCs), epiblast stem cells (EpiSCs) and induced pluripotent stem cells (iPSCs). Out of these, a suitable source of human stem cell therapy may be iPSCs because of averting the ethical controversy and enabling the development of patient-personalized therapies (Hockemeyer and Jaenisch, 2016). On the other hand, the pigs have anatomical and physiological similarities to humans, so they are suitable for preliminary studies to test human disease models (Nowak-lmialek et al., 2011; Kobayashi et al., 2017). Therefore, in the present study, porcine induced pluripotent stem cells (piPSCs) were used to differentiate into vascular ECs for stem cell-based therapy of vascular diseases.

piPSCs used in this study were constructed with Lenti-viral vectors encoding POU5F1, NANOG, SOX2, C-MYC, KLF4 and LIN28 (OSKMNL). The colonies were observed from piPSCs (OSKMNL) cultured in stem cell medium on the day 2. The activity of alkaline phosphatase (AP), which is commonly conventional marker of PSCs, was positively observed in colonies derived from piPSCs (OSKMNL) at passage 3 and 20. In order to differentiate into ECs, piPSCs (OSKMNL) were cultured in four differentiation media (APEL, APEL + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on 0.5% gelatin-coated plate for 8 days. The APEL-2 (albumin, polyvinyl alcohol, essential lipids) medium was commonly used in conjunction with a variety of cytokines to induce the differentiation of human induced pluripotent stem cells (hiPSCs) into ECs (Ng et al., 2008; Harding et al., 2017). On the contrary, it has been reported that endothelial cell growth basal medium-2 (EBM-2) is commonly used to differentiate human embryonic stem cells (hESCs) into ECs (Joddar et al., 2018). Moreover, adding 50 ng/mL of vascular endothelial growth factor (VEGF) induces the differentiation of functional endothelium in hESCs and hiPSCs (Nourse et al., 2010; Lin et al., 2012). In our previous studies, when porcine EpiSCs were cultured in EBM-2 differentiation medium in association with 50 ng/mL of VEGF on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days, differentiated cells derived from these cells was morphologically changed to the feature of ECs (Jeon et al., 2021; Shin et al., 2022).

To the in vitro differentiation of PSCs into any cell types (endoderm, mesoderm and ectoderm), the ectopic expression of pluripotency-associated genes such as OCT-3/4, SOX2 and NANOG must be down-regulated to avoid heterogenic induction in differentiation-induced cells. However, it has been shown in differentiation-induced cells that some phenotypic traits such as the inclination to generate a certain differentiated descendant are mixed with pluripotency gene expression originated from ESCs (Kalmar et al., 2009). Although we anticipated any cardiovascular features restraining from the expression of pluripotency-associated genes in the differentiated cells derived from piPSCs (OSKMNL), the continuous expression of pluripotency-associated genes in the differentiation-induced cells was a consequence of stochastic integration into the genome, which is permanently altered, of the host cells. Therefore, when the in vitro differentiation of piPSCs (OSKMNL) into ECs was induced in the differentiation culture media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days, it was truly expected that expression of the pluripotency-associated genes is reduced in the differenced cells. However, in the present study, pluripotency-associated markers were higher expressed in the four differentiation media when compared to those of piPSCs used as the control. In addition, protein expression of pluripotency-association markers (OCT-3/4, SOX2, and NANOG) was also observed similarly through immunostaining, western blotting and FACS. Putting these results together, the ectopic expression of pluripotency-associated genes was not down-regulated to avoid heterogenic induction in the differentiation-induced cells. They have been suggested that transcription factors needed to induce iPSCs are introduced by infecting differentiated cells with non-integrative methods using plasmids, Sendai virus, synthetic mRNAs and recombinant proteins (Telugu et al. 2010).

Finally, CD-31 as an endothelial-association marker was expressed commonly in early vascular development and capillary-like structures derived from endothelial cells on matrigel (Albelda, 1991; Gong et al., 2017; Jeon et al., 2021; Shin et al., 2022). However, the expression of CD-31 in the differentiated cells derived from of piPSCs (OSKMNL) cultured in four differentiation media was not observed through immunocytochemistry and wester blotting. Also, when compared to swine umbilical cord vein endothelial cell (SUVEC) as a pure vascular endothelial cell using a flow cytometer, it can be seen that the PE-signal shifted, but no change in the rest of the conditions. In our previous study, EBM-2 + VEGF combination showed ~27% CD-31 positive expression in porcine epiblast stem cells (pEpiSCs) (Jeon et al., 2021) but not in piPSCs.

Taken together, our present study indicates unsuccessful in vitro differentiation of piPSCs (OSKMNL) into ECs using the differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2 and EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days unlike the differentiation of porcine epiblast stem cells (pEpiSCs) into ECs. The expression of pluripotency-associated markers (OCT-3/4, SOX2, and NANOG) were observed in cells differentiated into the four differentiation media, and the endothelial-associated marker (CD-31) was not expressed. It can be seen that piPSCs (OSKMNL) are not able to differentiate into ECs under the four differentiation conditions. Therefore, it is necessary to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.

None.

Conceptualization, S-K.B., T-S.K., C.H. and J-H.L.; investigation and writing, I-W.L., D-K.M., H-G.L., Y-J.L., B-G.S. and J-H.L.; review and editing, I-W.L., D-K.M. and J-H.L.; supervision, C.H. and J-H.L.; funding acquisition, J-H.L. All authors have read and agreed to the published version of the manuscript.

This work was supported by the National Research Foundation of Korea funded by the Korean Government (2020R1l1A3072689), Republic of Korea. In-Won Lee, Yeon-Ji Lee and Bo-Gyeong Seo were supported by the scholarship from the BK21Plus Program, Ministry of Education, Republic of Korea.

The animal study was supervised by the Institutional Animal Care and Use Committee of Gyeongsang National University (IACUC GNU-210524-P0050) and used in accordance with regulation and guidelines of this committee.

Not applicable.

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No potential conflict of interest relevant to this article was reported.