Vascular endothelial cells (ECs) are composed of the inner surface of the endothelium or epithelial tissue that is in contact with the lumen of blood vessels. Vascular ECs play important roles in preventing platelet coagulation and maintaining blood fluidity (Pober and Sessa, 2007) and vascular homeostasis (Michiels, 2003). Due to the molecular interaction of vascular ECs, nitric oxidative (NO) as a free-radical scavenger inhibits platelet coagulation and regulates endothelial vasomotor function (Tousoulis et al., 2012; Gotlieb, 2018). Dysfunction of vascular ECs eliminates the synthesis of NO and causes a decrease in the bioavailability of NO (Faraci, 2005; Tousoulis et al., 2012). Factors such as increased oxidative stress and alterations in the extracellular matrix contribute to this age-related decline in endothelial function (Xiao et al., 2023). Additionally, imbalance in vascular homeostasis caused by aging, smoking and lack of physical activity gradually reduces the function of vascular ECs (Lai et al., 2021; Shin et al., 2022). This reduction, which affects a variety of physiological processes including wound healing and angiogenesis, may contribute to an increased risk of cardiovascular diseases and age-related conditions (Gotlieb, 2018).

Biomaterials would be alternative materials for creating artificial blood vessels, often used in medical applications such as vascular grafts or tissue engineering. These materials are needed to mimic the properties of natural blood vessels, including biocompatibility, mechanical strength and the ability to integrate with surrounding tissues. Pluripotent stem cells (PSCs), which be derived from embryonic sources or induced from adult cells, have the ability to differentiate into any cell type in the body, providing valuable resources for regenerative medicine and tissue engineering. They can be used to create scaffolds that support cell growth and differentiation. As one of biomaterials, PSCs could provide a conducive environment for differentiation into vascular ECs, facilitating tissue engineering and regenerative applications.

Embryonic stem cells (ESCs) were first established in mice (Evans and Kaufman, 1981) and subsequently cells with similar characteristics were described in humans (Tesar et al., 2007), and about fifteen years ago, ESCs were established in the rat (Buehr et al., 2008). Since then, numerous attempts have been made to establish ESCs lines in domestic animals, particularly pigs, but no authenticated success has been reported yet (Alberio et al., 2010; Hou et al., 2016). According to their origins, pluripotent ESCs are commonly divided into two cell types: embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). ESCs are derived from the inner cell mass (ICM) of a pre-implantation embryo at the blastocyst stage (Evans and Kaufman, 1981; Smith, 2001; Zhang et al., 2006), however, EpiSCs are derived from epiblast of a post-implantation embryo which has developed a further (Brons et al., 2007; Tesar et al., 2007; Nichols and Smith, 2009). In the mouse, EpiSCs, which closely resemble human ESCs in their colony morphology, are dependent upon the growth factor FGF2 and Activin/Nodal signaling pathways rather than leukemia inhibitory factor (LIF) needed to maintain their pluripotent state in mouse ESCs (Nichols and Smith, 2009; Alberio et al., 2010; Hanna et al., 2010). Also, pluripotent ESCs have been well characterized in the expression of representative pluripotency markers such as OCT-3/4, NANOG and SOX2.

As one of the adult stem cells, adipose-derived mesenchymal stem cells (A-MSCs) derived from adipocytes have the ability to maintain self-renewal and differentiate into various cell lineages such as endothelial, bone, muscle, fat, cartilage and epithelial cells (Gir et al., 2012; Ho and Yoon, 2019; Rautiainen et al., 2021). Unlike the acquisition of ESCs, A-MSCs were readily obtainable from stromal vascular fraction of white adipose tissue in large quantities by a minimally invasive approach of extraction with less pain (Bekhite et al., 2014; Dube et al., 2018). Vascular ECS differentiated from A-MSCs were directly differentiated through autocrine and paracrine functions to create new blood vessels (Hutchings et al., 2020; Zhang et al., 2021; Gan et al., 2022). Generally, vascular ECs undergo the angiogenesis process through stimulation of growth factors including vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), angiopoietin 1 (Ang 1) and hepatocytes growth factor (HGF) (Hattori et al., 2001; Xin et al., 2001; Rehman et al., 2004).

On the other hand, bone marrow-derived mesenchymal stem cells (BM-MSCs), which provide the microenvironment for hematopoiesis, possess the ability of multi-lineages differentiation including bone, cartilage, fat, muscle and pericyte cells (Tan et al., 2013; Rautiainen et al., 2021; Pharoun et al., 2024). Due to their abilities of self-renewing, paracrine effect and homing effect to move to damaged areas in response to injury signals, BM-MSCs have been extensively studied as one of therapeutic sources. For example, BM-MSCs were able to be differentiated into vascular ECs in culture medium supplemented with vascular endothelial growth factor (VEGF) in vitro (Pankajakshan et al., 2013; Ikhapoh et al., 2015; Gan et al., 2022). VEGF that promotes the angiogenesis process through PI3K signaling pathway in vascular ECs has the function of increased vascular permeability, which can alleviate atherosclerosis by improving low-density lipoproteins (LDL) permeability and maintaining blood cholesterol concentration (Cao et al., 2005; Duffy et al., 2000-2013; Zhang et al., 2011). Therefore, VEGF, which is involved in vascular endothelial cell proliferation, migration, sprouting and formation of tubule-like structure, may promote neovascularization and angiogenic capacity in tissue engineering using BM-MSCs (Eriksson and Alitalo, 1999; Neufeld et al., 1999).

This study was aimed at differentiating porcine epiblast stem cells (pEpiSCs), adipose-derived mesenchymal stem cells (pA-MSCs) and bone marrow-derived mesenchymal stem cells (pBM-MSCs), which are biomaterials, into vascular ECs as a treatment for vascular disease. Therefore, to examine the functionality of pEpiSCs, pA-MSCs and pBM-MSCs into vascular ECs, this study was conducted in the differentiation medium supplemented with VEGF.

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

Stem cells culture

Porcine epiblast stem cells (pEpiSCs) were cultured for these experiments as described in a previous study (Baek et al., 2019). Briefly, pEpiSCs were constructed from epiblast that isolated from in vivo fertilized embryos recovered on the day 9-9.5 after artificial insemination. Mouse embryonic fibroblast cells (MEFs) were originated from the placental fetus of a pregnant mouse on the day 11. MEFs were cultured in Dulbecco’s modified Eagle’s medium high glucose (DMEM-HG) containing 10% of fetal bovine serum (FBS), 1.5 mM of β-mercaptoethanol, 1% of MEM and 1% of penicillin/streptomycin. When approximately 80-90% of MEFs have grown in the culture dish, the cells were inactivated by 10 µg/mL mitomycin C for two and a half hours. Inactivated MEFs (iMEFs) were treated with 0.05% Trypsin/EDTA and 1.0 × 10⁶ cells were seeded onto 6-well plate. After 24 hours, pEpiSCs cultured on iMEFs with Dulbecco’s modified eagle’s medium (DMEM/F12) containing 20% of FBS, 1% of GlutaMAX, 1% of MEM, 1% of penicillin/streptomycin and 0.1 mM β-mercaptoethanol, 5 ng/mL basic fibroblast growth factor-2 (bFGF; R&D system) and 10 µM Y-27632 (Enzo) at 39℃ in 5% CO₂ for 3 days. pEpiSCs were passaged using mixture of 1 mg/mL Collagenase IV and 1 mg/mL Dispase for 10 min at 39℃.

In the case of mesenchymal stem cells, piglets less than 1-month old were used for recovering bone marrow or adipose according to standard surgical procedures. Porcine adipose-derived mesenchymal stem cells (pA-MSCs) and bone marrow-derived mesenchymal stem cells (pBM-MSCs) were recovered from the abdomen and femur of pigs. pA-MSCs were isolated from subcutaneous adipose tissue as described in a previously protocol (Bunnell et al., 2008). Briefly, the stem cells digested with 0.075% collagenase type I were separated by filtration through 100- and 40-µm cell strainers. On the other hand, pBM-MSCs were isolated as described in a previously protocol (Ock et al., 2010). These isolated cells were cultured in advanced Dulbecco’s Modified Eagle Medium (advanced DMEM; Gibco) containing 10% FBS, 1% GlutaMAX and 1% penicillin-streptomycin at 39℃ in 5% CO₂. Once approximately 90% of pBM-MSCs and pA-MSCs had grown in the culture dish, these cells were dissociated using 0.25% trypsin/ethylene-diamine-tetraacetic acid (EDTA) solution and then pelleted at 300 × g for 5 min.

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

Swine umbilical vein endothelial cells (SUVECs) were isolated and cultured for these experiments as described in a previous study (Lee et al., 2023). 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 (GIBCO, Grand Island, NY, USA), 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 stem cells into endothelial cells (ECs)

For in vitro differentiation of stem cells (pEpiSCs, pBM-MSCs and pA-MSCs) into vascular endothelial cells (ECs), these cells were passaged by manually picking the colonies and then seeded onto 0.5% gelatin-coated culture plates in differentiation medium [Endothelial Cell Basal Medium 2 (EBM-2; Lonza, Muenchensteinerstrasse, Basel, Switzerland) supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (VEGF; R&D Systems, Minneapolis, MN, USA)] on cultured plates coated with matrigel^® (CORNING, Corning, NY, USA) (1:40 dilution with DMEM/F-12 medium) at 39℃ for 8 days. These culture medium was changed every 2-3 days. Culture conditions for ECs differentiated from porcine stem cells represents in Table 1.

Table 1. Culture conditions for porcine embryonic stem cells and mesenchymal stem cells.

Name	Media	Supplementation
DMEM-HG	DMEM-HG, 10% FBS	1% MEM, 1.5 mM of β-mercaptoethanol
DMEM/F-12	DMEM/F-12, 20% FBS	1% MEM, 1% GlutaMax, 0.1 mM of β-mercaptoethanol, 5 ng/mL of bFGF, 10 μM of Y-27632
Advanced DMEM	Advanced DMEM, 10% FBS	1% GlutaMax
EBM-2 + VEGF	EBM-2, 2% FBS	4 μL/mL of FGF, 1 μL/mL of GA-1000, 1 μL/mL of IGF, 1 μL/mL of EGF, 1 μL/mL of ascorbic acid, 1 μL/mL of heparin, 0.4 μL/mL of hydrocortisone, 50 ng/mL of VEGF-165
M199 + ECGS	M199, 20% FBS	100 μg/mL of heparin, 30 μg/mL of ECGS

DMEM-HG, Dulbecco’s modified Eagle’s medium high glucose; MEM, minimum essential medium; DMEM/F-12, Dulbecco’s modified Eagle’s medium/Ham’s F-12; bFGF, basic fibroblast growth factor-2; Advanced DMEM, advanced Dulbecco’s modified Eagle’s medium; EBM-2, endothelial cell basal medium-2; FGF, fibroblast growth factor; GA-1000, gentamycin-1000; IGF, insulin like growth factor; EGF, epidermal growth factor; VEGF-165, vascular endothelial growth factor-165; M199, Medium 199; ECGS, endothelial cell growth supplement..

Magnetic-activated cells sorting (MACS)

Dynabeads^TM M-280 streptavidin Sheep anti-rabbit and CD-31/PECAM-1 antibody were mixed in 1:10 ratio and incubated at 4℃ for overnight. ECs differentiated from pEpiSCs, pBM-MSCs and pA-MSCs were washed with Dulbecco’s phosphate-buffered saline (D-PBS). The cells were detached in 5 mM of EDTA for 15 min at 39℃ and then centrifuged at 300 × g for 3 min. Collected cells were suspended in DMEM-HG medium. Magnetic beads labeled with CD-31/PECAM-1 antibody were washed a couple of times with 0.1% bovine serum albumin-DMEM-HG (0.1% BSA/DMEM-HG) using PolyATtract^® system 1000 magnetic separation stand (Promega, Z5410). Suspended cells in 0.1% BSA/DMEM-HG and magnetic beads labeled with the antibody were mixed and then rotated at room temperature for 1 h. Then, the mixtures were washed with 0.1% BSA/DMEM-HG to remove unlabeled cells using a magnetic stand five times. Sorted ECs were seeded on culture plates coated with 0.2% gelatin for the primary culture. EGM-2 and M199 containing 20% of FBS, 30 µg/mL of endothelial cell growth supplement (ECGS) and 100 µg/mL of heparin were used for the culture medium of sorted ECs.

Immunocytochemistry

Pluripotent stem cells (pEpiSCs, pA-MSCs and pBM-MSCs) cultured in the differentiation medium were fixed with 4% paraformaldehyde (PFA) for 20 min at 4℃ and then permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The cells were washed with PBS- T (1 mL TWEEN-20/L) a couple of 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 15 hours; OCT-3/4 (1:100; Santacruz, Dallas, TX, USA), NANOG (1:100; Abcam, Cambridge Biomedical Campus, Cambridge, UK), SOX2 (10 ng/mL; R&D System, Minneapolis, MN, USA) and CD-31/PECAM-1 (1:500; Novusbio, Centennial, CO, USA). After 15 hours, the cells were washed with PBS-T a couple of 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 (1:200; Invitrogen, Carlsbad, CA, USA), Alexa Fluor^® 555 Donkey Anti-Mouse IgG (1:200; Invitrogen, Carlsbad, CA, USA) and Goat anti-rabbit IgG, FSD^TM 488 (1:500; BioActs, Namdong-gu, Incheon, KO). 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 polymerase chain reaction PCR (qRT-PCR)

Total RNAs of cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs were extracted using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. 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 was executed using the GoTaq^® Master Mix with SYBR-Green chemistry (Promega, Madison, Wisconsin, USA) on the Rotor-Gene Q - Pure Detection system (QIAGEN, Hilden, Germany). The conditions were followed: pre-denaturation for 60 secs at 95℃, denaturation for 15 secs at 95℃ and extinction for 60 secs 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 quantitative real-time PCR represents in Table 2.

Table 2. Quantitative real-time polymerase chain reaction primer lists used in this study.

Gene	Sequence		Target size (bp)	Reference
	Forward	Reverse
18S	5’-TCG GAA CTG AGG CCA TGA TT-3’	5’-GAA TTT CAC CTC TAG CGG CG-3’	69	NR_046261.1
CD-31	5’-CGA GGT CTG GGA ACA AAG GG-3’	5’-GGT TTC CGT GTA TCA GGG TAC TT-3’	462	XM_013980836.2
OCT-3/4	5’-GGA TAT ACC CAG GCC GAT GT -3’	5’-GTC GTT TGG CTG AAC ACC TT-3’	68	NM_001113060.1
NANOG	5’-CCC GAA GCA TCC ATT TCC AG-3’	5’-GAT GAC ATC TGC AAG GAG GC-3’	86	DQ_447201.1
SOX2	5’-CAT GTC CCA GCA CTA CCA GA-3’	5’-GAG AGA GGC AGT GTA CCG TT-3’	66	NM_001123197.1
CD-45	5’-GCG CCA AGC AAA GTC AGA AA-3’	5’-CGG TAG GAG GCG TAC AAG TC-3’	79	XM_003130596.6

Flow cytometry (FACS)

pEpiSCs, pA-MSCs and pBM-MSCs cultured in the differentiation medium were washed with PBS (without Ca⁺⁺ or Mg⁺⁺) at room temperature. These stem 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.

Capillary-like structure formation assay

To confirm capillary-like structure formation of pEpiSCs, pA-MSCs and pBM-MSCs, these stem cells were cultured on matrigel, which be thawed at 4℃ for overnight and 50 µL added to matrigel on 96 wells plate. Plates coated with matrigel were incubated at 37℃ for 30 min. These stem cells were counted to 2 × 10⁴ and then seeded on matrigel with EBM-2 + 50 ng/mL VEGF. All images were acquired using Olympus fluorescence microscope (Olympus, DP70) and performed with the DP manager (Olympus, version 3. 1. 1. 208).

Three-dimensional spheroid sprouting assay

Three-dimensional spheroid sprouting of pEpiSCs, pA-MSCs and pBM-MSCs was performed as described in the previous report (Shin et al., 2022). These stem cells were separated into single cells with 0.05% trypsin/EDTA. Formation of spheroids were performed by using methocel solutions consisting of 3 g of methyl cellulose in 125 mL of M199 starvation medium. The numbers of single cells were counted to 500 cells per 1 spheroid in 25 µL droplet with 20% methocel solutions in each medium. Droplets were formed on inverted lid of 100 mm culture dishes and then incubated at 37℃ for 24 hours. Formed spheroids in droplets were collected from dish lids with PBS containing 10% FBS. Collected spheroids were centrifuged at 100 g for 5 min. For embedding in collagen of spheroids, collagen solution was mixed with acetic acid, 100 mg/mL of collagen I (Corning) and M199 in 4:4:1 ratio. The pH was adjusted to change the color of the solution to salmon pink by adding 1 mM HEPES and 0.2 M NaOH. Collagen solutions and 80% of methocel were mixed in 1:1 ratio and then added to spheroids. Mixtures were deposited to 24 well culture plates and then polymerized at 37℃ for 1 hour. When mixtures were polymerized, 11× ECGS in M199 medium was added to mixtures to induce ECs sprouting. After 24 hours, the spheroids in polymerized collagen mixtures were fixed in 4% PFA for 20 min. Then, mixtures were washed with PBS 2 times for 5 min and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Then mixtures were blocked in 3% BSA in PBS for 1 hour. Phalloidin in 3% BSA was treated to mixtures at 4℃ for 2 hours (1:250, Invitrogen, Cat. No. #A12379). After the staining of phalloidin, mixtures were washed with PBS two times for 10 min. All images were acquired using OPTIKA fluorescence microscope (OPTIKA, XDS-3FL4) and performed with the software (OPTIKA, vision pro).

Statistical analysis

Statistical significance was determined in the case of p < 0.01 and p < 0.05 with t-test by at least three replicates experiment using Graph Pad Prism software v7.00 (GraphPad).

Culture of pEpiSCs, pBM-MSCs and pA-MSCs for differentiation

As the previously published by our group (Baek et al., 2021), porcine epiblast stem cells (pEpiSCs 8 line) showing alkaline phosphatase negative (AP (-)) activity were cultured onto inactivated MEFs and then colonies derived from pEpiSCs began to form on the second day of culture (Fig. 1B). After manually separating the pEpiSCs, they were separated into dissociated single cells using a mixture of 1 mg/mL Collagenase IV and 1 mg/mL Dispase. In the case of mesenchymal stem cells, porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and adipose-derived mesenchymal stem cells (pA-MSCs) were recovered from the abdomen and femur of pigs, respectively. These stem cells were cultured in advanced DMEM supplemented with 10% FBS, 1% GlutaMAX and 1% penicillin-streptomycin at 39℃ in 5% CO₂. Once pBM-MSCs and pA-MSCs had grown to more than 90% of the culture dish, these stem cells were dissociated using 0.25% trypsin/EDTA.

Figure 1.In vitro differentiation of ECs derived from pEpiSCs, pBM-MSCs and pA-MSCs. (A) The schematic diagram presented in vitro differentiation of porcine epiblast stem cells (pEpiSCs), porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs) cultured in differentiation medium [Endothelial Cell Basal Medium-2 supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (EBM-2 + 50 ng/mL VEGF)] on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days. On day 8, ECs were separated from cell mixtures differentiated from pEpiSCs, pBM-MSCs and pA-MSCs using magnetic beads coated with endothelial cell marker (CD-31). After the cell sorting, ECs were cultured in the differentiation medium for the primary culture and then proliferated in M199 on 0.2% Gelatin. (B) (Left) alkaline phosphatase (AP) negative (-) images of a pEpiSCs colony on iMEF and AP negative (-) images of pBM-MSCs and pA-MSCs. (Right) images of dissociated pEpiSCs and pBM-MSCs and pA-MSCs. Scale bar = 100 µm.

In vitro differentiation of pEpiSCs, pBM-MSCs and pA-MSCs into ECs

To differentiate pEpiSCs into vascular endothelial cells (ECs) in vitro, this pluripotent stem cells PSCs) were manually harvested in colonies. Single cells dissociated from colony stem cells using a mixture of 1 mg/mL Collagenase IV and 1 mg/mL Dispase were seeded on plates coated with matrigel-coated petri dishes in differentiation medium (EBM-2) supplemented with 50 ng/mL of Vascular Endothelial Growth Factor (VEGF) for 8 days. This differentiation medium was exchanged with fresh culture every 2-3 days (Fig. 1A). Additionally, when the mesenchymal stem cells (pBM-MSCs and pA-MSCs) had grew to about 70-80% of confluence, these cells dissociated using 0.25% trypsin/EDTA were subjected to the identical differentiation conditions as pEpiSCs. At this time, in vitro differentiation into vascular ECs was induced in the differentiation medium containing 50 ng/mL of VEGF. Alkaline phosphorylation (AP) activity was observed negatively in pEpiSCs, and the activity of AP was also observed poorly in two mesenchymal stem cells (pBM-MSCs and pA-MSCs) (Fig. 1B).

Morphology of cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs

After culturing in an in vitro differentiation medium into vascular ECs containing 50 ng/mL of VEGF for 8 days, morphological changes in pEpiSCs, pBM-MSCs and pA-MSCs into were observed on the day 2, 5 and 8 (Fig. 2). From the 2nd day of differentiation culture, a long, elongated morphology, which is typical of vascular ECs, was observed in cells differentiated from pEpiSCs. Around day 5 and 8 of differentiation, it was observed that it changed from an elongated spindle-like morphology to a slightly thicker spindle-like morphology (Fig. 2). In the case of in vitro differentiation of pBM-MSCs, an elongated spindle-like morphology similar to that of pEpiSCs was observed on day 2 of differentiation. However, from the 5th day of the differentiation, structures such as round pebbles-like morphology began to appear, and on the 8th day, many morphologies such as round pebbles were distributed. Finally, an elongated spindle-like morphology similar to pEpiSCs was observed on the 2nd day of differentiation in the case of pA-MSCs. However, a mixture of pEpiSCs with elongated spindle-like morphologies and pBM-MSCs with round pebbles-like morphologies was observed on the day 5 of differentiation (Fig. 2).

Figure 2.Morphologies of cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs. Morphologies of porcine epiblast stem cells (pEpiSCs), porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs) cultured in differentiation medium (EBM-2 + 50 ng/mL VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) on the day 2, 5 and 8. Cells differentiated from pEpiSCs showed an elongated spindle-like morphology, whereas cells differentiated from pBM-MSCs showed a round pebble-like morphology. In the case of pA-MSCs, these two morphologies were mixed with each other on the day 2, 5 and 8, respectively. Scale bar = 200 µm.

Characterization of ECs differentiated from pEpiSCs, pBM-MSCs and pA-MSCs

The expression of CD-31, a specific marker for vascular ECs, was examined pEpiSCs, pBM-MSCs and pA-MSCs using immunofluorescence staining (Fig. 3A). Expression of CD-31 was observed in cells differentiated from porcine stem cells, but a significant increase in CD-31 expression was observed in cells differentiated from pEpiSCs (p < 0.01). In the case of cells differentiated from porcine mesenchymal stem cells (pBM-MSCs and pA-MSCs), CD-31 expression was significantly decreased in cells differentiated from pBM-MSCs and pA-MSCs compared to those from pEpiSCs (p < 0.05), but no significant difference was observed in cells differentiated from two porcine mesenchymal stem cells (pBM-MSCs and pA-MSCs) (Fig. 3B). Interestingly, in the flow cytometry analysis, no difference was observed in the expression of CD-31 between pEpiSCs, pBM-MSCs and pA-MSCs before differentiation, and cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs (Fig. 3C).

Figure 3.Expression of vascular endothelial cells marker (CD-31) protein in cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs. (A) Immunostaining images of vascular endothelial cells marker (CD-31) protein in porcine epiblast stem cells (pEpiSCs), porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs) cultured in differentiation medium (EBM-2 + 50 ng/mL VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days. Green: staining of CD-31 protein. Blue: staining of Hoechst 33342, Merge: CD-31 protein and Hoechst 33342 signals. Scale bar = 25 µm. (B) Relative mRNA levels of CD-31 in differentiated cells from pEpiSCs, pBM-MSCs and pA-MSCs cultured in EBM-2 + 50 ng/mL VEGF on cultured plates coated with matrigel^® for 8 days. Values presented as mean SEM. *p < 0.05, **p < 0.01 versus SUVEC. SUVEC were used as the control. (C) Flow cytometry of CD-31 expression in unsorted and sorted cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs, respectively.

Expression of pluripotency markers in ECs differentiated from pEpiSCs, pBM-MSCs and pA-MSCs

In order to maintain the pluripotency state of stem cells, pluripotency-related genes (OCT-3/4, NANOG and SOX2) must be continuously expressed. However, in order for pluripotent stem cells (PSCs) to differentiate into specific cells, the expression of the pluripotency-related genes must be reduced or suppressed. If PSCs remain after differentiation, there must be controlled because undifferentiated cells can differentiate into other or tumor cells. As shown in Fig. 4A, expression of OCT-3/4 protein was observed in all pEpiSCs, pBM-MSCs and pA-MSCs. However, after differentiation, OCT-3/4 protein expression was not observed in pEpiSCs and pBM-MSCs, but OCT-3/4 expression was observed in pA-MSCs (Fig. 4B).

Figure 4.Expression of pluripotency marker (OCT-3/4) protein in cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs. (A) Immunocytochemistry of pluripotency marker (OCT-3/4) protein in porcine epiblast stem cells (pEpiSCs), porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs), respectively. Green: staining of OCT-3/4 protein. Blue: staining of Hoechst 33342, Merge: OCT-3/4 protein and Hoechst 33342 signals. (B) Immunocytochemistry of pluripotency marker (OCT-3/4) protein in cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs cultured in EBM-2 + 50 ng/mL VEGF on cultured plates coated with matrigel^® for 8 days, respectively. Red: staining of OCT-3/4 protein. Blue: staining of Hoechst 33342, Merge: OCT-3/4 protein and Hoechst 33342 signals. Scale bar = 100 µm.

The expression patterns of pluripotency-related genes before and after differentiation of porcine stem cells were observed (Fig. 5). First of all, in the case of pre-differentiation, it was observed that the expression of pluripotency-related genes in all porcine stem cells was significantly increased compared to SUVEC (p < 0.05) (p < 0.01). Among porcine stem cells, the expression of pluripotency-related genes of pEpiSCs was significantly increased than that of two mesenchymal stem cells (pBM-MSCs and pA-MSCs) (p < 0.05) (Fig. 5A). Secondly, even after differentiation, the expression pattern of pluripotency-related genes in porcine stem cells was significantly reduced when compared to the control, SUVEC. Among porcine stem cells, the decrease was noticeable in pEpiSCs and pBM-MSCs (Fig. 5B). Interestingly, the expression of pluripotency-related genes in pA-MSCs was significantly increased than that of the two stem cells (pEpiSCs and pBM-MSCs) (p < 0.01).

Figure 5.Expressions of pluripotency-associated genes in cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs. (A) Relative mRNA levels of pluripotency-related genes (OCT-3/4, NANOG, SOX2) in porcine epiblast stem cells (pEpiSCs) cultured on iMEFs with DMEM/F-12 containing 20% of FBS, 1% of GlutaMAX, 1% of MEM, 1% of penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 5 ng/mL bFGF-2 and 10 µM Y-27632 for 3 days. Porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs) cultured in advanced Dulbecco’s Modified Eagle Medium (advanced DMEM) containing 10% FBS, 1% GlutaMAX and 1% penicillin-streptomycin, respectively. (B) Relative mRNA levels of OCT-3/4, NANOG, SOX2 in cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs cultured in differentiation medium (EBM-2 + 50 ng/mL VEGF) on cultured plates coated with matrigel^® for 8 days, respectively. Values presented as mean SEM. *p < 0.05, **p < 0.01 versus SUVEC. SUVEC were used as the control.

On the other hand, in this experiment, when porcine stem cells were differentiated into vascular ECs, the morphology of hematopoietic cells was observed rather than the morphology of vascular ECs in differentiated cells, and CD-45, a specific marker of hematopoietic cells, was observed in cells differentiated from porcine stem cells (Fig. 6A). Interestingly, it was found that when pBM-MSCs were differentiated, a significant increase in the expression of CD-45 in the differentiated cells was observed (p < 0.01). However, the expression of hematopoietic cell marker (CD-45) was significantly reduced in cells differentiated from pEpiSCs and pA-MSCs. These results showed that when pBM-MSCs differentiate into vascular ECs, differentiation into hematopoietic cells progresses further than vascular ECs (Fig. 6B).

Figure 6.Expression of hematopoietic marker (CD-45) and morphologies of CD-31 positive (+) sorted cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs. (A) Relative mRNA levels of hematopoietic marker (CD-45) in cells differentiated from porcine epiblast stem cells (pEpiSCs), porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs) cultured in differentiation medium (EBM-2 + 50 ng/mL VEGF) on cultured plates coated with matrigel^® for 8 days. Values presented as mean SEM. *p < 0.05, **p < 0.01 versus SUVEC. SUVEC were used as the control. (B) Morphologies of CD-31 positive (+) sorted endothelial cells (ECs) differentiated from in pEpiSCs, pBM-MSCs and pA-MSCs cultured in proliferative medium (M199 + 20% FBS + ECGS) on the day 2 and 6. Scale bar = 200 µm.

Capillary-like structure formation of ECs differentiated from pEpiSCs, pBM-MSCs and pA-MSCs

The capillary-like structure formation was performed to determine whether vascular ECs differentiated from each pEpiSCs, pBM-MSCs or pA-MSCs formed angiogenesis’s reorganization. This assay was performed to identify the functionality of vascular ECs in the conformation of typical vascular ECs. The ability to form capillary-like structures was measured by porcine stem cells-derived differentiated cells dispensed on the matrigel-coated culture dishes. As shown in Fig. 7, capillary-like structure formation was significantly expanded and widely broadened in cells differentiated from pEpiSCs, but it was unclear capillary-like structure formation in pBM-MSCs and pA-MSCs (p < 0.05). When comparing the capillary-like structure formation of the two mesenchymal stem cells (pBM-MSCs and pA-MSCs), pA-MSCs were induced to form partial capillary-like structure than cells differentiated from pBM-MSCs around 6 days after differentiation induction. These results indicated that the cells differentiated from pEpiSCs have reorganization of vessel tube capability as functional vascular ECs.

Figure 7.Formation of capillary-like structure of CD-31 positive (+) sorted cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs. (A) Formation of capillary-like structures of CD-31 positive (+) sorted cells differentiated from porcine epiblast stem cells (pEpiSCs), porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs) cultured in proliferative medium (M199 + 20% of FBS + ECGS) on culture plates coated with matrigel^® for 6 hours. Scale bar = 200 µm. (B) Branch points in cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs cultured in proliferative medium (M199 + 20% of FBS + ECGS) on culture plates coated with matrigel^® for 6 hours. *,**Values in same columns with different superscripts are significantly different (p < 0.05).

Three-dimensional spheroids sprouting of ECs differentiated from pEpiSCs, pBM-MSCs and pA-MSCs

The three-dimensional spheroids sprouting using a hanging drop protocol in collagen type I mixtures supplemented with ECGS for 24 hours was performed to determine whether angiogenic functions were formed under three-dimensional conditions of vascular ECs differentiated from each pEpiSCs, pBM-MSCs or pA-MSCs. As shown in Fig. 8, both porcine embryonic stem cells (pEpiSCs) and mesenchymal stem cells (pBM-MSCs and pA-MSCs) have well-structured spheroids. Extending out of spheroids after allowing spheroids to sprout was observed in all porcine stem cells. However, it had been presented that pEpiSCs are significantly better formed than mesenchymal stem cells (pBM-MSCs and pA-MSCs) in capillary-like structure formation that extend out of the spheroids for 24 hours (p < 0.05). In addition, when comparing mesenchymal stem cells (pBM-MSCs and pA-MSCs), there was no significant difference between the two mesenchymal stem cells, but it was observed that pA-MSCs are better formed than pBM-MSCs. Collectively, these results suggested that pEpiSCs-derived ECs have an excellent angiogenetic function as mature vascular ECs in three-dimensional conditions.

Figure 8.Three-dimensional spheroids sprouting of CD-31 positive (+) sorted cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs. (A) Three-dimensional (3D) spheroids derived from CD-31 positive (+) sorted cells differentiated from porcine epiblast stem cells (pEpiSCs), porcine bone marrow-derived mesenchymal stem cells (pBM-MSCs) and porcine adipose-derived mesenchymal stem cells (pA-MSCs) using the hanging drop protocol in collagen type I mixtures supplemented with ECGS for 24 hours. Scale bar = 100 µm. (B) Sprouts length was calculated by using Image J. *,**Values in same columns with different superscripts are significantly different (p < 0.05).

As the ageing population increases worldwide, several diseases associated with vascular disease have been continuously reported (Guo et al., 2022; Maier et al., 2023; Zhao et al., 2023). In particular, the occurrence of diseases related to cerebrovascular or cardiovascular vessels has been reported at a fairly high level (Luepker and Lakshminarayan, 2009; Nakai et al., 2022; Yang et al., 2023). Although the stent, which is a polymer material, is inserted into the blood vessels of patients with coronary artery diseases, the same problem occurs over time, causing problems in replacing or inserting new stents. Artificial blood vessels have emerged as treatments for these vascular diseases, but they are exclusively limited to patient care due to limited supply and the problem of having to use synthetic materials. Therefore, as an alternative to solving this problem, interest in supplying young and healthy new blood vessels using biomaterials such as pluripotent stem cells (PSCs) has been increasing (Liu et al., 2020; Prakash et al., 2023; Wang et al., 2024). The use of patient-derived biomaterials stem cells not only solves immune rejection but also has the advantage of being easily obtained from patients through non-invasive surgical methods. Stem cells, including embryonic stem cells and mesenchymal stem cells, are able to be excellent biomaterials that can be used to develop treatments for various human diseases.

The present study aimed to generate young and healthy vascular endothelial cells (ECs) through in vitro differentiation using porcine stem cells as biomaterials. In general, pigs have considerably higher anatomical and physiological similarities to humans than other livestock, and have the advantage of being able to obtain biomaterials easily (Pabst, 2020; Hou et al., 2022). Also, unlike other domestic animals, pigs have fewer zoonotic diseases with humans and are very vulnerable to diseases, so they can easily identify infectious diseases from pigs before using their biomaterials for treatment (Boneva et al., 2001; Lin et al., 2023). Therefore, in this study, porcine embryonic stem cells (pEpiSCs) with excellent differentiation ability and readily available mesenchymal stem cells (BM-MSCs and A-MSCs) were selected and used as biomaterials for generating vascular ECs.

In previously reported studies, they have been proved that the expression of pluripotency-related genes (OCT-3/4, NANOG and SOX2) in porcine EpiSCs, and even if passaged for a long period of time, pluripotency was not lost and the ability of stem cells to self-renew was sustained continuously (Baek et al., 2021; Baek et al., 2023). In addition, it was observed that dissociation into single cells, freezing and thawing procedures for ease of availability proceeded well without significant apoptosis (Baek et al., 2019). Interestingly, for each stem cells derived from same porcine EpiSCs, the activation of alkaline phosphorylation (AP) was different, showing a different pattern of pluripotency and expression of surface factors according to the AP activity (Chen et al., 2011; Baek et al., 2021). In this study, we used an AP negative (-) EpiSCs line because the ability to differentiate vascular ECs was better than the AP positive (+) EpiSCs line. In porcine mesenchymal stem cells (pBM-MSCs and pA-MSCs), pluripotent features different from embryonic stem cells (ECs) have been reported (Casado et al., 2012; Bharti et al., 2016; Ock et al., 2016). For example, both porcine mesenchymal stem cells generally showed low AP activity, but pA-MSCs had slightly better differentiation ability than pBM-MSCs, especially in the adipogenesis and osteogenesis pathways (Ock et al., 2010; Ock et al., 2016). Also, when morphological changes to vascular ECs were observed after differentiation of porcine stem cells into vascular ECs for 8 days of, elongated spindle-like morphology was observed in pEpiSCs and pA-MSCs, whereas round pebbles-like morphology was formed in cells differentiated from pBM-MSCs. Therefore, to summarize the above results, although the activity of AP was low in both porcine mesenchymal stem cells, it was found that pA-MSCs had better differentiation ability into vascular ECs than pBM-MSCs.

After in vitro differentiation from porcine stem cells to vascular ECs, the functionality of vascular ECs derived from differentiated cells was evaluated. In our group, they were reported the formations of capillaries-like structures and three-dimensional spheroids sprouting of vascular ECs after inducing the differentiation of porcine embryonic stem cells (pEpiSCs) into vascular ECS from the differentiation medium (EBM-2) to which 50 ng/mL of vascular endothelial growth factor (VEGF) was added (Jeon et al., 2021; Shin et al., 2022). In the early stages of development, VEGF and fibroblast growth factor (FGF) secreted by the endoderm would support the differentiation of mesodermal cells to angioblasts. In vitro, VEGF not only promotes vascular ECs proliferation but also capillary-like structures formation and three-dimensional spheroids sprouting (Risau and Flamme, 1995). However, the molecular mechanisms of VEGF in regulating migratory or sprouting of the activity of angioblasts remain largely unknown. In our studies, we reported that VEGF promotes capillary-like structure formation and three-dimensional spheroids sprouting in porcine stem cells, especially in epiblast stem cells (EpiSCs) (Jeon et al., 2021; Shin et al., 2022). Hematopoietic stem cells and vascular ECs are derived from blood vessel cells, which are a common precursor in yolk sac blood islands (Pardanaud et al., 1989). VEGF and its cellular receptor2 (Flk-1) were provided an excellent marker for blood vessel cells during early mouse development because in the population where Flk-1 is expressed, it produces hematopoietic and vascular ECs in mouse embryonic stem cells (Nishikawa et al., 1998; Schuh et al., 1999). CD-31 or Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa cell adhesion molecule that is highly expressed on the surface of ECs and to a lesser extent on a range of hematopoietic cells (DeLisser et al., 1993). Therefore, CD-31 is primarily used to demonstrate the presence of ECs, to measure vessel density or to quantify angiogenesis. In this study, as a result of confirming the expression of CD-31, a representative marker of vascular ECs, it was confirmed that the expression of CD-31 was significantly increased in cells differentiated from porcine embryonic stem cells (pEpiSCs) rather than in two mesenchymal stem cells (pBM-MSCs and pA-MSCs). Additionally, CD-45, a representative marker of hematopoietic stem cells, showed remarkably low expression in pEpiSCs and pA-MSCs in this study, but significantly increased in pBM-MSCs. These results confirmed that the functionality of vascular ECs when the differentiation ability of vascular ECs derived from pEpiSCs was better than that of pBM-MSCs and pA-MSCs.

We evaluated the differentiation ability of vascular ECs in porcine embryonic stem cells (pEpiScs) and porcine mesenchymal stem cells (pBM-MSCs and pA-MSCs) and the functionality of blood vessels in differentiated cells. In pEpiSCs, changes were observed in the morphology of vascular ECs, while pBM-MSCs showed different morphology. In addition, the expression of CD-31, a specific marker of vascular ECs, was significantly increased in pEpiSCs-derived differentiated cells than in cells derived from two mesenchymal stem cells, and capillaries-like structure formation and three-dimensional spheroids sprouting were also observed in functional evaluation.

None.

Y-J.L., C.H., and J-H.L.; data curation, Y-J.L., and J-H.L.; formal analysis, Y-J.L., I-W.L., and J-H.L.; investigation, Y-J.L., and B-G.S.; methodology, T-S.K., and S-K.B.; project administration, J-H.L.; resources, S-K.B.; supervision, C.H., and J-H.L.; writing-original draft, Y-J.L., and I-W.L.; writing-review & ediring, J-H.L., and C.H.

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.

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