Functional Characterization of Endothelial Cells Differentiated from Porcine Epiblast Stem Cells

Endothelial cells (ECs), lining blood vessels’ lumen, play an essential role in regulating vascular functions. As multifunctional components of vascular structures, pluripotent stem cells (PSCs) are the promising source for potential therapeutic applications in various vascular diseases. Our laboratory has previously established an approach for differentiating porcine epiblast stem cells (pEpiSCs) into ECs, representing an alternative and potentially superior cell source. However, the condition of pEpiSCs-derived ECs growth has yet to be determined, and whether pEpiSCs differentiate into functional ECs remained unclear. Changes in morphology, proliferation and functional endothelial marker were assessed in pEpiSCs-derived ECs in vitro. pEpiSCs-derived ECs were subjected to magnetic-activated cell sorting (MACS) to collect CD-31+ of ECs. We found that sorted ECs showed the highest proliferation rate in differentiation media in primary culture and M199 media in the subculture. Next, sorted ECs were examined for their ability to act as typical vascular ECs through capillary-like structure formation assay, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and three-dimensional spheroid sprouting. Consequently, pEpiSCs-derived ECs function as typical vascular ECs, indicating that pEpiSC-derived ECs might be used to develop cell therapeutics for vascular disease.


Introduction
Endothelial cells (ECs), which constitute the lumen of blood vessels in the body, play a critical role in modulating vascular functions [1]. They are implicated in thrombosis and platelet adhesion, immunological and inflammatory responses, and vascular tone and blood flow regulation [2][3][4]. Endothelial dysfunction has been linked to a wide range

In Vitro Differentiation of Endothelial Cells from Porcine Epiblast Stem Cells
pEpiSCs were cultured on iMEFs for 3 days, then detached stem cell colonies using manual picking. The pEpiSCs were differentiated into endothelial cells (ECs) in 50 ng/mL of VEGF included EGM-2 with VEGF excluded endothelial cell growth medium-2 SingleQuots ® . Differentiation has proceeded for 8 days on culture plates coated with matrigel ® (1:40 dilution with DMEM/F-12 medium) at 39 • C.

Magnetic-Activated Cells Soring (MACS)
Dynabeads™ M-280 streptavidin Sheep anti-rabbit and CD31 antibody were mixed in 1:10 ratio and incubated at 4 • C for overnight. ECs differentiated from pEpiSCs were washed with Dulbecco s phosphate-buffered saline (D-PBS). The cells were detached in 2 mM of ethylene-diamine-tetraacetic acid (EDTA) for 15 min at 39 • C and then centrifuged at 300× g for 3 min. Collected cells were suspended in 1% bovine serum albumin-DMEM medium. Magnetic beads labeled with CD-31 antibody were washed three times with 1% Cells 2022, 11, 1524 4 of 13 BSA/DMEM using PolyATtract ® system 1000 magnetic separation stand (Promega, Z5410). Suspended cells in 1% BSA/DMEM and magnetic beads labeled with the antibody were mixed and then rotated at room temperature for 1 h. Then, the mixtures were washed with 1% BSA/DMEM 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, M199 supplemented with 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.

Proliferation Assay
Cells were seeded on culture plates coated with 0.2% gelatin and then counted the cell numbers for 5 days. Additionally, cell proliferation rates were examined with the expression of Ki-67 as a representative proliferation nuclear marker [43].

Flow Cytometry Analysis
Cells were washed with DPBS and then treated with 2 mM EDTA/PBS at 37 • C for 10 min. After centrifugation at 300× g for 3 min, collected purified ECs were suspended in stain buffer consisting of 2% BSA in PBS. The cells were stained with phycoerythrin (PE) conjugated CD-31 antibodies for 1 h at room temperature in the dark. The cells were suspended in stain buffer and then analyzed by FACSverse™ (BD Biosciences).

Immunocytochemistry
Cells were fixed with 4% paraformaldehyde (PFA) for 20 min. The cells were treated with the blocking solution (5% BSA/PBS-T) for 1 h, and then incubated with CD-31 antibody at 4 • C for 16 h, followed by Alexa Fluor ® 546 Goat Anti-Rabbit IgG antibody. Hoechst 33342 was used to nucleus counterstain. All images were acquired using the LEICA fluorescence microscope (LEICA, DM 2500) and performed with the Leica Application Suite (LAS; LEICA, version 3.8).

Quantitative Polymerase Chain Reaction
Total RNA was extracted using RNeasy Plus Mini Kit following the manufacturer's methods. Extracted RNAs were synthesized into cDNA using the Revoscript™ RT Premix. Quantitative real-time polymerase chain reaction (q-PCR) was performed using the GoTaq ® SYBR Master Mix with Rotor-Gene Q-Pure Detection system (QIAGEN). The primer list used for quantitative real-time polymerase chain reaction is Table 1. The gene expression was quantified relative to the reference gene (18S).

Three-Dimensional Spheroid Sprouting Assay
Three-dimension spheroid sprouting of purified ECs was performed as described in the previous study [44]. Cells were separated into single cells with 0.05% trypsin/EDTA. Spheroids were formed using methocel solutions consisting of 3 g of methyl cellulose in 125 mL of M199. 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 the inverted lid of 100 mm culture dishes and then incubated at 37 • C for 24 h. Droplets formation of spheroids 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 and M199 in a 4:4:1 ratio. Collagen solutions and 80% of methocel were mixed in a 1:1 ratio and then added to spheroids. Mixtures were deposited to 24 well culture plates and then polymerized at 37 • C for 1 h. When mixtures were polymerized, 330 ug/mL ECGS in M199 medium was added to mixtures to induce ECs sprouting. After 24 h, the spheroids in polymerized collagen mixtures were fixed in 4% PFA for 20 min. Phalloidin was stained in mixtures (1:250, Invitrogen, A12379). All images were acquired using OPTIKA fluorescence microscope (OPTIKA, XDS-3FL4) and performed with the software (OPTIKA, vision pro). Sprouts length was calculated using the ImageJ software.

Capillary-Like Structure Formation Assay
To capillary-like structure formation, cells were cultured on Matrigel, thawed at 4 • C for overnight and 50 µL added to Matrigel on 96 wells plate. Plates coated with Matrigel were incubated at 37 • C for 30 min. Cells were counted to 2 × 10 4 and then seeded on Matrigel. All images were acquired using an Olympus fluorescence microscope (Olympus, DP70) and performed with the DP manager (Olympus, version 3.1.1.208).

Dil-Acetylated-LDL Uptake Assay
Cells were added 2 µg/mL of Dil-Ac-LDL and then incubated at 37 • C for 4 1 2 h. The cells were fixed with 4% PFA for 10 min and then washed with PBS. Hoechst 33342 was used for nuclear staining. All images were explored using the LEICA fluorescence microscope (LEICA, DM 2500) and performed with the Leica Application Suite (LAS; LEICA, version 3.8).

Statistical Analysis
Graph Pad Prism software v7.00 (GraphPad) was used to analysis of data. Relative mRNA levels of OCT-3/4, NANOG, SOX2, quantification of Ki-67 positive cells in culture of differentiation media, sprouts length, branch points and quantification of Dil-Ac LDL uptake assay were analyzed in triplicate and data were presented as means ± SEM. Oneway or two-way ANOVA used statistical significance between groups. P < 0.05 was considered statistically significant.

Separating Endothelial Cells Differentiated from pEpiSCs
Endothelial cells (ECs) were differentiated from porcine epiblast stem cells (pEpiSCs) using the method described in our previous study [38]. We sought to separate ECs solely from differentiated pEpiSCs by using magnetic-activated cell sorting (MACS) with CD-31 antibody, an endothelial cell marker ( Figure 1A), and the expression of CD-31 was analyzed by flow cytometry and immunofluorescence. CD-31 expression was found in about 28% of the unsorted cell population, whereas 100% in the sorted cell population ( Figure 1B). Furthermore, unsorted cells showed partial CD-31 expression, but sorted ECs showed most CD-31 expression by immunofluorescence ( Figure 1C). pEpiSCs did not express at all. To evaluate pluripotency of sorted ECs, gene expression of pluripotency markers such as OCT-3/4, NANOG and SOX2 were measured. Comparison of the pluripotency in pEpiSCs, unsorted ECs and sorted ECs revealed significant changes. These changes showed that pluripotency markers were significantly decreased in sorted ECs compared to pEpiSCs ( Figure 1D). These results indicate that MACS-based cell sorting is sufficient for separating ECs solely and that sorted ECs lost their pluripotency. as OCT-3/4, NANOG and SOX2 were measured. Comparison of the pluripotency in pEp-iSCs, unsorted ECs and sorted ECs revealed significant changes. These changes showed that pluripotency markers were significantly decreased in sorted ECs compared to pEp-iSCs ( Figure 1D). These results indicate that MACS-based cell sorting is sufficient for separating ECs solely and that sorted ECs lost their pluripotency.

Proliferation of pEpiSCs-Derived ECs
For the primary culture of sorted ECs, these ECs were cultured using various media for a couple of days (Table 2). Sorted ECs were not primarily survived and proliferated in EGM-2 or M199 while growing well in differentiation media ( Figure 2A). Photographs were obtained on the day using a phase-contrast microscope, with representative photographs shown. Interestingly, after subculture of these cells, sorted ECs were grown well in M199. The proliferation rate of subculture of ECs were evaluated for five days. The cell growth in each culture condition was compared to differentiation media as the control. As the results, EGM-2 and EGM-2-EV culture showed lower proliferation than control otherwise, M199 showed a significantly increased proliferation rate than control on days four and five. Also, the cells were stained with Ki-67 and showed the highest staining level when cultured in M199 compared to other media ( Figure 2B). These results suggested that early passage of sorted ECs are needed differentiation media for stabilization and then, M199 is the best condition for growth and proliferation.

Proliferation of pEpiSCs-Derived ECs
For the primary culture of sorted ECs, these ECs were cultured using various media for a couple of days (Table 2). Sorted ECs were not primarily survived and proliferated in EGM-2 or M199 while growing well in differentiation media ( Figure 2A). Photographs were obtained on the day using a phase-contrast microscope, with representative photographs shown. Interestingly, after subculture of these cells, sorted ECs were grown well in M199. The proliferation rate of subculture of ECs were evaluated for five days. The cell growth in each culture condition was compared to differentiation media as the control. As the results, EGM-2 and EGM-2-EV culture showed lower proliferation than control otherwise, M199 showed a significantly increased proliferation rate than control on days four and five. Also, the cells were stained with Ki-67 and showed the highest staining level when cultured in M199 compared to other media ( Figure 2B). These results suggested that early passage of sorted ECs are needed differentiation media for stabilization and then, M199 is the best condition for growth and proliferation.

Angiogenic Function of pEpiSCs-Derived ECs by Three-Dimensional Spheroid Sprouting
Typical ECs have a function of angiogenesis, which is to form new vessels by various signaling. To confirm the angiogenesis capacity of differentiated cells in three-dimensional conditions, spheroids spouting assay derived from three types of cells (pEpiSCs, unsorted ECs and sorted ECs) (Figure 3). pEpiSCs and sorted ECs formed well spheroids, but unsorted ECs did not form spheroids. After allowing the spheroids to sprout, capillary-like structures sprouted out of spheroids were formed in sorted ECs ( Figure 3A). However, no branched spheroids were displayed from pEpiSCs and unsorted ECs coexisting with undifferentiated PSCs and differentiated ECs. Additionally, sprouting out of three-dimensional spheroids derived from the sorted ECs were identified with fluorescence staining ( Figure 3B,C). Collectively, these results suggested that pEpiSCs-derived ECs have an angiogenetic function as mature vascular endothelial cells in three-dimensional conditions.

Angiogenic Function of pEpiSCs-Derived ECs by Three-Dimensional Spheroid Sprouting
Typical ECs have a function of angiogenesis, which is to form new vessels by various signaling. To confirm the angiogenesis capacity of differentiated cells in three-dimensional conditions, spheroids spouting assay derived from three types of cells (pEpiSCs, unsorted ECs and sorted ECs) (Figure 3). pEpiSCs and sorted ECs formed well spheroids, but unsorted ECs did not form spheroids. After allowing the spheroids to sprout, capillarylike structures sprouted out of spheroids were formed in sorted ECs ( Figure 3A). However, no branched spheroids were displayed from pEpiSCs and unsorted ECs coexisting with undifferentiated PSCs and differentiated ECs. Additionally, sprouting out of threedimensional spheroids derived from the sorted ECs were identified with fluorescence

Vessel Organization of pEpiSCs-Derived ECs Using Capillary-like Structure Formation
Capillary-like structure formation was performed to evaluate angiogenesis's reorganization in vascular ECs. This assay was conducted to identify the functional capability of sorted ECs as acts typical ECs. The ability to form a capillary-like structure was assessed by seeding pEpiSCs, differentiated endothelial cells on matrigel-coated plates (Figure 4). As a result, sorted ECs started to form capillary-like structures for two hours. Capillarylike structures from sorted ECs were gradually expanded and then widely broadened. By contrast, unsorted ECs presented unclear capillary-like structure formation because differentiated ECs and undifferentiated pEpiSCs were mixed ( Figure 4A,B). Interestingly, single cells derived from pEpiSCs colonies formed partial capillary-like structures. These results show that pEpiSCs-derived ECs have reorganization of vessel tube capability as functional vascular endothelial cells.

Vessel Organization of pEpiSCs-Derived ECs Using Capillary-like Structure Formation
Capillary-like structure formation was performed to evaluate angiogenesis's reorganization in vascular ECs. This assay was conducted to identify the functional capability of sorted ECs as acts typical ECs. The ability to form a capillary-like structure was assessed by seeding pEpiSCs, differentiated endothelial cells on matrigel-coated plates (Figure 4). As a result, sorted ECs started to form capillary-like structures for two hours. Capillarylike structures from sorted ECs were gradually expanded and then widely broadened. By contrast, unsorted ECs presented unclear capillary-like structure formation because differentiated ECs and undifferentiated pEpiSCs were mixed ( Figure 4A,B). Interestingly, single cells derived from pEpiSCs colonies formed partial capillary-like structures. These

Acetylated Low Density Lipoprotein Uptake of pEpiSCs-Derived ECs
ECs maintain homeostasis of cholesterol concentration in the blood vessels by uptaking acetylated low-density lipoprotein (Ac-LDL) [45]. To evaluate the function of Ac-LDL uptake in pEpiSCs, unsorted ECs or sorted ECs were examined with Dil-Ac-LDL ( Figure  5). As a result, Dil-Ac-LDL uptake was detected only in differentiated endothelial cells (unsorted ECs and sorted ECs). Unsorted ECs showed little uptake of Dil-Ac-LDL, especially sorted ECs were shown the highest uptake of Dil-Ac-LDL. These results revealed that differentiated endothelial cells have a capacity of Ac-LDL uptake as functional property of endothelial cells.

Acetylated Low Density Lipoprotein Uptake of pEpiSCs-Derived ECs
ECs maintain homeostasis of cholesterol concentration in the blood vessels by uptaking acetylated low-density lipoprotein (Ac-LDL) [45]. To evaluate the function of Ac-LDL uptake in pEpiSCs, unsorted ECs or sorted ECs were examined with Dil-Ac-LDL ( Figure 5). As a result, Dil-Ac-LDL uptake was detected only in differentiated endothelial cells (unsorted ECs and sorted ECs). Unsorted ECs showed little uptake of Dil-Ac-LDL, especially sorted ECs were shown the highest uptake of Dil-Ac-LDL. These results revealed that differentiated endothelial cells have a capacity of Ac-LDL uptake as functional property of endothelial cells.

Acetylated Low Density Lipoprotein Uptake of pEpiSCs-Derived ECs
ECs maintain homeostasis of cholesterol concentration in the blood vessels by uptaking acetylated low-density lipoprotein (Ac-LDL) [45]. To evaluate the function of Ac-LDL uptake in pEpiSCs, unsorted ECs or sorted ECs were examined with Dil-Ac-LDL ( Figure  5). As a result, Dil-Ac-LDL uptake was detected only in differentiated endothelial cells (unsorted ECs and sorted ECs). Unsorted ECs showed little uptake of Dil-Ac-LDL, especially sorted ECs were shown the highest uptake of Dil-Ac-LDL. These results revealed that differentiated endothelial cells have a capacity of Ac-LDL uptake as functional property of endothelial cells.

Discussion
Technologies for the production of a vessel-, tissue-, organ-, disease-, and further patient-specific ECs will become a fundamental necessity for research of molecular target validation, high-throughput drug screening and ECs-based cell therapy, including the engineering of clinically applicable engineered vascular tissue grafts. Although primary ECs have several limitations, such as restricted scalability and high probability of karyotypic defects, ECs have been used in various disease models to explore vascular dysfunction. Because of their indefinite self-renewal and high pluripotency, pluripotent stem cells (PSCs), which include embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), and induced pluripotent stem cells (iPSCs), are the promising therapeutic strategy for human degenerative diseases. However, owing to the harsh ethical and limitation of culture expansion to utilize human ESCs (hECS) for regenerative medicine, many attention has been diverted to non-primate species such as pig retained anatomical and physiological similarities with humans [46]. Accordingly, porcine PSCs were of considerable significance for human degenerative diseases therapy [47].
We recently reported an in vitro differentiation protocol in which pEpiSCs were incubated stem cell culture medium on a feeder layer of mitomycin-treated mouse embryo fibroblasts (MEFs) for three days. Then, the condition changed to EBM-2-EV supplemented with 50 ng/mL VEGF on matrigel for eight days to induce ECs differentiation [38]. We observed differentiation efficiencies of approximately 27% for CD31-positive cells. Such protocols are undoubtedly scalable; however, strategies to select pure ECs with culture conditions that allow pure ECs to proliferate effectively and further functional validation of pEpiSC-derived ECs into typical vascular ECs would be essential for therapeutic application in a clinical trial. Applying the separation of ECs from the heterogeneous mixtures, pEpiSCs-derived cells, including differentiated or undifferentiated ECs were adopted in magnetic-activated cell sorting (MACS) using CD31 antibody. Although the protocol achieved the approach yield of about 27% ECs, 100% purified ECs could be identified through flow cytometry analysis by MACS sorting These were achieved by generating a more efficient amount that is not limited by the current scalable culture conditions of primary ECs.
For the widespread application of ECs, it has been fundamentally important to establish an efficient ECs proliferation system. However, sorted ECs were not proliferated effectively in Endothelial Cell Growth Medium-2 BulletKit (EGM-2) as a specialized ECs growth media. It was reported that adding 50 ng/mL of VEGF to EGM-2-EV supports the ECs survival for the primary culture until the primary sorted cells start growing at the normal proliferation rate [48]. When the required growth factors were not added to the culture medium, proliferation of endothelial progenitor cells (EPCs) proceeded slowly and cell death occurred lastly [49]. For such reasons, it was necessary to culture the sorted ECs in differentiation media until the primary passage. In general, VEGF plays an essential role in the survival and proliferation of ECs by activating of PI3K/Akt/forkhead signaling pathway in scalable suspension culture [50][51][52]. After the primary culture in differentiation media, the M199 culture system-induced most significant effect on the proliferation of ECs. Therefore, it is reasonable that the requirement of cytokines is different during the ECs proliferation. Collectively, the purified ECs were identified to exhibit the greatest effect on VEGF for the primary culture and the M199 culture system for the proliferation. We further confirmed the expression of Ki67, a marker of cell proliferation, through fluorescence staining.
Considering the requirement of practical grade for the vascular disease therapy, functional assessment of generated pEpiSCs-derived ECs were evaluated applying three different assays. ECs enriched by surface marker selection would provide a safer cell resource. Expression of CD-31 as an ECs-specific surface marker was observed strongly in early vascular development and capillary-like structures derived from ECs on Matrigel [53,54]. Although a previous report described ECs enrichment from human ESCs with a selection of CD-31+ expression [55], the purity of enriched ECs in that study was lower (~20%) than ours (~30%) [38]. On the other hand, PSCs differentiation usually occurs within multicellular, three-dimensional structures called embryoid bodies (EBs). However, in the current study, ECs sorted by selecting CD-31+ expression assembled networks of capillary-like structures, whereas pEpiSCs and unsorted ECs differentiated from pEpiSCs were barely formed. As well as, pEpiSCs-derived ECs solely networks showed much branching points compared with networks from pEpiSCs or unsorted differentiated ECs. The sorted ECs on Matrigel were attached and wrapped around in a way that is reminiscent of angiogenesis.
Additionally, to characterize the phenotypic nature of the ECs derived from pEpiSCs, a functional method that involves measuring Ac-LDL uptake using the fluorescent probe Dil (Dil-Ac-LDL) was performed. The sorted ECs were brilliantly fluorescent, whereas the fluorescent intensity of pEpiSCs and unsorted ECs were barely detectable. Finally, the three-dimensional spheroid sprouting of sorted ECs using the hanging drop protocol cultured in collagen type IV mixtures supplemented with ECGS for one day was examined. Nascent capillary-like structures out of the three-dimensional spheroid were formed in the sorted ECs, elucidating the vessel formation. These suggests that the sorted ECs by selected CD-31+ expression were fully differentiated and functionally competent.

Conclusions
The protocol described here offers the first opportunity to generate purified ECs from pEpiSCs with well-set up culture conditions for proliferation, which show the functionality of typical vascular ECs. Functional tests revealed that the generated ECs might be used in vitro assays to examine angiogenesis or cellular responses to various vascular diseases. Additionally, the ability to generate functional-ECs in sufficient quantities for cell therapy techniques may allow these purified pEpiSCs-derived ECs to be used in regenerative treatments.