Isolation of Vascular Wall Mesenchymal Stem Cells from the Thoracic Aorta of Adult Göttingen Minipigs: A New Protocol for the Simultaneous Endothelial Cell Collection

Simple Summary It has been widely demonstrated that blood vessels are sources of multipotent progenitor cells, including mesenchymal stem cells. These stem cellular populations persist throughout adulthood and can be isolated from both microvascular and large vessels. Increasing evidence suggests that vascular stem cells, together with other cell populations residing in blood vessels, such as endothelial cells, are involved in physiological and pathological vascular remodeling. In the present paper, we described, for the first time, a new improved method to isolate a pure population of vascular wall cells showing a preserved mesenchymal tri-lineage differentiative potential from thoracic aorta of Göttingen Minipigs, preserving and also collecting endothelial cells. Considering the increasing interest in the use of Göttingen Minipigs as an animal model for cardiovascular diseases, the results obtained in the present research open the way to plan in vitro vascular remodeling experiments by using in co-culture system vascular mesenchymal stem cells and endothelial cells. Abstract Two main classes of perivascular multipotent populations have been described: the microvascular pericytes and the vascular wall mesenchymal stem cells (VW-MSCs). VW-MSCs are isolated from large vessels in many species and they participate in vascular remodeling together with other cellular components such as endothelial cells. Considering that the Göttingen Minipigs are widely used in Europe as a translational model in the field of cardiovascular diseases, the aim of the present research was to isolate VW-MSCs from the adult aorta of Göttingen Minipigs while preserving and also collecting endothelial cells. The results obtained in the present research demonstrated that this new protocol allows us to obtain a pure population of VW-MSCs and endothelial cells. VW-MSCs from Göttingen Minipigs responded fully to the MSC minima international criteria, being positive to CD105, CD90, and CD44 and negative to CD45 and CD34. Moreover, VW-MSCs presented a differentiative potential towards osteogenic, chondrogenic, and adipogenic lineages. Overall, the present protocol, preserving the viability and phenotypic features of the two isolated populations, opens future possibilities of using minipig VW-MSCs and endothelial cells in in vitro vascular remodeling studies.


Introduction
It is now widely known that embryonic blood vessels are home to stem cells niches identified as natural precursors of mesenchymal stem cells; moreover, it has been demon-The antibodies used for the immunofluorescence (IF) and flow cytometry analyses (FC) are listed in Table 1 and were purchased from AbD Serotec (Kidlington, UK); Abcam (Cambridge, UK), Biolegend (San Diego, CA,USA) and Sigma-Aldrich (St. Louis, MO, USA).

Animal Description
To reduce the number of animals utilized, tissue samples were collected from animals enrolled in an experimental lactation study approved by the Italian Ministry of Health as dictated by D.Lgs 26/2014 (approval n • 32/2021-PR). All procedures were performed in compliance with ARRIVE guidelines. At the end of the experimental trial, sows, acquired by Ellegaard Göttingen Minipigs A/S (Dalmose, Denmark), were group housed with a light/dark cycle of 12:12 and hand fed with a standard commercial diet (Micropigs 9AB20; Mucedola srl, Settimo Milanese Italy). Pens were equipped with both chewable and wooden environmental enrichments. On the day of sacrifice, animals were sedated intramuscularly (IM) with 5 mg/kg of tiletamine/zolazepam (Zoletil, Virbac, Carros, France) and euthanized upon barbiturate overdose (Sodium thiopental 60 mg/kg; Pentothal sodium, MSD Animal Health srl, Madison, NJ, USA). Thoracic aortic traits (11 ± 2 cm; n = 3) were surgically removed and collected from 3 Göttingen Minipigs.

Histological Examination
Portions of 1 cm long portions from the aorta, pre and post enzymatic digestion, were embedded in OCT and frozen in isopentane cooled in liquid nitrogen. Ten-micrometerthick sections were cut with a Leica CM1950cryostat (Leica, Wetzlar, Germany), then left to adhere to a microscope slide and stained with hematoxylin and eosin (H&E) according to the standard procedure. Images were obtained using a Nikon digital camera (DS-Qi2 Monochrome Digital Microscope Camera) installed on a Nikon epifluorescence microscope Eclipse E600 and analyzed with NIS-Elements BR Ver5.30.00 digital image software (Nikon, Tokyo, Japan).

Cell Isolation
pAEC and VW-MSC isolation from minipigs (mpAECs and mpVW-MSCs, respectively) were obtained by modifying two methods previously developed and described ( Figure 1) for the isolation of porcine aortic endothelial cells (pAECs) and porcine vascular wall mesenchymal stem cells (pVW-MSCs) [22,43] from hybrid commercial pigs. The explanted vessel was cleaned before being moved to the laboratory. Two additional washes were performed using Endothelial Medium plus 1× antibiotic-antimycotic (ECm 1% anti-anti), before transferring the vessel under a laminar flow hood.
All excess tissue was removed from the vessels and the aorta was cannulated. The lumen of the vessel was gently washed twice with ECm 1% anti-anti to remove the residual blood and then was filled with 0.2% collagenase IA solution and incubated at 38.5 °C. To recover the endothelial layer, collagenase solution containing cellular suspension was collected after 20 min of incubation in a sterile 50 mL tube, and the enzymatic reaction was stopped with an equal volume of 10% FBS. Then, the vessel was washed with 15 mL ECm (1% anti-anti) to collect the residual cell clusters. The cellular suspensions were centrifuged at 500× g for 10 min. The supernatant was discarded, and the cellular pellet was resuspended in ECm containing 10% FBS and 1× antibiotic-antimycotic solution and seeded in a 75 cm 2 tissue culture flask (BD Primaria, BD Bioscience (Haryana, India)). The aorta was refilled with 0.2% collagenase IA solution and incubated for an additional 2 h at 38.5 °C. Collagenase solution containing cellular suspension was then recovered in a 50 mL tube, and the vessel was washed twice with DPBS and 1× antibiotic-antimycotic to collect the residual cell clusters. Collagenase enzymatic activity was then stopped by addition of an equal volume of 10% FBS. The cellular suspensions were centrifuged at 500×g for 10 min. The supernatant was discarded, and the pellet was resuspended in high glucose (hg) DMEM, to which a 10% FBS and 10× antibiotic-antimycotic solution (hgDMEM-10×) was added and seeded in a 75 cm 2 tissue culture flask (BD Primaria, BD Bioscience).

Cell Expansion
mpAECs at the first passage were detached and expanded for at least three passages in EC medium containing 5% FBS and 1% antibiotic-antimycotic solution. The explanted vessel was cleaned before being moved to the laboratory. Two additional washes were performed using Endothelial Medium plus 1× antibiotic-antimycotic (ECm 1% anti-anti), before transferring the vessel under a laminar flow hood.
All excess tissue was removed from the vessels and the aorta was cannulated. The lumen of the vessel was gently washed twice with ECm 1% anti-anti to remove the residual blood and then was filled with 0.2% collagenase IA solution and incubated at 38.5 • C. To recover the endothelial layer, collagenase solution containing cellular suspension was collected after 20 min of incubation in a sterile 50 mL tube, and the enzymatic reaction was stopped with an equal volume of 10% FBS. Then, the vessel was washed with 15 mL ECm (1% anti-anti) to collect the residual cell clusters. The cellular suspensions were centrifuged at 500× g for 10 min. The supernatant was discarded, and the cellular pellet was resuspended in ECm containing 10% FBS and 1× antibiotic-antimycotic solution and seeded in a 75 cm 2 tissue culture flask (BD Primaria, BD Bioscience (Haryana, India)). The aorta was refilled with 0.2% collagenase IA solution and incubated for an additional 2 h at 38.5 • C. Collagenase solution containing cellular suspension was then recovered in a 50 mL tube, and the vessel was washed twice with DPBS and 1× antibiotic-antimycotic to collect the residual cell clusters. Collagenase enzymatic activity was then stopped by addition of an equal volume of 10% FBS. The cellular suspensions were centrifuged at 500× g for 10 min. The supernatant was discarded, and the pellet was resuspended in high glucose (hg) DMEM, to which a 10% FBS and 10× antibiotic-antimycotic solution (hgDMEM-10×) was added and seeded in a 75 cm 2 tissue culture flask (BD Primaria, BD Bioscience).

Cell Expansion
mpAECs at the first passage were detached and expanded for at least three passages in EC medium containing 5% FBS and 1% antibiotic-antimycotic solution.
mpVW-MSCs at the first passage were detached and expanded in PGM medium to maintain their multipotent potential. Doubling times were calculated for both cell populations as indicated previously [44]. The freezing protocol for both mpAECs and mpVW-MSCs consisted of detaching cells at 70% of confluence using 0.25% Trypsin EDTA, washing them in DPBS, and counting and suspending them at a concentration of 0.5 × 10 6 cells/mL in a freezing medium consisting of 10% dimethylsulfoxide (DMSO)+ 90% FBS. Cells were dispensed in cryovials tubes and were slowly cooled in a Mr. Frosty freezing container placed in a freezer at −80 • C before transferring to the nitrogen liquid biobank the following day.

Cell Characterization by Flow Cytometry Analysis
To confirm the endothelial or mesenchymal immunophenotype, flow cytometry analysis was performed. Briefly, 2 × 10 5 cells were resuspended in 100 µL of phosphate-buffered saline (PBS) and incubated for 1 h at 4 • C in the dark with appropriate fluorochromeconjugated antibodies at the titers reported in Table 1. Unstained controls, to evaluate the inherent background for autofluorescence, were obtained by omitting primary antibodies. After incubation, cells were washed twice and resuspended in 200 µL of PBS, then analyzed with MacsQuant Analyzer10 (Miltenyi Biotec, Bergisch Gladbach, Germany). For CD34 staining, after the first incubation with the primary antibody, cells were washed and incubated with PE-conjugated secondary antibody (Table 1) for 40 min at 4 • C in the dark. Data were analyzed using Flowlogic™ software (https://flowlogic.software/) (Inivai Technologies, Mentone, VIC, Australia).

mpAEC In Vitro Angiogenesis Assay
To verify the mpAECs ability to create a capillary-like network, an in vitro angiogenesis assay was carried out using 8-slide chambers (BD Falcon Bedford, MA, USA) coated with 100 µL of undiluted Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher, Waltham, MA, USA). Extracellular matrix coating was carried out 1 h before the cell seeding in a humidified incubator at 38.5 • C and 5% CO 2 ; then, 8 × 10 5 mpAECs were seeded and incubated for 18 h. Images were acquired at 0, 3 and 18 h using a digital camera installed on a Nikon phase contrast microscope and analyzed by Image J 64 open software (National Institutes of Health, Bethesda, MD, USA). Then, mpAECs were gently washed with DPBS and fixed with 4% of paraformaldehyde, and immunohistochemistry staining for CD31 was performed as previously described [23].

mpVW-MSC Cell Adhesion Assay
To verify mpVW-MSCs ability to adhere to each other, forming a spheroid [45], the hanging drop method was used; 5 mL of DPBS was placed at the bottom of a Petri dish to create a hydration chamber, then drops of 6 × 10 4 cells in 10 µL of PGM medium were seeded on the cover of the dish. Then, the cover was overturned and incubated at 5% CO 2 and 38.5 • C. After 24 h, spheroids were checked for morphology and dimensions by an Eclipse E600 epifluorescence microscope equipped with a Nikon digital camera and ACT-2U software for image capturing (Nikon, Tokyo, Japan).

Mesenchymal Trilineage Differentiation Potential
To verify mpVW-MSCs ability to differentiate in the three mesenchymal lineage, mpVW-MSCs were cultured with a StemPro Adipogenesis Differentiation Kit, a Stem-Pro Osteogenesis Differentiation Kit, a StemPro Chondrogenesis Differentiation Kit (all purchased from GIBCO-Life Technologies), or in standard culture conditions with PGM medium (control cells) for 21 days. After that, the cells were stained with OilRedO, Alizarin Red, and Alcian Blue (all purchased by Sigma-Aldrich) as indicated by the manufacturer's protocol.

Statistical Analysis
Doubling time data were analyzed by a one-way analysis of variance (ANOVA) followed by a post hoc Tukey comparison test (p < 0.05) (GraphPad Prism 5 software). Flow cytometry data were analyzed with a Student's t test comparing the MFI of the negative control and the MFI of single stained cells (p < 0.05) in each cell population.

mpAECs and mpVW-MSCs Isolation
Sections from the minipig aorta wall before enzymatic digestion showed all the normal three layers: intima, media, and adventitia. The intima appeared as a flattened unicellular layer of endothelial cells (Figure 2a). After the two consecutive collagenase treatments, the endothelial cell layer, including the basal lamina, was detached and approximately one-sixth of the tunica media was digested (Figure 2b). After 24 h (Figure 2c), cells collected from the first digestion time (20 min) were attached to the plastic flask and they showed a typical endothelial morphology by growing flat in small island; after approximately 1 week, the cells reached the confluence, forming a compact monolayer (Figure 2d,e).

Statistical Analysis
Doubling time data were analyzed by a one-way analysis of variance (ANOVA) followed by a post hoc Tukey comparison test (p < 0.05) (GraphPad Prism 5 software). Flow cytometry data were analyzed with a Student's t test comparing the MFI of the negative control and the MFI of single stained cells (p < 0.05) in each cell population.

mpAECs and mpVW-MSCs Isolation
Sections from the minipig aorta wall before enzymatic digestion showed all the normal three layers: intima, media, and adventitia. The intima appeared as a flattened unicellular layer of endothelial cells (Figure 2a). After the two consecutive collagenase treatments, the endothelial cell layer, including the basal lamina, was detached and approximately one-sixth of the tunica media was digested (Figure 2b). After 24 h (Figure 2c), cells collected from the first digestion time (20 min) were attached to the plastic flask and they showed a typical endothelial morphology by growing flat in small island; after approximately 1 week, the cells reached the confluence, forming a compact monolayer ( Figure  2d,e).

Figure 2.
Representative images of thoracic aorta segments before enzymatic digestion with the typical three layers: the tunica intima (TI), the tunica media (TM), and the tunica adventitia (TA) (a); after two consecutive collagenase digestion treatments, the tunica intima was dissolved while the tunica media was partially digested (b); (a,b) scale bar = 1 mm. Representative images of small endothelial cellular islands after 24 h of isolation (c) and after reaching confluence (d,e). Representative images of VW-MSCs with elongated shape morphology at ~25% confluence (f) and the typical spindle shape fibroblast-like morphology at full confluence (g,h); scale bar 100 µm. Representative images of thoracic aorta segments before enzymatic digestion with the typical three layers: the tunica intima (TI), the tunica media (TM), and the tunica adventitia (TA) (a); after two consecutive collagenase digestion treatments, the tunica intima was dissolved while the tunica media was partially digested (b); (a,b) scale bar = 1 mm. Representative images of small endothelial cellular islands after 24 h of isolation (c) and after reaching confluence (d,e). Representative images of VW-MSCs with elongated shape morphology at~25% confluence (f) and the typical spindle shape fibroblast-like morphology at full confluence (g,h); scale bar 100 µm.
The cells collected after the second digestion time (2 h) and after the selective protocol described previously [22] displayed a stellate-or elongate-shaped morphology with centrally placed oval or round nuclei ( Figure 2f); they grew slowly and reached confluence approximately after 3 weeks, showing a spindle shape fibroblast-like morphology (Figure 2g,h).

mpAEC and mpVW-MSC Characterization
The three primary mpAEC cell lines were successfully expanded in EC medium for three passages. They maintained the typical endothelial morphology (Figure 3a); the mean doubling time of the three primary cell lines was 35.30 ± 4.63 h. Cytometric analysis showed that all the cellular population were clearly positive to CD31 (Figure 3b) and cells seeded on an extracellular matrix showed the ability to create a capillary-like network. In particular, three hours after cell seeding, mpAECs were already aligned to form a network (Figure 3c). The capillary-like network was fully formed after 18 h (Figure 3d), and cells clearly expressed the endothelial marker VE-cadherin (Figure 3e,f). No differences were observed among the three primary cellular lines.
The cells collected after the second digestion time (2 h) and after the selective protocol described previously [22] displayed a stellate-or elongate-shaped morphology with centrally placed oval or round nuclei ( Figure 2f); they grew slowly and reached confluence approximately after 3 weeks, showing a spindle shape fibroblast-like morphology ( Figure  2g,h).

mpAEC and mpVW-MSC Characterization
The three primary mpAEC cell lines were successfully expanded in EC medium for three passages. They maintained the typical endothelial morphology (Figure 3a); the mean doubling time of the three primary cell lines was 35.30 ± 4.63 h. Cytometric analysis showed that all the cellular population were clearly positive to CD31 (Figure 3b) and cells seeded on an extracellular matrix showed the ability to create a capillary-like network. In particular, three hours after cell seeding, mpAECs were already aligned to form a network (Figure 3c). The capillary-like network was fully formed after 18 h (Figure 3d), and cells clearly expressed the endothelial marker VE-cadherin (Figure 3e,f). No differences were observed among the three primary cellular lines.  The three primary mpVW-MSC lines cultured in no selective medium showed a typical spindle shape, fibroblast-like morphology and occasionally spherical formations sprouted from the cellular monolayer (Figure 4a). When cultured using the hanging drop technique, mpVW-MSCs were able to form compact spheroids with a mean diameter of 770.40 ± 69.79 µm (Figure 4b); no significant differences were observed among the three primary cellular lines. mpVW-MSCs expanded in PGM medium showed a typical perivascular mesenchymal cell morphology: a small cell body with thin and long cellular extensions (Figure 4c,d). No differences were observed among the three primary cellular lines ( Figure S1). The three primary mpVW-MSC lines cultured in no selective medium showed a typical spindle shape, fibroblast-like morphology and occasionally spherical formations sprouted from the cellular monolayer (Figure 4a). When cultured using the hanging drop technique, mpVW-MSCs were able to form compact spheroids with a mean diameter of 770.40 ± 69.79 µm (Figure 4b); no significant differences were observed among the three primary cellular lines. mpVW-MSCs expanded in PGM medium showed a typical perivascular mesenchymal cell morphology: a small cell body with thin and long cellular extensions (Figure 4c,d). No differences were observed among the three primary cellular lines ( Figure S1). A flow cytometric analysis showed a typical MSC profile. In alignment with the criteria for MSC characterization, the cell populations were positive for the mesenchymal stemness markers CD105, CD90, and CD44 and for the specific vascular wall MSC marker CD56, whereas they were completely negative for the hematopoietic markers CD45 and CD34 ( Figure 5). A flow cytometric analysis showed a typical MSC profile. In alignment with the criteria for MSC characterization, the cell populations were positive for the mesenchymal stemness markers CD105, CD90, and CD44 and for the specific vascular wall MSC marker CD56, whereas they were completely negative for the hematopoietic markers CD45 and CD34 ( Figure 5).

mpVW-MSC Trilieaneage Potential
mpVW-MSCs were able to differentiate towards osteo, chondro, and adipocyte phenotypes. The cells displayed osteogenic (Alizarin Red stain Figure 6a,c), chondrogenic (Alcian Blue stain Figure 6e,g), and adipogenic (OilRedO stain Figure 6i,m) differentiation potentials after 21 days of treatment with differentiating culture media ( Figure 6). Cells grown in pro-osteogenic medium for 21 days formed a typical center of bone enucleation that was positive to Alazarin red (Figure 6a,c); no positivity was observed in control cells (Figure 6b,d). mpVW-MSCs grown in pro-condrogenic medium clumped together, forming long bundles and secreting extracellular matrix, and were positive to Alcian Blue (Figure 6e,g). No positivity stain was observed in control cells (Figure 6f,h). pVW-MSCs grown in pro-adipogenic medium demonstrated the intercytoplasmic presence of fat vesicles that were positive for oil red staining (Figure 6i,k). No fat vesicles were observed in control cells (Figure 6j,l).

mpVW-MSC Trilieaneage Potential
mpVW-MSCs were able to differentiate towards osteo, chondro, and adipocyte phenotypes. The cells displayed osteogenic (Alizarin Red stain Figure 6a,c), chondrogenic (Alcian Blue stain Figure 6e,g), and adipogenic (OilRedO stain Figure 6i,m) differentiation potentials after 21 days of treatment with differentiating culture media ( Figure 6). Cells grown in pro-osteogenic medium for 21 days formed a typical center of bone enucleation that was positive to Alazarin red (Figure 6a,c); no positivity was observed in control cells (Figure 6b,d). mpVW-MSCs grown in pro-condrogenic medium clumped together, forming long bundles and secreting extracellular matrix, and were positive to Alcian Blue (Figure 6e,g). No positivity stain was observed in control cells (Figure 6f,h). pVW-MSCs grown in pro-adipogenic medium demonstrated the intercytoplasmic presence of fat vesicles that were positive for oil red staining (Figure 6i,k). No fat vesicles were observed in control cells (Figure 6j,l).
The cross-talk between MSCs and endothelial cells in the vascular network is a known reality and the potential effects of this interaction are also well known [70]. In the presence of inflammatory stimuli, MSCs are able to increase the self-repair activity of endothelial cells, and this action appears to be carried out by increasing production of soluble factors; therefore, MSC transplantation is considered as a potential way to slow down the development of atherosclerosis [71]. Tissue engineering studies stress the need for autologous cell-based approaches, but clarification of processes that support the innate cellular capacity to remodel and form vasculature is still needed. In 3D eterotypic cultures, endothelial cell sprouting is dependent from MSC position in EC-MSC co-culture spheroids evidencing that the communication between different cell populations can define network formation [72].
In terms of the development of ex vivo and in vitro models, and to encourage the use of methods complementary to the in vivo ones, it could be of high interest to have cells of different types but belonging to the same subject, which could reconstitute in vitro multi-cellular models that can recapitulate the animal from which they were derived.
In the present study, we reported the simultaneous isolation of vascular wall mesenchymal stem cells and endothelial cells from the thoracic aorta of adult Göttingen Minipigs. The protocol described in the present research was developed from two distinct protocols for the isolation of endothelial cells [43] and VW-MSCs from the thoracic aorta of hybrid commercial pigs [22]. The new optimized protocol allows to isolate mpVW-MSCs resident in the first layers of the tunica media, as described for those cells isolated from commercial hybrid pigs [22]. mpMSCs showed a fibroblast-like morphology when cultured with a non-selective medium, as described by the International Society for Cellular Therapy [73] concerning the minimal criteria for defining multipotent mesenchymal stromal cells. Occasionally, in the first stage of growth, spheroid cellular formations spontaneously sprouted; the ability to form compact spheroids is a well-demonstrated ability of MSCs grown in particular conditions of no attached culture. In the present research, the mpVW-MSC's ability to grow in a spheroid structure was confirmed when cells were cultured by the hanging drop technique. However, when mpVW-MSCs were cultured in a specific medium for multipotent pericytes, the cell morphology changed towards a phenotype characteristic of perivascular cells with a small cell body, thin and long cellular extensions [23]. A flow cytometric analysis showed a typical MSC profile in line with the criteria for vascular MSC characterization and in agreement with those observed in hybrid commercial pigs. Overall, the results obtained demonstrated that the protocol applied guaranteed the isolation of a pure population of VW-MSCs from Göttingen Minipigs.
Moreover, the present protocol allowed the isolation of endothelial cells from the tunica intima, preserving their viability and phenotype. Our previous protocol did not permit to collect endothelial cells (EC), but damaged them after excessive vessel manipulation and enzymatic digestion times, while mpAECs obtained with this new protocol were viable and exhibited the endothelial peculiar feature to form a capillary-like network if cultured on an extracellular matrix, confirming what was previously shown for pAECs [74].

Conclusions
In this paper, we described for the first time the isolation of mesenchymal stem cells from the thoracic aorta of Göttingen Minipigs by using an improved method that preserves the endothelial cell population. The ability to isolate both endothelial cells and vascular mesenchymal stem cells from the same vessel will be useful in planning in vitro 3D heterotypic experiments for studying the contribution of these cellular populations and the cell-cell interactions in physiological and pathological vascular remodeling.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.