Parallel Isolation and Characterization of Porcine Smooth Muscle, Endothelial and Mesenchymal Stromal Cells for Bioengineering Applications

There is signicant interest in the pig as the animal model of choice for organ transplantation and the study of tissue engineering (TE) products and applications. Currently, efforts are being taken to bioengineer solid organs to reduce donor shortages for transplantation. For complex organs such as the lung, heart, and liver, the vasculature represents a fundamental feature. Thus, to generate organs with a functional vascular network, the different cells constituting the building blocks of the blood vessels should be procured. However, due to species' specicities, porcine cell isolation, expansion, and characterization are not entirely straightforward compared to human cell procurement. Here, we report the establishment of simple and suitable methods for the isolation and characterization of distinct porcine cells for bioengineering purposes. We successfully isolated, expanded and characterized porcine bone marrow-derived mesenchymal stromal (pBM-MSC), aortic smooth muscle (pASMC), and umbilical vein endothelial cells (pUVEC). We demonstrated that the three cell types showed specic immunophenotypical features. Moreover, we demonstrated that pBM-MSC could preserve their multipotency in vitro, and pUVEC were capable of maintaining their functionality in vitro. These cultured cells could be further expanded and represent a useful cellular tool for TE purposes (i.e., for recellularization approaches of vascularized organs or in vitro angiogenesis studies). Porcine Bone Marrow derived-Mesenchymal Stem/Stromal Cells; pEGM: Porcine Endothelial Growth Medium; pUVEC: Porcine Umbilical Vein Endothelial Cells; RM: Regenerative Medicine; 𝛼 SMA: alpha Smooth Muscle Actin; SM22 𝛼 : Smooth Muscle 22 alpha; SMC: Smooth Muscle Cells.


Introduction
To date, organ transplantation represents the only available and de nitive treatment for patients suffering from end-stage organ disease. This procedure effectively increases patient survival but is limited due to a dramatic shortage of organ donors [1]. This chronic scarcity of donated human organs for transplantation has motivated considerable interest in the production of biocompatible and fully functional bioengineered organs. This goal seems to be within reach through organ decellularization and recellularization technologies [2][3][4]. The resulting bioengineered organs can be additionally recellularized using other cell types and further matured to create more functional organs ready to be transplanted [5].
To restore the full function of an organ, it is fundamental that all components are correctly bioengineered since the overall function of an organ depends on the successful integration of its constituents (e.g., epithelia, mesoderm, parenchyma, and vasculature) [6][7][8]. The vasculature plays a crucial role as it represents the primary point of communication between each organ or tissue and the rest of the body. It is also vital to guarantee the delivery of oxygen and nutrients to the cells and remove cellular waste products. This aspect is critical in regenerative medicine approaches, especially for whole organ bioengineering, since oxygen delivery would be limited to a few hundred µm by simple gas diffusion in non-vascularized tissue [7]. This would undoubtedly result in cell/tissue necrosis, limiting the in vitro generation of organs and survival post-transplantation [8]. Moreover, EC play an active role in orchestrating the processes involved in tissue repair [9][10][11][12][13][14][15] and produce a non-thrombogenic barrier [16], which are critical features in the generation and engraftment procedures of bioengineered organs.
Thus, this study aimed to establish appropriate methods for the e cient and reproducible isolation, characterization, and expansion of distinct porcine cell types (EC, SMC, and MSC) in parallel for organ scaffold recellularization, as well as for other tissue engineering applications, i.e., in vitro angiogenesis studies. The rationale behind the use of porcine cells is due to the present and future usage of the pig as the animal model of choice for the transplantation of bioengineered livers [17]. Nevertheless, porcine and human cell procurement, expansion, and characterization are not precisely equivalent, and any developed procedures need to be extensively investigated and tested due to interspecies differences. Hence, in this work, we report practical and straightforward strategies for the parallel isolation of pure populations of cells from different biological samples. When expanded using the conditions described in this study, the isolated cells could actively proliferate, maintaining viability during the culture. Also, we identi ed a combination of appropriate antibodies and functional assays, allowing for a feasible characterization of the immunophenotypical and functional identity of these cells. Overall, considering the relevance of EC, SMC, and MSC in tissue engineering and regenerative medicine, and the usefulness of the pig as an animal model in this eld, the methods reported herein may represent a highly suitable and straightforward tool for obtaining pure cultures of primary porcine cells with well-de ned phenotypical and functional features for bioengineering applications, as showed in the experimental owchart presented [ Figure 1].

pBM-MSC isolation and culture
Porcine BM MNC separation was achieved with Histopaque 1.077 gradient centrifugation. The MNC fraction was seeded with DMEM/FBS-MSC culture medium in tissue culture-treated 152cm 2 Petri dishes coated with 0.2% gelatin to improve cell adhesion. pBM-MSC adhered to the bottom of the dish and the non-adherent cells were removed during the rst media change. Discrete cell colonies were evident at 3-5 days after the initial seeding and pBM-MSC population showed an elongated broblast-like appearance [ Figure 2A], which is more evident in cells organized in colonies, as reported in the literature [20,21].
Cultures were heterogeneous and constituted by different subpopulations of cells, such as small polygonal cells interspersed with characteristic spindle-shaped cells [ Figure 2B], as reported for human BM-MSC [22][23][24]. The number and size of the colonies increased progressively to reach con uence (80%) 10-12 days after the initial seeding.

Phenotypical and functional characterization of pBM-MSC
The immunophenotypical features of pBM-MSC were determined by ow cytometry using a panel of surface markers, which have been described as representative of mesenchymal cell lineage (CD90), other antigens generally expressed by MSC (CD29, CD44), and lineage markers (CD11b, CD14, CD31, CD34, CD45). Essentially, all pBM-MSC expressed CD29, CD44, and CD90. However, the expression of endothelial marker CD31 and other markers present in hematopoietic cells like CD11b, CD14, CD34, and CD45 were absent [ Figure 3A]. Furthermore, we assessed the expression of a set of surface markers (CD29, CD44, and CD90) by immuno uorescence [ Figure 3B]. The cell staining result corroborated the data obtained by ow cytometry analysis. Thus, the data generated from both ow cytometry and immuno uorescence analysis allows us to state that pBM-MSC showed signi cant phenotypical features of MSC.
Moreover, we tested the trilineage differentiation capability of pBM-MSC into adipocytes, osteoblasts, and chondrocytes. This was con rmed by standard induction protocols using commercial differentiation kits.
Adipogenic differentiation was evaluated by Oil Red O staining [ Figure 4A and D]. Some cells in the negative control with expansion medium could differentiate into adipocytes, even if to a relatively smaller extent than the cells treated with the adipogenic differentiation medium. This is a common effect in con uent cultures of MSC.
Nonetheless, Oil Red O staining revealed that pBM-MSC was capable of successfully differentiate to adipocytes. The osteogenic differentiation capability of pBM-MSC was evaluated by ALP/Von Kossa staining [ Figure 4B and E]. The negative control, cultured in expansion medium, only showed a slight heterogeneous red staining induced by ALP [ Figure 4B], while the cells treated with osteogenic differentiation medium signi cantly differentiated, as demonstrated by the presence of black stains indicating calcium deposits and more intense red staining which suggests the presence of osteogenic progenitor cells in the culture. Finally, the chondrogenic differentiation ability of pBM-MSC was determined by Alcian Blue staining. The negative control, cultured in expansion medium, did not show any sign of differentiation [ Figure 4C], while the cells cultured with chondrogenic differentiation medium stained in dark blue, suggesting glycosaminoglycans' presence, a fundamental component of cartilage [ Figure 4F]. Therefore, we demonstrated that pBM-MSC presented relevant mesenchymal features, including the expression of mesenchymal markers (CD29, CD44, and CD90) and the ability to differentiate into adipocytes, osteoblasts, and chondrocytes in vitro. pASMC isolation and culture Cells were isolated by enzymatic digestion of aortic tunica media after removing the endothelial cell fraction resident in the tunica intima, seeded in bronectin-coated Petri dishes, and cultured in DMEM/FBS. The primary pASMC were attached to the culture dishes and distinguishable 48 hours after isolation [ Figure 5A]. The selected pASMC population had a spindle-shaped broblast-like appearance [ Figure 5B-D]. The number and size of the colonies increased progressively to reach con uence (90%) 10-12 days after the initial seeding.
Moreover, we assessed the expression of a panel of intracellular and extracellular markers, including smooth muscle markers (SM22 , SMA, and Caldesmon) and other antigens commonly expressed by SMC (CD29, CD44, CD90) by immuno uorescence analysis. All the tested markers were highly expressed by pASMC [ Figure 6B]. Notably, the most expressed markers were the contractile SMC markers SM22 and SMA, reported as contractile phenotype-associated markers in SMC [25].
Thus, considering the data generated from both ow cytometry and immuno uorescence analysis, we can state that the isolated SMC from the porcine aorta showed phenotypical features characteristic of SMC.
pUVEC isolation and culture The cells were isolated by enzymatic digestion, seeded in 1% (v/v) gelatin-coated Petri dishes, and cultured in a porcine endothelial growth medium (pEGM). The primary pUVEC were selected by their adhesive properties. Cell cultures were heterogeneous and constituted of different subpopulations of cells, including cobblestone-like shaped cells and elongated cells. To remove the contaminant cells from the culture we selected the CD31-positive cell fraction by cell sorting [ Figure 7]. This allowed to obtain pure cultures of pUVEC which have been used for further experiments.
Moreover, we assessed the expression of endothelial markers (CD31, CD105, CD144, Tie-2), other antigens commonly expressed by EC (CD44), and vimentin, an intermediate lament of the cytoskeleton which plays a critical role in the physiological endothelial mechano-response and inherent to the endothelial phenotype [27,28]. Notably, the most common markers for EC (CD31, CD105, CD144, and Tie-2) were highly expressed by pUVEC, as well as Vimentin [ Figure 8B].
To further demonstrate the endothelial phenotype of pUVEC, we tested the functionality of the cultured cells with the DiI-Ac-LDL uptake assay. Indeed EC, but not SMC, can internalize and metabolize Ac-LDL, which is detectable using a labeled form of the molecule, the probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI), as reported in the literature [29]. The DiI-Ac-LDL uptake assay showed that pUVEC signi cantly internalized the dye, as demonstrated by the associated red uorescence detectable using a conventional uorescence microscope [ Figure 9]. Therefore, considering the results obtained from ow cytometry, immuno uorescence analysis, and the functional assay, we demonstrated that pUVEC isolated from porcine umbilical vein showed a robust endothelial phenotype.

Discussion
To date, the only de nitive treatment for end-stage organ disease is orthotopic transplantation.
Conventional surgical practices typically utilize organs from living or deceased donors for transplantation. However, the de cit between the number of patients waiting for a life-saving transplant and the number of organs available for transplantation has grown substantially in recent years. As a result, new options such as tissue engineering and regenerative medicine are gaining traction to overcome organ shortage.
Decellularization is a promising new approach in regenerative medicine that allows researchers to generate three-dimensional biological scaffolds by selective removal of all the cellular and nuclear components of an organ without signi cantly modifying the architecture and biochemistry of the vasculature, which can be used as an easy route to deliver cells throughout the entire organ [2,30,31]. Complete recellularization of the whole vascular network of an acellular scaffold represents a huge bottleneck towards the translation of bioengineered organs to the clinic. Blood vessels, such as arteries and veins, consist of three layers: tunica intima, mainly composed of EC; tunica media, which consists of connective tissue and SMC; and tunica adventitia, composed mainly of connective tissue. Capillaries and sinusoids are composed of a single layer of attened EC and a basal lamina with few pericytes [32]. Therefore, EC, SMC, and pericytes/MSC must be considered fundamental bricks to rebuild the vasculature while using the preserved native architecture of the acellular organ as a guiding blueprint. In this regard, and to thoroughly investigate this, the pig has been consistently used in the past years as one of the animal models of choice for regenerative medicine and transplantation research [17,33]. Thus, isolating porcine vascular cells for future organ bioengineering applications is paramount to advancing the eld without increasing the experimental complexity by crossing xenogeneic immune barriers using human cells transplanted in pigs [33].
Hence, to de ne optimal cell sources for organ revascularization, we isolated the three main cellular components of porcine blood vessels, expanded under conventional bidimensional culture conditions, and characterized them by their immunophenotype and functionality. We found that all the three cell types isolated and cultured could maintain speci c phenotypical features, as demonstrated by ow cytometry and immuno uorescence analysis and cell functionality, as shown through pBM-MSC trilineage differentiation and pUVEC DiI-Ac-LDL uptake assays. Additionally, we con rmed that our culture medium containing 10% FBS MSC Quali ed induced robust cell proliferation providing adequate support for pBM-MSC expansion [34]. However, it is essential to highlight that there is still a lack of consensus regarding the de nition of the optimal markers and methods to verify the identity of pBM-MSC [21,[35][36][37]. Thus, considering the existing literature, we tested a panel of markers to determine the phenotype of pBM-MSC. Many molecules involved in cell adhesion and ECM proteins, cytokines, and growth factor receptors are expressed by MSC and are all associated with their functions and cell interactions within the bone marrow [38,39]. We showed that cultured pBM-MSC were adherent to plastic, with characteristic spindle-shaped morphology and expressed speci c mesenchymal surface markers (CD90, CD29, and CD44), while they did not express lineage markers (CD11b, CD14, CD31, CD34, CD45), as reported in the literature [21,[40][41][42][43]. A general lack of consensus also exists about the optimal differentiation conditions for pBM-MSC [21]. Thus, we performed in vitro differentiation of pBM-MSC into adipocytes, osteoblasts, and chondrocytes using commercial differentiation kits to obtain further information regarding the functionality of pBM-MSC. As these kits are formulated for human MSC, we followed the manufacturer's instructions, and we differentiated the porcine cells for 21 days. The multipotency of pBM-MSC has been demonstrated, as the cells could differentiate into the three lineages, con rming other reported results [20,21,34,40].
Moreover, it has been described that adipogenic differentiation kinetics of pBM-MSC are different from human MSC, and fat accumulates initially in the adipocytes but is secreted afterward. This behavior is observed in pBM-MSC but not in human MSC. Therefore, the measurement of adipogenic differentiation by Oil Red O staining may underestimate the actual fat formed by pBM-MSC [21]. From our results, we could show that pBM-MSC can be successfully differentiated using these human MSC formulated commercial kits.
Vascular SMC are present in distinct phenotypic states in blood vessels. Indeed, they exhibit different morphological and functional properties within the same blood vessel [44,45] and different blood vessels, e.g., arteries and veins [46]. SMC show a high degree of plasticity and the ability to switch from a contractile to a synthetic phenotype in response to environmental stresses and vascular injury. This plasticity creates a complex diversity among SMC and is associated with changes in morphology, proliferation and migration rates, and the expression of different marker proteins. Contractile SMC express SM22 , SMA, Caldesmon, smooth muscle calponin, smooth muscle myosin heavy chain, and smoothelin, among other markers, while synthetic SMC express osteopontin, collagen I, moesin, plateletderived growth factor A and other markers [25]. Cell morphology represents an essential parameter for the de nition of SMC phenotype. Contractile SMC are elongated, spindle-shaped cells, while synthetic SMC are epithelioid or rhomboid with a cobblestone-like morphology [47,48]. In this study, we have e ciently isolated vascular SMC from the porcine aorta, which retained important phenotypic features of contractile SMC when in culture. Indeed, pASMC showed a characteristic spindle-shaped morphology and expressed speci c muscle markers (SM22 , SMA, Caldesmon) and mesenchymal markers (CD90, CD29, CD44) while the expression of lineage markers were absent (CD11b, CD14, CD31, CD34, CD45, CD144). In addition, the cells expressed CD56, which is considered a natural killer, neuronal, and muscle marker but has also been described as a marker of a subset of MSC derived from human bone marrow [49][50][51]. SMA is the most general marker of SMC lineage, and it is highly expressed in spindle-shaped SMC, while SM22 , smoothelin, calponin, and Caldesmon are late differentiation markers abundantly expressed in spindle-shaped SMC [52][53][54]. CD44 is a cell-surface receptor of hyaluronate, which has been described on various cell types that play a role in ECM binding, cell adhesion, cell-cell interactions, and cell migration [55,56]. The expression of this mesenchymal marker is reported in vascular cells derived from the porcine aorta [57] and rat aortic SMC [58]. Also, our results are consistent with the results showed by Zaniboni et al. that reported that vascular cells derived from the tunica media of porcine aorta expressed CD56 and CD90, while conversely, the cells did not express the hematopoietic markers CD34 and CD45 [57]. Our results show that pASMC express CD29 is also consistent with several published reports, demonstrating that integrin beta 1 (i.e., CD29) is predominant in vascular SMC in vivo and cultured SMC [59,60]. Therefore, we have shown that pASMC isolated from the aorta's tunica media showed speci c mesenchymal surface markers. Moreover, the cells exhibited an elongated, fusiform, spindle-shaped morphology and expressed CD29, SM22 , SMA, and Caldesmon, which are marker proteins of the contractile phenotype of SMC.
EC are the main component of the endothelium, which serves as a permeable barrier for blood vessels and regulates blood ow. EC are present in the whole vascular system, from the heart to capillaries, and control the exchanges of materials and the transit of white blood cells in and out of the bloodstream. Furthermore, EC are involved in regulating blood ow, coagulation, endothelial permeability, vascular tone, and vascular remodeling in responses to physiological and pathological stimuli. In terms of recellularization of decellularized scaffolds, a complete re-endothelialization of the acellular vascular network is required to avoid thrombosis after transplantation, induced by the interaction between ECM proteins and the plasma clotting factors and platelets. In this study, we were able to isolate vascular EC from the porcine umbilical vein and expand them under conventional bidimensional culture conditions. The isolated primary cultures were composed of heterogeneous cell populations with different morphology, similar to the morphology reported for their human equivalents [61] and Chrusciel and colleagues' work [62]. After that, we characterized the cells by ow cytometry and immuno uorescence analysis to de ne the phenotypical identity of expanded pUVEC. We showed that pUVEC expressed speci c endothelial markers (CD31, CD105, CD144, Tie-2) and mesenchymal markers (CD29, CD44, Vimentin), while the expression of CD34 was absent. . Further, we assessed the functionality of pUVEC by DiI-Ac-LDL uptake assay and showed that cultured pUVEC were able to internalize the uorescent-labeled-DiI-Ac-LDL with success. These results suggest that pUVEC could maintain speci c endothelial immunophenotypical features and functionality when isolated and cultured under these de ned conditions.
In conclusion, this study provides suitable and e cient methods for the parallel isolation of pBM-MSC, pASMC, and pUVEC and convenient and straightforward cell culture procedures for cell expansion under conventional bidimensional conditions valuable and rigorous characterization of the cultured porcine cells.
Overall, our results showed that it is possible to isolate, expand and characterize pBM-MSC, pASMC, and pUVEC successfully using our reported methods. Thus, the obtained cells could be further expanded and could represent a valuable cellular tool for tissue engineering purposes, i.e., for recellularization approaches of any vascularized organ or for angiogenesis studies using co-culture assays to understand cell interactions under physiological conditions in bidimensional and three-dimensional cultures.

Animal tissues
Bone marrow and aorta tissues were obtained from male and female domestic piglets weighing 5-7 kg. All animals (n=4) were housed at the Experimental Surgery Department under approved conditions and the tissues were harvested after intraoperative approved euthanasia. Umbilical cords were obtained from

Isolation and cultivation of pASMC
The porcine aorta was procured from 5-7 kg piglets. pASMC were isolated using a modi ed version of the protocol published by Beigi and colleagues [18]. In a laminar ow hood and sterile conditions, the aorta was rinsed with cold calcium, and magnesium-free PBS supplemented with 1% (v/v) P/S to remove all residual blood. The aortic tube was dissected to eliminate the external elastic lamina and remove the tunica adventitia. One extremity of the vessel was ligated with a surgical 2-0 silk suture (B. Braun Melsungen, Germany) and checked for leaks with PBS. Next, the aorta was lled with dispase II protease (Sigma-Aldrich, Merck, Germany) (2,5 U/ml in DMEM) supplemented with 1% (v/v) P/S, and the other end of the tube was ligated. The resulting vessel was incubated for 1 hour at 37°C in a humidi ed atmosphere of 5% CO 2 . Then, the enzyme solution from the aorta was collected using an equal volume of DMEM/FBS, and removed to eliminate the endothelial cell fraction of the aorta. After this, the tissue was cut open and placed in a Petri dish with the tunica intima facing up and scraped away any remaining EC using a cell scraper (ThermoFisher Scienti c, USA). The tunica media layer was cut into 1-2 mm pieces, placed in a 50ml conical centrifuge tube (Corning, USA), and incubated for 1 to 1.5 hours in 0.2% (w/v) collagenase type I (Sigma-Aldrich, Merck, Germany) in DMEM + 1% (v/v) P/S at 37°C with agitation at 210 rpm on an orbital shaker (Innova 40, New Brunswick Scienti c, USA). This collagenase solution was then discarded and replaced with 0.1% (w/v) collagenase type I in DMEM + 1% P/S and incubated for 5 hours at 37°C with agitation at 230 rpm on an orbital shaker (Innova 40, New Brunswick Scienti c, USA).
After 5 hours, the cell-enzyme solution was deactivated with an equal volume of DMEM/FBS and ltered through a 100 µm and then a 40 µm cell strainer (Corning, USA) to eliminate large pieces of undigested aortic tissue. The collected cell suspension was centrifuged, and the pellet was resuspended in DMEM/FBS. The obtained pASMC were seeded at 6,000-14,000 cells/cm 2 depending on the yield of isolated cells in 152cm 2 Petri dishes coated with 5µg/ml bronectin (Sigma-Aldrich, Merck, Germany) in DMEM/FBS at 37°C in a humidi ed atmosphere of 5% CO 2 . The medium was replaced with a fresh medium after 48 hours from the initial seeding and changed every three days. Once con uent (90%), cells were either frozen or sub-cultured following cell detachment with 0.05% (v/v) trypsin/0.02% (v/v) EDTA and trypsin inactivation with DMEM/FBS and seeded at 6,000 cells/cm 2 in 152cm 2 Petri dishes coated with 0.2% (v/v) gelatin in DMEM/FBS and let at 37°C in a humidi ed atmosphere of 5% CO 2 . Passage 3-5 cells were used in experiments. The photos were captured with an inverted optical microscope (Leica DMI3000B/Nikon Digital Camera Dxm1200F).

Isolation and cultivation of pUVEC
Porcine umbilical cords were procured from swine fetuses at 72-80 days post-insemination. The cells were isolated from the umbilical vein by enzymatic digestion with 0.05% trypsin/0.02% EDTA, following an adapted protocol of Davis and co-workers [19]. After isolation, the cells were seeded at 15,000 cells/cm 2 in 152cm 2 Petri dishes coated with 1% (v/v) gelatin in porcine endothelial growth medium (pEGM) (Cell Applications, USA) and incubated at 37°C in a humidi ed atmosphere of 5% CO 2 . The medium was replaced with a fresh medium after 48 hours from the initial seeding and changed every two days. Once con uent (100%), cells were either frozen or sub-cultured following cell detachment with 0.05% trypsin/0.02% EDTA and trypsin inactivation with DMEM/FBS and seeded between 3,000 and 6,000 cells/cm 2 in 152cm 2 Petri dishes coated with 1% (v/v) gelatin with pEGM and incubated at 37°C in a humidi ed atmosphere of 5% CO 2 . The medium was changed every 5 days. To remove any contaminant cells from the culture, the cells were selected by cell sorting. Brie y, the cells were harvested, pelleted, counted, resuspended in basic ow cytometry buffer (FC Buffer: 2% FBS in PBS) and incubated with phycoerythrin (PE) conjugated antibody anti-CD31 (Clone: LCI-4, Bio-Rad, USA). The cells were washed three times with FC Buffer, centrifugated and resuspended in basic sorting buffer (1% Bovine Serum Albumin (BSA) (Linus, Cultek, Spain) + 1mM EDTA in PBS + 1% P/S) for the following cell separation. Cell sorting was carried out using a BD FACSJazz cell sorter (Becton Dickinson, USA). Then, the cells were seeded between 3,000 and 6,000 cells/cm 2 in 152cm 2 Petri dishes coated with 1% gelatin with pEGM and let at 37°C in a humidi ed atmosphere of 5% CO 2 . The medium was changed every 5 days. Generally, passage 5-8 cells were used in the experiments. The pictures were captured with an inverted optical microscope (Leica DMI3000B/Nikon Digital Camera Dxm1200F).

Phenotypical characterization of pBM-MSC, pASMC and pUVEC
Flow cytometry and immuno uorescence assay were used to assess the expression of a panel of speci c extracellular and intracellular markers of pBM-MSC, pASMC, and pUVEC. The list of antibodies used for cell characterization is shown in Table 1. A minimum of 10,000 events were collected for ow cytometry, and cells were analyzed on a BD FACSCalibur ow cytometer using Cell Quest Software (Becton Dickinson, USA). All data were analyzed using FlowJo Software (FlowJo, Becton Dickinson, USA).
The ow cytometry data of pBM-MSC, pASMC, and pUVEC was con rmed by immuno uorescence staining in 24-wells plates (ThermoFisher Scienti c, USA). The cells were cultured under static conditions in an expansion medium at 37°C in a humidi ed atmosphere of 5% CO 2 . pBM-MSC were seeded at 3,000 cells/cm 2 in 24-wells plates coated with 0.2% gelatin, and the expansion medium was changed every 5 days until reaching con uence (80-90%). pASMC were seeded at 6,000 cells/cm 2 in 24-wells plates coated with 0.2% (v/v) gelatin at 37°C, and the expansion medium was changed every 5 days until reaching con uence (80-90%). Finally, pUVEC were seeded at 4,000 cells/cm 2 in 24-wells plates coated with 1% (v/v) gelatin, and the expansion medium was changed every 5 days until reaching con uence (100%). After xation with 4% (v/v) paraformaldehyde for 15 minutes, the cells were immunostained with several primary antibodies (Table 1) and an appropriate secondary antibody. The photos were captured with a uorescence microscope (Leica DMI3000B/Nikon Digital Camera Dxm1200F) and processed using ImageJ 1.51s software. Table 1 List of the distinct antibodies used for the immunophenotypical characterization of pBM-MSC, pASMC, and pUVEC. Brie y, for adipogenic and osteogenic differentiation assays the cells were cultured in 24-wells plates coated with 0.2% (v/v) gelatin at 10,000 cells/cm 2 in expansion medium (DMEM/FBS) until con uence (80%) at 37°C in a humidi ed atmosphere of 5% CO 2 . Then, the expansion medium was replaced with a differentiation medium, which was changed every 3 days for 21 days.

Competing interests' statement
The authors declare that they have no competing interests.

Funding
Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology (FCT) (UIDB/04565/2020) and FCT through the project PTDC/EQU-       DiI-Ac-LDL uptake assay of pUVEC cultured under static conditions with pEGM culture medium. The cells that internalized DiI-Ac-LDL are shown as red cells. Scale bar: 50 µm.