Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics

Cytotherapy, 2010; 12: 275–287

ISSN 1465-3249 print/ISSN 1477-2566 online © 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)DOI: 10.3109/14653241003596679

∗These authors contributed equally to this work.Correspondence: Pasquinelli Gianandrea, MD, Associate Professor, Clinical and Surgical Pathology, Department of Radiological and Histocytopatho-logical Clinical Sciences, Policlinico S. Orsola, via Massarenti 9, Bld 11, 40138, Bologna, Italy. E-mail: [email protected]

(Received 7 October 2009; accepted 4 January 2009)

ORIGINAL ARTICLE

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics

GIANANDREA PASQUINELLI 1 ∗, ANNALISA PACILLI 2 ∗, FRANCESCO ALVIANO 3 , LAURA FORONI 2 , FRANCESCA RICCI 4 , SABRINA VALENTE 2 , CATIA ORRICO 1 , GIACOMO LANZONI 3 , MARINA BUZZI 4 , PIER LUIGI TAZZARI 4 , PASQUALEPAOLO PAGLIARO 4 , ANDREA STELLA 2 & GIAN PAOLO BAGNARA 3

1 Clinical and Surgical Pathology, Department of Radiological and Histocytopathological Clinical Sciences, S. Orsola Hospital, University of Bologna, Bologna, Italy, 2 Chair of Vascular Surgery, Department of Specialistic Surgical and Anaesthesiological Sciences, S. Orsola Hospital, University of Bologna, Bologna, Italy, 3 Department of Histology, Embryology and Applied Biology, University of Bologna, Bologna, Italy, and 4 Cardiovascular Tissue Bank, Transfusion Medicine Service, S. Orsola Hospital, Bologna, Italy

Abstract Background aims. The presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue. Methods. Resident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitr o study of the stemness features and multipotency of these multidistrict MSC populations. Results. Notch-1 � , Stro-1 � , Sca-1 � and Oct-4 � cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothe-lial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high effl ux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis. Conclusions. Overall, these fi ndings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

Key Words: human artery wall , leiomyogenesis , multiorgan donors , multipotent mesenchymal stromal cells

models of ischemia by becoming incorporated in newly formed capillaries; these cells are therefore implicated in vascular injury repair via differentiation into fully competent resident vascular cells (4–10). However, BM-derived cells are not the only players in vascular homeostasis; other studies indicate that stem cells reside in both animal (11–14) and human vessels (15–18). These cells may participate not only in physiologic renewal, providing rapid targeted

Introduction

Cell proliferation and vascular wall (VW) remodeling are the fi rst responses by arteries to pathologic envi-ronmental stress (1–3). In adult life, bone marrow (BM)-derived stem cells and progenitors are mobi-lized continuously into the circulating blood to target areas of tissue injury. BM mesenchymal stromal cells (MSC) and circulating endothelial progenitor cells (EPC) contribute to angiogenesis in experimental

276 G. Pasquinelli et al.

rescue for regeneration after injury by giving rise to new endothelial cells (EC) and smooth muscle cells (SMC), but also in pathologic conditions. Consis-tent with this view, the existence of a capillary-rich vasculogenic zone has been identifi ed in adult human arteries; in this area it has been postulated that MSC may be present amidst the endothelial progenitors and hematopoietic stem cells (16). This hypothesis is supported by several studies claiming to have found an in vivo reservoir of MSC in post-natal tissues (19,20). Growing evidence does indeed indicate a developmental relationship between pericytes and MSC (20–24); moreover, it has been suggested that pericytes may represent the MSC in situ counterpart (20,24). The presence of multipotent progenitors is not restricted to microvasculature; VW-resident MSC have already been isolated from human large vessels such as the adult thoracic aorta (17), pulmonary artery (18), saphena (15) and umbilical veins (25).

We have demonstrated previously that the thoracic aorta harvested from multiorgan donors yields MSC endowed with angiogenic ability (17); here we investi-gated whether, according to the embryologic develop-ment of the aorta, MSC also reside in more proximal and peripheral arterial segments collected from healthy donors. Notch-1-, Stro-1-, stem cell antigen-1 (Sca-1)- and Oct-4-expressing cells were distributed along the previously described arterial wall vasculogenic niche, thus supporting the view that embryonic-like cells may persist in the adult life. We also provide evidence that MSC derived from the VW have consistent stem cell properties; they express genes involved in the control of stemness and cohere with embryonic stem cell-like features; they show a high effl ux of the DNA-binding dye Hoechst 33342, and are clonogenic and able to grow as spheroids under appropriate culture condi-tions; they display a wide differentiation potential that encompasses angiogenic, adipogenic, osteogenic, chondrogenic and leiomyogenic commitment. Inter-estingly, this study also extends the potential clinical use of freshly banked homografts to the fi elds of cell therapy, regenerative medicine and tissue engineer-ing. Finally, the multipotency of VW MSC could have a role in the morphogenesis of atherosclerosis, a possibility that still remains to be determined.

Methods

Arterial samples, cell culture and fl ow cytometry characterization

Human arterial segments of aortic arches (AA; n � 2), thoracic aortas (TA; n � 4) and femoral arteries (FA; n � 3) from heart-beating donors were harvested, processed for immunohistochemical examination and used for cell isolation as described previously

(17). The mean donor age was 39 years; two were females and six males.

Immunohistochemistry

To localize in situ embryonic-like VW MSC, 5-μm sections of vessels were stained with the following monoclonal antibodies (MAb): Oct-4 (1:100; Chemicon Int., Tamecula, CA, USA), Stro-1 (1:100; R&D Systems, Milan, Italy), Sca-1 (1:10; Ceder-lane Laboratories, Hornby, Canada) and Notch-1 (1:50, clone c-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antigen–antibody reaction was revealed using a Novolink polymer detection system (Novolink, Newcastle, UK).

Cell isolation

VW MSC were obtained after mechanical mincing and enzymatic digestion of segments with 0.3 mg/mL liberase type II (Roche, Milan, Italy) in serum-free Dulbecco’s modifi ed Eagle medium (DMEM; Lonza, Basel, Switzerland) at 37°C for 4 h using a rotor apparatus.

Immunophenotyping

Flow cytometry analysis was performed to assess the cell phenotype of VW MSC at passages 3–5 using the following MAb: anti-CD29–fl uorescein isothiocya-nate (FITC), CD31–phycoerythrin (PE), CD34–PE, CD146–PE, CD44–FITC, CD45–allophycocyanin (APC), CD73–FITC, CD90–phycoerythrin-cyanine 5 (PC5), CD105–PE, CD166–PE (all from Beckman Coulter, Fullerton, CA, USA), von Willebrand Fac-tor (vWF; clone F8/86; Dako Cytomation, Glostrup, Denmark), Stro-1 (clone STRO1; R&D Systems), Notch-1 (clone c-20; Santa Cruz Biotecnology) and Oct-4 (clone c-10; Santa Cruz Biotecnology). The following secondary MAb were used after cell stain-ing with unlabeled primary MAb: anti-mouse IgG–APC (Beckman Coulter) and anti-rabbit IgG–FITC (Dako Cytomation). To reveal vWF and Oct-4, the cells were fi xed and permeabilized with an Intrapep Kit (Beckman-Coulter) and subsequently incubated with anti-mouse IgG–FITC (Dako Cytomation). Samples were analyzed using a Cytomics FC500 fl ow cytometer equipped with two lasers (Beckman Coulter). Results were analyzed using CXP software (Beckman Coulter).

Hoechst side population labeling

To investigate the side population (SP) phenotype among VW MSC populations, cells at 3–5 passages were labeled with Hoechst 33342. Briefl y, cells were

Multidistrict human multipotent vascular cells 277

suspended in room-temperature Hanks’ balanced saline solution (HBSS) containing 2% fetal bovine serum (FBS) and 2 mM HEPES buffer (SP buf-fer) at 5 � 10 6 cells/mL. Cells were pre-warmed to 37°C and Hoechst 33342 added to a fi nal concentra-tion of 5 μg/mL. Cells were incubated for 90 min, centrifuged and resuspended in cold SP buffer at 5 � 10 6 /mL. Cells were then kept on ice and analyzed within 6 h of labeling. Propidium iodide was added to the cells at 2 μg/mL immediately prior to analysis. For some experiments, cells were pre-incubated with the ABCG2 inhibitor verapamil (100 μmol/L) at 37°C for 30 min prior to Hoechst 33342 addition.

Bell-shaped nuclei

The presence of bell-shaped nuclei was investigated using DAPI staining in immunofl uorescence (IF). All vascular populations were recovered at passage 4 and plated at 3 � 10 3 /cm 2 on collagen I-coated slide chambers (BD Labware, Franklin Lakes, NJ, USA); cells were fi xed in 2% paraformaldehyde and mounted with Pro-long anti-fade with DAPI mounting solution (Molecular Probes, Milan, Italy). Samples were observed under a Leica fully auto-mated inverted fl uorescence microscope DMI6000 B using a DAPI fi lter (Leica Microsystems, Milan, Italy). At least 200 nuclei/sample were counted on a minimum of 10 High-Power Field (HPF). The percentage/sample of cells having bell-shaped nuclei was determined by counting the total number of bell-shaped nuclei and calculating the percentage of cells with this characteristic out of the total number of counted nuclei. The count was performed independently by two operators on the same samples and then a mean of the individual results was calculated.

Clonogenic assay

To establish colony-forming units cells, VW MSC at passage 5 were plated in cell culture Petri dishes (Corning, New York, NY, USA) coated with collagen I (Sigma, Milan, Italy) at approximately 1–10 cells/cm 2 . Colony growth was visualized directly on plates after crystal violet staining; the percentage of clonogenic cell subsets was estimated by counting colonies derived from all seeded cells.

In vitro spheroid formation and characterization

The stem cell ability to form spheres is a well known acquisition (26–28). To determine whether VW MSC had this growth capacity, adherent VW MSC at passage 5 were suspended in culture medium and plated at 2 � 10 4 cells/well in ultralow attachment

24-well plates. After a few days, numerous spheres were observed under light microscopy. The spheres were recovered and processed for IF microscopy, transmission electron microscopy (TEM) and reverse transcription (RT)–polymerase chain reaction (PCR) gene expression analysis.

IF analysis

Single spheres were embedded in a 2% agar gel, mounted on a cork dish with a small amount of OCT mounting medium and frozen in liquid isopentane cooled in liquid nitrogen at –160°C. Frozen blocks were stored at –80°C until use. Immunofl uorescent stain-ing was performed on 6-μm thick sections cut with a cryostat at –20°C. Frozen sections were dried at room temperature and fi xed in cold acetone. After a second drying step, the sections were washed and incubated with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) to block unspecifi c reactive sites. Immunostaining was performed by labeling the samples with MAb against CD133 (1:80; Miltenyi Biotec, Bergisch Gladbach, Germany), CD34 (clone QBEND-10, 1:80; Dako Cytomation), c-kit (1:200; Santa Cruz Biotechnology), Stro-1 (1:100; R&D Sys-tems), Sca-1 (1:10; Cederlane Laboratories), Oct-4 (1:100; Chemicon), CD44 (clone G44-26, 1:50; BD Labware) and Notch-1 (1:50, clone c-20; Santa Cruz Biotechnology) for 45 min at 37°C and then incu-bating with FITC-conjugated polyclonal rabbit anti-mouse immunoglobulins for 45 min at 37°C. Finally, coverslips were mounted with Pro-long anti-fade with DAPI mounting solution and the samples observed under a Leica fl uorescence microscope DMI6000 B using appropriate fi lters.

Transmission electron microscopy

Single spheres were recovered and fi xed in 2.5% phosphate-buffered glutaraldehyde for 24 h at 4°C, followed by post-fi xation with 1% buffered osmium tetroxide (OsO 4 ) for 1 h at room temperature. Then the samples were dehydrated in a graded series of ethanol and embedded in epoxy resin; ultrathin sections were counterstained with lead citrate and uranyl acetate and observed with a transmission electron microscope Tecnai 12 (FEI Company, Milan, Italy).

RT-PCR gene expression analysis

We investigated which regulatory pathways were pre-dominantly expressed in stem cell-forming spheres among c-kit, Notch-1, BCRP-1, BMI-1, SOX-2, Oct-4, CD133 and KDR genes. Total RNA was extracted from spheroids using the RNA extracting


fi ve-hundred cells/cm 2 were plated on collagen I-bio-coated culture six-well plates (BD Labware) in SmGM-2 (Lonza). After 24 h, the medium was changed with fresh SmGM-2 plus 10 ng/mL Transforming Growth Factor beta 1 (TGF-β1) (Sigma) and 5 ng/mL Platelet Derived Growth Factor BB (PDGF-BB) (Sigma). Cells were cultured for 14 days, feeding the medium every 3 days. Cell controls were cultured in SmGM-2 without additional growth factors. RT-PCR and IF were used to investigate the gene modulation and pro-tein expression of smooth muscle-specifi c markers such as alpha smooth muscle actin (ASMA; 1:9000, clone 1A4; Dako Cytomation), high caldesmon (h-CALD; clone h-CD, 1:75; Dako Cytomation) and calponin (CALP; clone CALP, 1: 40; Dako Cytomation). TEM was used to describe changes in the contractile appa-ratus of induced cells compared with the non-induced counterpart.

Results

Major human elastic and muscular arteries contain cells with the morphologic and immunophenotype properties of MSC

From anatomically distinct segments we succeeded in isolating cells with morphologic and immunophe-notypical properties of MSC; primary cell cultures were established from two AA, four TA and three FA, thus demonstrating that MSC populations are

TRI reagent (Ambion Applied Biosystems, Austin, TX, USA) according to the manufacturer’s instruc-tions. RT reactions were performed in a 40-μL volume with 4 μg total RNA, and the RT-PCR with 1 μL of this solution. PCR primers are listed in Table I and were purchased from Invitrogen, San Giuliano Milanese (MI), Italy; RT reagents were furnished by Ambion Applied Biosystems and PCR reagents by Qiagen (Milan, Italy). The PCR products were sepa-rated by electrophoresis on a 2% agarose gel with 1� Tris–acetate–EDTA buffer, stained with ethidium bromide and photographed under ultraviolet light.

Multilineage differentiation potential

VW MSC taken at passages 3–5 were differentiated towards mesoderm-type cells such as adipocytes, osteo-blasts, chondrocytes and leiomyocytes. In vitro adipo-genic and osteogenic commitments were performed as described by Alviano et al . (29). To demonstrate adipo-genic and osteogenic differentiation, the cultures were stained with Oil red O and von Kossa, respectively; all samples were also analyzed by TEM. Chondrogenic induction was performed using a commercial medium (Lonza) and pellets were formalin-fi xed, embedded in paraffi n and immunostained for type II collagen (anti-human collagen type II, 1:200 in 1% BSA–PBS; Chemicon Int.); other samples were processed for ultrastructural investigation. Leiomyogenesis was induced using a home-made medium. Three- thousand

Table I. Primers for RT and PCR.

Gene Primers sequence Amplicon length (bp) Temp. (°C)

Oct-4 REV 5′-CCACATCGGCCTGTGTATAT-3′ 380 60FW 5′-CTCCTGGAGGGCCAGGAATC-3′

BMI-1 REV 5′-CATTGCTGCTGGGCATCGTAAG-3′ 369 62FW 5′-GGAGACCAGCAAGTATTGTCCTTTTG-3′

BCRP-1 REV 5′-CTGAGCTATAGAGGCCTGGG-3′ 652 60FW 5′-GTTTATCCGTGGTGTGTCTGG-3′

CD133 REV 5′-GTACAACACTACCAAGGACAAGGCGT-3′ 337 62FW 5′-CTGGGGCTGCTGTTTATTATTCTG-3′

KDR REV 5′-TTTGTCACTGAGACAGCTTGG-3′ 555 62FW 5′-TATAGATGGTGTAACCCGGA-3′

c-kit REV 5′-CATACAAGGAGCGGTCAACA-3′ 275 57FW 5′-GTCTCCACCATCCATCCATC-3′

SOX-2 REV 5′-GCGCCGCGGCCGGTATTTAT-3′ 208 60,5FW 5′-ACCGGCGGCAACCAGAAGAACAG-3′

Notch-1 REV 5′-TGGCATCAGCTGGCACTCGTCC-3′ 496 62FW 5′-CCGGCTGGTCAGGGAAATCGTG-3′

β2-microglobulin REV 5′-ATCTTCAAACCTCCATGATG-3′ 114 58FW 5′-ACCCCCACTGAAAAAGATGA-3′

ASMA REV 5′-GTGATCTCCTTCTGCATTCGGT-3′ 372 60FW 5′-CTGAGCGTGGCTATTCCTTC-3′

CALP2 REV 5′-GCAGGTCTGAGGGTCTGAAG-3′ 470 60FW 5′-CCAGTCAACCAAGGGTCTGT-3′

CALD REV 5-GCTGCTTGATGGGTCGATTTGA-3′ Low: 744 60FW 5′-GTCACCAAGTCCTACCAGAAGA-3′ High: 1508


were stained with Hoechst 33342 in the presence or absence of verapamil and processed for fl ow cytometry analysis. We found that approximately the 1% of all VW MSC possessed the SP pheno-type; effl ux of Hoechst 33342 dye was inhibited by verapamil, confi rming the specifi city of the staining (Figure 3).

Other features of more undifferentiated stem cells are the presence of bell-shaped nuclei and their clonogenic ability, both features related to symmetric divisions occurring during the cell cycle. Under a DAPI fi lter, the total number of bell-shaped nuclei was counted and later compared with the total number of seeded cells. Regardless of the vascular source, we found that a percentage of 0.30 � 0.05% of VW MSC presented bell-shaped nuclei (Figure 4A), a morphologic characteristic of embryonic-like stem cells. This result suggested that primary cultures of VW MSC are heteroge-neous even though they present similar growth and immunophenotypical characteristics; as indicated by DAPI identifi cation of cells with bell-shaped nuclei, a small proportion of hierarchically higher cells exists; this fi nding was confi rmed by other in vitro assays aimed at evaluating more established stemness characteristics of VW MSC.

To investigate the clonogenic ability of VW MSC, single cells were seeded in a 100-mm Petri dish via limited dilution. At this low density culture condi-tion, we noted that cells initially displayed a particu-lar phenomenon known as the shrink effect, a typical suffering response to non-optimal growing condi-tions: cells exhibited membrane wrinkling and fl at-tening. Only stress-resistant cells managed to survive in such an unfavorable contest. We found that 0.36 � 0.03% of total VW MSC retained a clo-nogenic ability in adult life (Figure 4B, C); again, the clono genic assay showed that, regardless of the arterial origin or similarities in growth and immunophenotypical characteristics, VW MSC were heterogeneous and contained a subset of more undif-ferentiated cells according to the stage of embryologic

present in arteries and have different in vivo extents and architecture, i.e. an elastic and muscular typol-ogy. Depending on the starting amount of arterial tissue used to establish the cultures, confluence was reached within 5 days. The cells were spindle-shaped and had a tendency to grow in a whorled pattern . No signifi cant differences, as judged by morphologic, immunophenotype and functional analyzes, were observed among cultures generated from arterial segments leading to different anatomical districts or architectures; because the amount of tissue avail-able for the study varied considerably, comparative analysis to establish MSC yield differences between arterial sources was not possible.

Regardless of the vascular source, cells derived from the human arterial wall had an immunophenotype profi le consistent with that of MSC; examples of fl ow cytometry surface molecule single analysis are shown in Figure 1. All the populations expressed typical mesen-chymal antigens such as CD29, CD44, CD90, CD73, CD105 and CD166; in contrast, hematopoietic lin-eage (CD45), hematopoietic progenitor (CD34) and endothelial cell (CD31, vWF and CD146) markers were negative. Stemness markers, i.e. Oct-4, Notch-1 and Stro-1, were intensely expressed.

We have already reported that CD117-positive cells are mainly located between media and adventi-tia layers in adult TA (17). To strengthen further the concept that in vivo this area exerts a niche-like function, we accomplished an immunohistochemi-cal study of stemness markers on arterial wall sec-tions for each district. Regardless of vascular segment, we found that Notch-1-, Stro-1- and Sca-1-positive cells were distributed along the previously described vasculogenic niche (16); also small, rounded and Oct-4-positive cells appeared to localize in this area (Figure 2).

A subset of human VW MSC has stem cell properties

A defi ning property of stem cells is low fl uores-cence after staining with Hoechst 33342. VW MSC

Figure 1. Flow cytometry analysis of VW MSC immunophenotype. AA-, TA- and FA-derived cells exhibit a similar immunophenotype without any relevant differences regarding their in vivo distribution.


hematopoietic progenitor, CD133 and CD34 mark-ers (data not shown). RT-PCR analysis confi rmed stem cell gene expression and demonstrated that, at passage 5, VW MSC lost the expression of BCRP-1 and BMI-1, previously described at passage 3 in TA MSC (17), whilst the expression of c-kit and Oct-4 persisted during extended subculture (Figure 4F, G). TEM analysis of spheres showed undifferentiated cells with large, irregularly shaped nuclei, dispersed chromatin and prominent nucleoli; the cytoplasm

development. This intrinsic ability is in agreement with current views about the clonal origin of some diseases such as tumors.

When VW MSC were seeded in ultralow attach-ment plates, they formed agglomerates that were able to generate growth-forming spheres (Figure 4D). Cells constituting spheres expressed stem cell markers, including c-kit, Stro-1, Notch-1 and Sca-1, and mes-enchymal markers, such as CD44, as documented by IF analysis (Figure 4E), whereas they were negative for

Figure 2. Perivascular niche. Immunohistochemical analysis of the arterial wall to localize resident progenitor cells expressing Sca-1, Notch-1, Stro-1 and Oct-4 molecules.

Figure 3. Identifi cation of arterial SP cells. Hoechst 33342 (Ho) and propidium iodide (PI) staining of VW MSC (left panel) as visualized by fl ow cytometry. Addition of verapamil selectively prevented Ho exclusion from SP cells (right panel).


small dense mitochondria, as well as an elevated endocytic activity (Figure 6B).

VW MSC also showed a good capacity for chondrogenic differentiation. Light microscopy on high-density cell cultures revealed that cells were embedded in an abundant extracellular matrix; immunohistochemistry revealed the presence of human type II collagen, which is typically found in joint cartilage (Figure 6A); again, the presence of a proteoglycan-rich matrix was shown by Alcian blue staining in induced cells but not in controls (data not shown). Ultrastructural analysis confi rmed the presence of abundant extracellular matrix containing mature collagen fi bers and proteoglycan particles (Figure 6B) exclusively in induced cells. Unlike adi-pogenesis and chondrogenesis, the osteogenic dif-ferentiation potential was not so effi cient. With light microscopy, von Kossa staining showed a calcium-rich mineralized matrix but the staining was less diffusely distributed than expected (Figure 6A). How-ever, TEM revealed multiple foci of electron-dense fi brillary deposits that were decorated with needle-shaped hydroxyapatite crystals (Figure 6B). Regard-ing the leiomyogenic differentiation potential, gene expression analysis showed increased levels of ASMA and h-CALD genes compared with controls. Again, stimulation with TGF-β1 and PDGF-BB caused de novo expression of the smooth muscle-specifi c

contained mitochondria and profi les of rough endo-plasmic reticulum that, in some cells, were distended by the presence of moderately dense material; in the intercellular space we occasionally found collections of extracellular membrane vesicles and primary cilia documenting intense extracellular membrane traffi c; primitive intercellular junctions were occasionally seen, and gap junctions; the outer cell layer had unexpectedly smooth surfaces, with randomly spaced long and thin fl oating fi lopodia (Figure 5).

Human VW MSC have multilineage differentiation potential

Cells were cultured under specifi c experimental conditions and exhibited a similar ability to differ-entiate along mesenchymal lineages. All VW MSC showed high adipogenic potential, although this ability was more intense when VW MSC were isolated from TA and FA. VW MSC treated with adipogenic media showed the presence of mul-tiple cytoplasm vacuoles that increased in size and number with the time of induction. Controls, in contrast, retained their morphology and did not display signifi cant cytoplasm vacuoles. As seen in Figure 6A, vacuoles stained intensely with the lipid dye Oil red O. By TEM, VW MSC contained mul-tiple, confl uent lipid droplets in the cytoplasm and

Figure 4. Stemness characteristics of VW MSC. (A) Bell-shaped nuclei observed in IF; (B) colony-forming units obtained from single-seeded cells observed under light microscopy and (C) stained with crystal violet dye. (D) In vitro spheroid formation in ultralow attachment plates. (E) Immunophenotype of spheres investigated by immunofl uorescent assay for stem cell antigens and the mesenchymal marker CD44. (F) RT-PCR analysis of stemness genes expressed by cells constituting spheres and (G) densitometric estimate of PCR products normalized with house-keeping gene product β 2-microglobulin.


The upshot is that the identifi cation and characteriza-tion of the progenitors involved in physiologic homeo-stasis and pathologic vascular remodeling is an issue of great interest, one that may improve current knowl-edge of vascular diseases and may provide useful strategies that can be transferred to the fi elds of repair and egenerative medicine and tissue engineering.

In previous studies we have demonstrated that the ‘vasculogenic zone’ (16) of human TA and FA has spe-cifi c immunohistochemical characteristics that make it unique in the arterial wall and congenial with a niche-like function (31–33). We have also documented the angiogenic properties of TA-derived MSC (17). Here we investigated their stem cell features and multipotent abilities, extending our observations to AA and FA. We therefore analyzed fresh elastic and muscular arteries from healthy multiorgan donors from which cells with a plastic-adherent growth and a fi broblast-like mor-phology were obtained. These cells had the typical antigen expression pattern of culture-expanded MSC; they were positive for a mixture of stemness (Stro-1, Notch-1 and Oct-4) and mesenchymal (CD44, CD90, CD105, CD73, CD29 and CD166) markers, and never expressed hematopoietic or endothelial cell markers. We named these cells VW MSC. As VW MSC are recovered from entire human arterial segments, we

marker CALP2 (Figure 7A). Comparing smooth muscle marker expression levels, no signifi cant differ-ence was seen among VW MSC derived from various arterial sources. TEM of induced cells showed a myofi -broblast phenotype; the cells were spindle-shaped and contained abundant peripherally arranged contractile fi laments along with profi les of rough endoplasmic reticulum, and well-organized fi bronexus junctions were seen. Control cells, in contrast, maintained an undifferentiated mesenchymal phenotype (Figure 7B–D). IF analysis on induced and uninduced cells confi rmed the RT-PCR results; in fact, smooth muscle-specifi c proteins were up-regulated after leiomyogenesis induction (Figure 8).

Discussion

Circulating endothelial cell (26), smooth muscle (4) and fi brocyte progenitors (27) have been identifi ed and their potential role in VW homeostasis and dis-ease is becoming recognized. Some researchers have unequivocally demonstrated the presence of VW-resident EPC in humans (16,28). Less is known about resident smooth muscle progenitors, although ani-mal studies have identifi ed different putative candi-dates for smooth muscle regeneration (13,14,19,30).

Figure 5. TEM analysis of spheres. Undifferentiated status of cells constituting spheres. (A) Primary cilium (PC); (B) nucleus (N) with prominent nucleoli (Nu) and rough endoplasmic reticulum (rER); (C) primitive intercellular junction (J); (D) extracellular membrane vesicles (V); (E) deeply cleaved nucleus; (F) smooth surface of outer cells.


human VW MSC collected from different vascular segments contain a subset of ancestral cells with an SP phenotype.

Furthermore, we seeded cells in low-attachment plates to establish whether they were capable of suspension growth. In these conditions, neural and cancer stem cells prove able to form colonies, and VW MSC did so effi ciently. The stemness of cells forming spheres was confi rmed by IF and molecular analysis, while electron microscopy determined their undiffer-entiated condition. Comparing IF analysis and RNA expression, we observed that cell spheroids expressed Notch-1, Stro-1, c-kit and Oct-4; Notch-1 was the most expressed gene, thus suggesting a major role in regulating the stemness and undifferentiated status of VW MSC. To explain the lower expression level of Oct-4, we hypothesized that a subset of VW MSC had more ancestral characteristics; this is consistent with results provided by SP, clonal and bell-shaped nuclei analysis. As expected, TEM showed that VW MSC are undifferentiated mesenchymal cells. Inter-cellular junctions were rare and always of the primi-tive type; primary cilia were also present and probably

evaluated the expression of stemness markers directly on tissue sections by immunohistochemistry. In agree-ment with the niche-like function of the vasculogenic zone, we found that Notch-1-, Stro-1-, Sca-1- and Oct-4-positive cells were located in this area. Post-natal perivascular stem cells are believed to be descendants of cells derived from the embryonic dorsal aorta, called mesoangioblasts (34); the presence of Oct-4-positive cells in adult multidistrict vascular segments reinforces this hypothesis.

However, phenotype characterization is not per se suffi cient to give to a cell population the property of stemness; thus we explored other features com-mon to all stem cells. It is well known that the ABC transporters and breast cancer resistance protein (BCRP/ABCG2), are highly expressed in a popula-tion of primitive stem cells, SP. SP cells were origi-nally discovered in BM by their capacity to exclude rhodamine 123 and Hoechst dye 33342; however, extensive research has also revealed their presence in other non-hematopoietic tissues (35). Hoechst-positive SP cells have been found in healthy arteries of the media of adult mice (13). Here we show that

Figure 6. Multilineage potential of VW MSC. (A) Evaluation of adipogenic, osteogenic and chondrogenic differentiation. The images were originally taken at � 20. (B) TEM analysis of classical mesenchymal commitments. Arrows indicate the presence of lipid droplets after adipogenesis; osteoid matrix and multiple hydroxiapatite crystals in osteogenesis; collagen fi bers after chondrogenesis. Uninduced cells retained their undifferentiated mesenchymal features.


in vitro mesengenic property of MSC deriving from additional human vascular sources was investigated; VW MSC from different vascular segments were able to commit towards adipogenic, osteogenic, chondrogenic and leiomyogenic lineages.

As for adipogenesis, we observed a progres-sive increase in cytoplasmic lipid vacuoles in all populations; however, the extent of adipogenesis was higher in VW MSC deriving from TA and FA than in assays performed on cells deriving from AA. Although we have no explanation for this difference, we may assume that it has a relationship with the embryo-logic origin of the ascending aorta, which is under the control of neuroectodermal progenitors (31).

VW MSC exhibited a good propensity for chon-drogenic but less for osteogenic commitment. Mark-ers of cartilaginous differentiation, such as type II collagen and proteoglycans, were detected with immu-nohistochemical, histochemical and ultrastructural

infl uenced the balance of stem and progenitor cell proliferation versus differentiation (36). One interest-ing characteristic of more ancestral cells is their clo-nogenic ability; accordingly, the clonogenic potential of single VW MSC was assessed in limited-dilution experiments and a percentage of 0.36 cells displayed this ability. In line with recent reports (37,38), we investigated the nuclear morphology of VW MSC as additional evidence of stemness. Regardless of the vascular source, 0.30 � 0.05% VW MSC showed bell-shaped nuclei. Taken together, these results document the stemness and embryonic-like quality of VW MSC populations.

MSC are considered to be a subset of post-natal perivascular stem cells deriving from multipotent mes-enchymal precursors, i.e. mesoangioblasts, which are localized in the embryonic dorsal aorta (34). Human MSC deriving from the pulmonary artery (18) and saphena vein (15) are multipotent. Consequently, the

Figure 7. Leiomyogenic differentiation of VW MSC. (A) RT-PCR of smooth muscle markers; β 2-microglobulin was used as control gene. (B – D) TEM analysis of leiomyogenesis. In induced samples (C, D), features of myofi broblastic differentiation, e.g. basal membrane, contractile fi laments and fi bronexus, are present, while control samples retain undifferentiated aspects (B). BM, basal membrane; CF, contractile fi laments; FN, fi bronexus.


are phenotypically heterogeneous (44) and that cells involved in remodeling injured arteries apparently include smooth muscle cells from the media as well as adventitial fi broblasts (30).

Our study confi rms the recent suggestion that the MSC compartment is more widely distributed than previously thought; it further highlights that MSC are resident in arteries of both elastic and muscular architecture. Changes in the immunophenotype and plasticity are presumably correlated with the specifi c in vivo vascular location and may also be a conse-quence of their embryologic origin (31). This sug-gests that MSC functional roles are at least partially organ and tissue specifi c.

The feasibility of isolating MSC from arterial samples collected from multiorgan donors in quali-fi ed tissue bank facilities and of storing them in good cell and tissue practice-certifi ed cell factories could prompt use of them in cardiovascular research and clinical applications. Indeed, VW MSC can be consid-ered a natural candidate for the realization of biosyn-thetic arterial substitutes and cell therapy, especially in the case of vascular diseases where conventional treatments are failing. Future studies establishing the in vivo regenerative and repair potential of VW MSC in animal models of vascular diseases will no doubt seek answers to this still unexplored fi eld.

Acknowledgement

This work was supported by Fondazione Fanti Melloni and Fondazione del Monte (AS, GP) and Programma di Ricerca Regione Emilia-Romagna-Università

assays. The results from von Kossa staining sug-gested a failed commitment; however, TEM revealed that ossifi cation corresponded to a stage preceding the formation of trabecular bone. After 3 weeks of induction, VW MSC did in fact synthesize an osteoid matrix enriched with multiple hydroxiapatite crystals. Presumably the exposure time was not suffi cient to promote VW MSC to complete ossifi cation because of their low innate propensity for osteogenesis.

Regarding leiomyogenesis, a preliminary evalu-ation was performed to establish an appropriate induction media; we fi nally used SmGM-2 supple-mented with PDGF-BB (5 ng/mL) and TGF-β1 (10 ng/mL), two well-noted leiomyogenic inducible factors (39–41). When stimulated with leiomyogenic media, VW MSC increased their levels of ASMA and h-CALD transcripts and proteins and showed de novo expression of CALP, all specifi c markers of human smooth muscle cells. These results were confi rmed by TEM, which demonstrated contractile fi laments in induced VW MSC. The concomitant presence of rough endoplasmic reticulum and well-organized fi bronexus allowed us to categorize the differentiat-ing cells as myofi broblasts. According to Eyden et al. (42), electron microscopy detection of cell-to-matrix junctions enables us to identify myofi broblasts and distinguish them from other ASMA-expressing cyto-types, including pericytes. This observation suggests that, in VW, myofi broblasts derive not only from smooth muscle cell-to-myofi broblast transition (43) but also from local resident mesenchymal cells, VW MSC being the possible source. This is consistent with the concept that vascular smooth muscle cells

Figure 8. Immunofl uorescent analysis of leiomyogenesis. Protein expression modulation of smooth muscle markers such as ASMA, h-CALD and CALP in induced and uninduced cells.


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di Bologna, Area 1-b ‘Cell Therapy of Heart Fail-ure’ (GP). We gratefully acknowledge Annamaria Ricciardi for her technical assistance and Ralph Nis-bet for his editing support.

Disclosures

None.

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Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics