Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells

Abstract

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α-actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10(+), Sox17(+)) and a glia marker (S100β(+)). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.

Fig. 1

a–r Representative immunocytochemical staining of smooth muscle cell (SMC) differentiation markers. Rat (rSMC), murine (mSMC) and bovine (BASMC) cells were cultured in 0.5 % (a–c, g–i, m–o) and 5 % (d–f, j–l, p–r) fetal bovine serum (FBS)-supplemented medium for 3 days before immunocytochemistry was performed. Cells stained positively (red) for smooth muscle myosin heavy chain (SM-MHC), calponin1 (CNN1) and smooth muscle α-actin (SMA). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale Bars are 50 μm (a–f, m–r) and 10 μm (g–l). Data are representative of three independent wells. s, t Representative immunoblot analysis of SMC differentiation markers, i.e. smooth muscle myosin heavy chain (SM-MHC in s) and calponin1 (CNN1 in t). Equal loading was confirmed by Ponceau S staining. Data are representative of two independent experiments

Fig. 2

a–u Representative immunocytochemical staining of SMC differentiation markers, neural stem cell markers and mesenchymal stem cell (MSC)-like markers (green). Rat aortic multipotent vascular stem cells (MVSCs) were isolated by explant and cultured in MVSC maintenance media before being analysed by immunofluorescence microscopy for SMC markers (a–c SM-MHC), neural stem cell markers (d–f Sox10, g–i Sox17, j–l S100β) and MSC (Mesenchymal) markers (m–o CD44, p–r CD29, s–u CD146). Nuclei were stained with DAPI (blue). Scale Bars are 50 μm. v Representative immunoblot analysis of SMC differentiation markers (SMA, CNN1, SM-MHC) and neural stem cell markers (Sox10, Sox17 and S100β) in MVSCs. Equal loading was confirmed by Ponceau S staining. Data are representative of two independent experiments

Fig. 3

a–i Representative immunocytochemical staining (green) of SMC differentiation markers and neural stem cell markers during MVSC transition to vascular smooth muscle cells (vSMCs). MVSCs were cultured in maintenance media or differentiation media (DMEM supplemented with 10 % FBS) for 10 days before being examined for SMC differentiation markers (a, b SMA, d, e SM-MHC, g, h CNN1). Confocal immunofluorescence images for SM-MHC (c), SMA (f) and CNN1 (i). Scale Bars are 20 μm and 50 μm. j–l Representative flow cytometry analysis of Sox10 (j), Sox17 (k) and S100β (l) in MVSCs cultured in DMEM supplemented with 10 % FBS for 10 days with antibodies against Sox10, Sox17 and S100β (open curves negative IgG control cells, red filled curves cells stained with antibodies against Sox10, Sox17 and S100β). m–o Representative confocal immunofluorescence images of Sox10 (m), Sox17 (n) and S100β (o). Nuclei were stained with DAPI (blue). Bar 100 nm. Data are representative of three individual slides

Fig. 4

a–p Representative immunocytochemical staining (green) of SMC differentiation markers and neural stem cell markers in rSMCs and BASMCs. Rat rSMCs and BASMCs were grown in normal media supplemented with 5 % FBS (a–d, i–l) or 0.5 % FBS (e–h, m–p) for 3 days before being analysed for Sox10 and SM-MHC (arrows localisation of Sox10 primarily to the nucleus). Nuclei were stained with DAPI (blue). Scale Bars are 25 μm. Data are representative of at least three individual slides. q–u Representative immunoblot analysis (q) and immunocytochemical staining (r–u) of Sox10 and SM-MHC in fresh isolates from murine carotid artery vessels and primary rat vascular aortic smooth muscle cells (rSMCs) in culture, generated by either enzymatic dispersal or explant Magnification x 100. Human carotid artery vascular smooth muscle cells served as an SMC control. Equal loading was confirmed by Ponceau S staining of membrane and by D-glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) levels. Data are representative of three independent experiments

Fig. 5

a–h Representative immunocytochemical staining of expression of Sox10 and SM-MHC in mSMCs. Murine SMCs were grown in normal media containing 0.5 % FBS (a–d) or 5 % FBS (e–h) for 3 days before being analysed for Sox10 and SM-MHC (arrows localisation of Sox10 primarily to the nucleus). Nuclei were stained with DAPI (blue). Scale bars are 25 μm. i, j Representative flow cytometry analysis of mSMCs cultured for 3 days in normal media before analysis with specific antibodies against Sox10 and SM-MHC, respectively (open curves negative IgG control cells, blue curves cells stained with specific antibodies). Data are representative of three individual experiments. k, l Representative immunoblot analysis of neural stem cell markers Sox17 and Sox10, respectively, in SMCs. Equal loading was confirmed by Ponceau S staining. Data are representative of two independent experiments

Fig. 6

Quantitative real-time polymerase chain reaction (qRT-PCR) of neural stem cell marker and SMC differentiation marker mRNA levels in a representative vSMC line. Murine SMCs were cultured in DMEM that was serum-rich (5 % FBS) or serum-deprived (0.5 % FBS) for 3 days before mRNA levels of SMC differentiation markers SMA, MHC, CNN1 and smoothelin (SMO), of neural stem cell markers Sox10, Sox17 and S100β and of the Notch target gene Hey1 were measured by qRT-PCR. GAPDH was used to normalize gene expression. The Ct values were obtained and analysed by using the Relative Expression Software Tool (ReST) to determine the relative levels of transcripts. Data are means± SEM and are representative of three independent wells. *P<0.05 when compared with 0.5 % FCS

Fig. 7

Adipogenic potential of vSMCs in vitro. The ability of MSCs, MVSCs, rSMCs and mSMCs to differentiate to adipocytes was determined following treatment with adipocyte differentiation media for 14 days. Adipocyte differentiation was determined by both Oil Red O and LipidTOX staining of lipid droplets (arrows cells positive for adipocyte characteristics). Data are representative of three independent wells Magnification x 40

Fig. 8

Osteogenic potential of SMCs in vitro. The ability of MSCs, MVSCs and rSMCs to differentiate to osteoblasts was determined following treatment with osteogenic differentiation media for 21 days. Osteoblast differentiation was determined by measuring calcium deposition with Alizarin Red (arrows cells positive for osteoblast characteristics). Scale bars are 25 μm. Data are representative of three independent wells

Fig. 9

Effects of transforming growth factor-β1 (TGF-β1) and Notch inhibition by γ-secretase inhibitor (DAPT) on Sox10 and SM-MHC levels in a representative vSMC line, namely mSMCs. The expression of Sox10 and SM-MHC was determined by immunocytochemistry. Cells were grown in media containing 10 % FBS supplemented with (a–j TGF-β1 (1 ng/ml) or (k–t) 10 xM DAPT for 3 days. Scale bars are 25 μm. Ratio of Sox10:SM-MHC (sm-2)-positive cells in untreated and in (u) TGF-β1-treated and (v) DAPT-treated cells. Data are representative of three independent wells