Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods

Abstract

Even though umbilical cord arteries are a common source of vascular smooth muscle cells, the lack of reliable marker profiles have not facilitated the isolation of human umbilical artery smooth muscle cells (HUASMC). For accurate characterization of HUASMC and cells in their environment, the expression of smooth muscle and mesenchymal markers was analyzed in umbilical cord tissue sections. The resulting marker profile was then used to evaluate the quality of HUASMC isolation and culture methods. HUASMC and perivascular-Wharton's jelly stromal cells (pv-WJSC) showed positive staining for α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as α-SMA+ , SM-MHC+ , CD10- cells, which are additionally negative for endothelial markers (CD31 and CD34). Enzymatic isolation provided primary HUASMC batches with 90-99 % purity, yet, under standard culture conditions, contaminant CD10+ cells rapidly constituted more than 80 % of the total cell population. Contamination was mainly due to the poor adhesion of HUASMC to cell culture plates, regardless of the different protein coatings (fibronectin, collagen I or gelatin). HUASMC showed strong attachment and long-term viability only in 3D matrices. The explant isolation method achieved cultures with only 13-40 % purity with considerable contamination by CD10+ cells. CD10+ cells showed spindle-like morphology and up-regulated expression of α-SMA and SM-MHC upon culture in smooth muscle differentiation medium. Considering the high contamination risk of HUASMC cultures by CD10+ neighboring cells and their phenotypic similarities, precise characterization is mandatory to avoid misleading results.

Keywords: CD10; Characterization; Human umbilical artery smooth muscle cells (HUASMC); Isolation; Markers; Wharton’s jelly stromal cells.

Fig. 1

Immunohistochemical staining of umbilical cord tissue. IgG control staining was documented as overview (a) showing an umbilical artery embedded in Wharton’s jelly and (b) in detail at larger magnification. The dotted line marks the border between the Wharton’s jelly and the vascular region. The endothelial marker CD34 is exclusively expressed by endothelial cells (c) CD10 antibody stains stromal cells outside the umbilical blood vessel (d) CD90 (e) vimentin (f) and α-SMA (g) are additionally localized in vascular regions, but only vimentin is expressed in endothelial cells. Desmin is preferentially expressed in the muscular and external layer of the umbilical cord vessels, as well as in stromal cells of the perivascular Wharton’s jelly (asterisks, h), and the sub-amniotic region. SM-MHC is highly restricted to umbilical vessels. Except in endothelial cells, it is found in all layers of the umbilical vessels and in the cells of their immediate surrounding (asterisks, i). Scale bar 200 µm; UCV, umbilical cord vessel; WJ, Wharton’s jelly

Fig. 2

Flow cytometry characterization of the cells obtained by enzymatic digestion derived from 11 umbilical cords shows that on average 95 % cells are CD10−/CD31−, 4 % are CD10+ , 1 % are CD31+ (a, n = 11). In detail, analysis of two representative cell batches with flow cytometry (b) and western blotting (c) showing that CD10−/CD31− cells are SM-MHC and α-SMA positive

Fig. 3

Calcein AM staining of HUASMC embedded in Matrigel. In the first 2 weeks (a) cells remained mostly rounded and form minor aggregates. After 1 month of culture (b) cell clusters were larger and sprouted to connect with other clusters or other cells. Scale bar 150 µm

Fig. 4

Staining for α-SMA (a, c) and SM-MHC (b, d) was negative in cells in P2 from the explant method (95 % CD10+) cultured in SMC growth medium (a, b) and positive after 6 days in Medium 231 supplemented with SMDS (c, d). Scale bar 50 µm