Pericyte-like differentiation of human adipose-derived mesenchymal stem cells: An in vitro study

Abstract

BACKGROUND Adipose-derived mesenchymal stem cells (ASCs) are characterized by long-term self-renewal and a high proliferation rate. Under adequate conditions, they may differentiate into cells belonging to mesodermal, endodermal or ectodermal lineages. Pericytes support endothelial cells and play an important role in stabilizing the vessel wall at the microcirculation level. The loss of pericytes, as occurs in diabetic retinopathy, results in a breakdown of the blood-retina barrier (BRB) and infiltration of inflammatory cells. In this context, the use of pericyte-like differentiated ASCs may represent a valuable therapeutic strategy for restoring BRB damage. AIM To test in vitro strategies to obtain pericyte-like differentiation of human ASCs (hASCs). METHODS Different culture conditions were tested: hASCs cultured in a basal medium supplemented with transforming growth factor β1; and hASCs cultured in a specific pericyte medium (PM-hASCs). In a further sample, pericyte growth supplement was omitted from the PM. In addition, cultures of human retinal pericytes (hRPCs) were used for comparison. Pericyte-like differentiation of hASCs was tested by immunocytochemical staining and western blotting to evaluate the expression of α-smooth muscle actin (α-SMA) and neural/glial antigen 2 (NG2). Interactions between human retinal endothelial cells (hRECs) and different groups of hASCs were investigated in co-culture experiments. In these cases, the expression of typical junctional proteins such as vascular endothelial-Cadherin, zonula occludens-1 and Occludin were assessed in hRECs. In an in vitro model of the BRB, values of trans-endothelial electrical resistance were measured when hRECs were co-cultured with various groups of pretreated hASCs. The values observed were compared with co-cultures of hRECs and hRPCs as well as with cultures of hRECs alone. Three-dimensional co-cultures of hRECs and hRPCs or pericyte-like hASCs in Matrigel were designed to assess their reciprocal localization. RESULTS After 3-6 d of culture, α-SMA and NG2 immunocytochemistry showed that the closest pericyte-like phenotype was observed when hASCs were cultured in Pericyte Medium (PM-hASCs). In particular, α-SMA immunoreactivity, already visible at the basal level in pericytes and ASCs, was strongly increased only when transforming growth factor was added to the culture medium. NG2 expression, almost undetectable in most conditions, was substantially increased only in PM-hASCs. Immunocytochemical results were confirmed by western blot analysis. The presence of pericyte growth supplement seems to increase NG2 expression rather than α-SMA, in agreement with its role in maintaining pericytes in the proliferative state. In co-culture experiments, immunoreactivity of vascular endothelial-Cadherin, zonula occludens-1 and Occludin was considerably increased in hRECs when hRPCs or PM-hASCs were a...

Effects of High Glucose Concentration on Pericyte-Like Differentiated Human Adipose-Derived Mesenchymal Stem Cells

“…The MSC nature of ASCs used in the present study was verified in previous works, where cells of the same stock were investigated [ 25 ]. In particular, they were immuno-positive for typical MSC markers (CD44, CD73, CD90 and CD105), and immunonegative for hematopoietic stem cell markers (CD14, CD34 and CD45).…”

“…Indeed, beneficial effects were reported in rodent models of DR following intravitreal injections of native or TGF-β1 pretreated ASCs [ 14 ]. Furthermore, in a previous investigation we were able to induce a pericyte-like differentiation by growing ASCs in a culture medium specifically designed for pericytes [ 25 ]. In our opinion, this strategy offers the advantage of obtaining pericyte-like ASCs still in the proliferative stage, more suitable to restore a functional BRB.…”

“…By intravitreal injection of native or pretreated ASCs, retinal capillary dropout was prevented and the revascularization of the central retina was observed. In our previous in vitro investigation, a pericyte-like differentiation of ASCs was achieved by growing ASCs in a culture medium specifically designed for pericytes (PM) [ 25 ]. As a result, overexpression of smooth muscle actin α (α-SMA) and neural/glial antigen 2 (NG2) was obtained, as well as their typical peritubular localization.…”

Potential therapeutic applications of mesenchymal stem cells for the treatment of eye diseases

“…Main results are summarized in Table 1 . A successful pericyte-like differentiation of ASCs was achieved by using specific culture media, as demonstrated by the expression of typical pericyte markers [α-smooth muscle actin, nerve/glial-antigen 2, platelet-derived growth factor receptor (PDGFR)] as well as by their perivascular localization in three-dimensional cultures[ 19 , 20 ]. In an in vitro model of the BRB, pericyte-like ASCs were also able to reinforce endothelial junctions, as indicated by the increased expression of VE-Cadherin, zone occludens-1 and occludin.…”

“…In an in vitro model of the BRB, pericyte-like ASCs were also able to reinforce endothelial junctions, as indicated by the increased expression of VE-Cadherin, zone occludens-1 and occludin. In these experiments, also trans-endothelial electrical resistance values were increased[ 20 ]. It is also suggested that a high glucose (HG) concentration does not affect the pericyte-like functions of ASCs[ 21 , 22 ].…”