Donor-dependent variances of human adipose-derived stem cells in respect to the in-vitro endothelial cell differentiation capability

Abstract

Human adipose-derived stem cells (ASC) have been shown to differentiate into mature adipocytes and to play an important role in creating the vasculature, necessary for white adipose tissue to function. To study the stimulatory capacity of ASC on endothelial progenitor cells we used a commercially available co-culture system (V2a - assay). ASC, isolated from lipoaspirates of 18 healthy patients, were co-cultured for 13 d on endothelial progenitor cells. Using anti CD31 immunostaining, cells that had undergone endothelial differentiation were quantified after the defined co-cultivation period. Endothelial cell differentiation was observed and demonstrated by an increase in area covered by CD31+ cells compared with less to no endothelial cell differentiation in negative and media-only controls. Enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) in supernatant medium collected during the co-cultivation period revealed elevated VEGF levels in the co-culture samples as compared with ASC cultures alone, whereas no increase in adiponectin was detected by ELISA. These findings help to provide further insights in the complex interplay of adipose derived cells and endothelial cells and to better understand the diversity of ASCs in respect of their stimulatory capacity to promote angiogenesis in vitro.

Keywords: adipose stem cell; angiogenesis; cell-cell interaction; endothelial cell; vasculogenesis.

Figure 1.

Representative light microscopy pictures of adipogenically and osteogenically differentiated ASCs (10xmagnification) a) intracellular oil red stained lipid droplets at day 7 post induction of differentiation. b) Extracellular calcium deposits stained with alizarin red as marker for osteogenic differentiation on day 24 of differentiation c) Undifferentiated cells.

Figure 2.

Phenotypic analysis of ASCs by immunfluorescence staining of markers established for mesenchymal stem cells and adipose derived stem cells (CD44, CD90, CD73 and CD 29). Confirmation of lineage specificity by antibody staining against CD31, CD14 and MHC-class II.

Figure 3.

a) Percentage of Cell surface expression of 4 mesenchymal stem cell markers in addition to lineage specific markers on ASCs (n = 5) cultivated in ECGM-Media at P1 until confluence and evaluated by flow cytometry. b) Mean values and standard deviation of 5 representative experiments.

Figure 4.

Boxplot showing areas covered by samples, VEGF positive control, and medium only and negative controls. The difference between samples and non-positive control was statistically significant (p < 0, 05).

Figure 5.

(see previous page). Different differentiation patterns of V2a-cells ASC co-cultures. Left: View of entire well after anti-CD31 immunostaining with CD31-positive cells stained dark blue/violet. Middle: Native picture (enlarged from left image, compare yellow rectangle) of the area used for quantitative analysis. Right: 8-bit image created by ImageJ in final quantitative analysis step; the dark area was measured as a percentage of total image area. A, B, and C were co-culture wells with ASC from 3 different patients. D was a representative positive control well with VEGF added to medium. E was medium-only control. F was a negative-control well with Suramin added to medium. Differences in the differentiation patterns both between samples A-C and between samples and controls are obvious. Furthermore, the similar appearance of medium-only (E) and negative control (F) is clear. Percentages of area covered by CD31+ cells: A 43.2%, B 12.7%, C 34.0%, D 23.2%, E 6.4%, F 5,7%.

Figure 6.

VEGF levels of 5 representative samples in ng/ml over the course of 12 day cocultivation period.

Figure 7.

Mean VEGF levels during the co-cultivation period blotted against percentage of well surface covered by CD31+ cells. No correlation could be found.