Dermal fibroblasts display similar phenotypic and differentiation capacity to fat-derived mesenchymal stem cells, but differ in anti-inflammatory and angiogenic potential

Abstract

Background: Mesenchymal stem cells (MSCs) are multipotent stem cells able to differentiate into different cell lineages. However, MSCs represent a subpopulation of a more complex cell composition of stroma cells contained in mesenchymal tissue. Due to a lack of specific markers, it is difficult to distinguish MSCs from other more mature stromal cells such as fibroblasts, which, conversely, are abundant in mesenchymal tissue. In order to find more distinguishing features between MSCs and fibroblasts, we studied the phenotypic and functional features of human adipose-derived MSCs (AD-MSCs) side by side with normal human dermal fibroblasts (HNDFs) in vitro

Methods: AD-MSCs and HNDFs were cultured, expanded and phenotypically characterized by flow cytometry (FC). Immunofluorescence was used to investigate cell differentiation. ELISA assay was used to quantify angiogenic factors and chemokines release. Cultures of endothelial cells (ECs) and a monocyte cell line, U937, were used to test angiogenic and anti-inflammatory properties.

Results: Cultured AD-MSCs and HNDFs display similar morphological appearance, growth rate, and phenotypic profile. They both expressed typical mesenchymal markers-CD90, CD29, CD44, CD105 and to a minor extent, the adhesion molecules CD54, CD56, CD106 and CD166. They were negative for the stem cell markers CD34, CD146, CD133, CD117. Only aldehyde dehydrogenase (ALDH) was expressed. Neither AD-MSCs nor HNDFs differed in their multi-lineage differentiation capacity; they both differentiated into osteoblast, adipocyte, and also into cardiomyocyte-like cells. In contrast, AD-MSCs, but not HNDFs, displayed strong angiogenic and anti-inflammatory activity. AD-MSCs released significant amounts of VEGF, HGF and Angiopoietins and their conditioned medium (CM) stimulated ECs proliferation and tube formations. In addition, CM-derived AD-MSCs (AD-MSCs-CM) inhibited adhesion molecules expression on U937 and release of RANTES and MCP-1. Finally, after priming with TNFα, AD-MSCs enhanced their anti-inflammatory potential; while HNDFs acquired pro-inflammatory activity.

Conclusions: AD-MSCs cannot be distinguished from HNDFs in vitro by evaluating their phenotypic profile or differentiation potential, but only through the analysis of their anti-inflammatory and angiogenic properties. These results underline the importance of evaluating the angiogenic and anti-inflammatory features of MSCs preparation. Their priming with inflammatory cytokines prior to transplantation may improve their efficacy in cell-based therapies for tissue regeneration.

Figure 1

AD-MSCs and HNDFs display similar capacity to differentiate into adipogenic, osteogenic and cardiomyogenic cell lineages. The Figure shows the capacity of AD-MSCs and HNDFs to differentiate toward the adipogenic, osteogenic and cardiomyogenic cell lineages in the presence of lineage-specific induction factors. In (A) is shown the positive staining of AD-MSCs and HNDFs for Oil Red O and AP, indicating differentiation into adipogenic and osteogenic cell lineages respectively (magnification 20×). In (B) note the positive immunofluorescence staining of AD-MSCs and HNDFs for Desmin, MyoA and TNP-C cardiomyogenic markers (magnification 10×).

Figure 2

AD-MSCs, but not HNDFs, produce high levels of angiogenic factors. ELISA-tests were performed to detect VEGFa, TGF-β1, HGF, PDGF, Ang-1 and Ang-2 angiogenic factors released by AD-MSCs and HNDFs in the CM. Note that AD-MSCs, compared to HNDFs, release very high quantities of VEGFa, HGF and Ang-1. Data are the absolute values expressed as mean ± SD of the secreted factor per 10⁶ cells after 72 hrs of incubation. The background values of angiogenic factors contained in EGM control medium (supplemented with 10% FCS and 50 ng/ml bFGF) were subtracted. Tests were run in triplicate and repeated twice. ** p < 0.01 versus HNDFs release

Figure 3

AD-MSC-CM possess a higher capacity to stimulate HUVECs and HMECs proliferation and tube formations compare to HNDFs-CM. The effect of AD-MSCs-CM and HNDFs-CM on proliferation of HUVECs (A) and HMECs (B) was tested by adding different dilutions to EGM control medium. As a positive growth control, we used VEGFa 10 ng/ml. AD-MSCs-CM addition had a great capacity to stimulate the proliferation of HMECs (B) and to a minor extent HUVECs (A), while HNDFs-CM addition did not affect ECs proliferation at any dilution tested. In (C) and in (D) are shown the capacity of AD-MSCs-CM and HNDFs-CM to induce HMECs tube formation respectively. AD-MSCs-CM and HNDFs-CM were added at a ratio 1:1 with EGM control medium (Magnification 10×). Note that AD-MSCs-CM not only increased HMECs tube formation on day 2, but also delayed their regression on day 5. The columns in (A) and (B) are mean ± SD of three independent experiments run in triplicate. *p < 0.05 and **p < 0.01 versus CTRL.

Figure 4

AD-MSCs, but not HNDFs, reduced expression of AMs on U937. AD-MSCs-CM and HNDFs-CM were tested on the adhesion molecules expression of the U937 monocyte cell line stimulated or not with TNFα (25 ng/ml × 12 hrs). Mean Fluorescent Intensity (MFI) of each marker expression was evaluated by FACS and values are the mean ± SD of independent experiments performed with 3 different preparations of AD-MSCs-CM and HNDFs-CM. In (A) the expression of CD54/ICAM-1, (B) the expression of CD44, (C) the expression of CD42L/L-selectin and in (D) the expression of CD49d/VLA-4. Note that, both AD-MSCs-CM and HNDFs-CM did not affect basal adhesion molecules expression on U937. However, AD-MSCs-CM, but not HNDFs-CM (at 1:1), inhibited CD54/ICAM-1, CD44 and CD42L/L-selectin up-modulation produced by TNFα stimuli. CD49d expression was not affected by either AD-MSCs-CM or HNDFs-CM. * p < 0.05 and **p < 0.01 versus CTRL.

Figure 5

Addition of AD-MSCs-CM, but not HNDFs-CM, to U937 blocked production of RANTES and MCP-by U937. ELISA-kits were used to quantify the production of RANTES and MCP-1 by U937 under basal culture conditions, in the presence of inflammatory stimuli LPS(1 μg/ml) (A and B) and TNFα (25 ng/ml) (C and D) and in the presence of AD-MSCs-CM and HNDFs-CM. In (A) RANTES and in (B) MCP-1 production are blocked in a dose dependent manner by the addition of AD-MSCs-CM but not by HNDFs-CM under basal or in the presence of LPS. Similarly, in (C) RANTES and in (D) MCP-1 release is inhibited by the addition of AD-MSCs-CM to U937 stimulated or not with TNFα for 12 hrs. The columns in the figure are mean ± SD of three independent experiments run in triplicate. *p < 0.05 and **p < 0.01 versus CTRL.

Figure 6

Priming of AD-MSCs and HNDFs with TNFα induce an opposite effect on RANTES and MCP-1 release by U937. AD-MSCs and HNDFs were primed for 12 hrs with TNFα 25 ng/ml. Thereafter cells were washed and further incubated for 24 hrs. At the end of incubation, CMs were collected from primed AD-MSCs (P-AD-MSCs-CM) and primed HNDFs (P-HNDFs-CM) and tested on U937 chemokines production. Note that, while the addition of P-AD-MSCs-CM to U937 completely blocked RANTES and MCP-1 release, in an opposite manner, P-HNDFs-CM improved their release. The columns in the figure are mean ± SD of two independent experiments run in triplicate. ° p < 0.05 and °° p < 0.01 versus HNDF-CM, *p < 0.05 and **p < 0.01 versus AD-MSCs-CM, + p < 0.05 and ++ p < 0.01 versus CTRL.