Fibroblasts and Mesenchymal Stromal/Stem Cells Are Phenotypically Indistinguishable

Abstract

Background/aims: Human mesenchymal stromal/stem cells (MSCs), derived from many different tissues, are characterized by a fibroblast-like morphology, the expression of certain cell surface markers and their ability to differentiate into adipocytes, chondrocytes and osteoblasts. A number of studies have shown that MSCs share many characteristics with fibroblasts; however, there is no well-defined set of phenotypic characteristics that could distinguish between these 2 types of cells.

Methods: We used 4 well-established human fibroblast strains from 3 different tissue sources and several human MSC strains from 2 different tissue sources to compare the phenotypic and immunological characteristics of these cells.

Results: Fibroblast strains had a similar morphology to MSCs, expressed the same cell surface markers as MSCs and could also differentiate into adipocytes, chondrocytes and osteoblasts. Also, similar to MSCs, these fibroblasts were capable of suppressing T cell proliferation and modulating the immunophenotype of macrophages. We also show that MSCs deposit extracellular matrices of collagen type I and fibronectin, and express FSP1 in patterns similar to fibroblasts.

Conclusions: Based on currently accepted definitions for cultured human MSCs and fibroblasts, we could not find any immunophenotypic property that could make a characteristic distinction between MSCs and fibroblasts.

Figure 1. Fibroblasts and MSCs display similar morphologies

Representative micrographs at 50x magnification of the different fibroblast strains compared to BM and Ad MSCs. Scale bar = 200μm.

Figure 2. Fibroblasts express cell surface markers that define MSCs

The graph shows the percent of cells expressing the indicated cell surface markers. Bars represent means ±SE for three independent experiments. Representative flow cytometry histograms are shown in Supplemental Figure 1.

Figure 3. Fibroblasts can differentiate into adipocytes, chondrocytes, and osteoblasts

Fibroblasts were grown in adipogenic, chondrogenic, and osteogenic medias for 21, 24, and 21 days and stained with oil red O, Alcian blue, and Alizarin red S, respectively. BM and Ad MSCs were differentiated the same way and used as positive controls. The micrographs are representative images at 200x magnification for adipocytes, 100x magnification for chondrocytes, and 100x magnification for osteoblasts. Negative controls are BM MSCs cultured for the same lengths of time with normal MSC growth media, fixed and stained the same way.

Figure 4. Fibroblasts express HLA-DR when stimulated with IFNγ

Fibroblasts were plated in a 6-well plate and stimulated with 100 IU/mL IFNγ for 4 days and subsequently probed for HLA-ABC, HLA-DR, CD80, and CD86. BM and Ad MSCs were used as positive controls. Bars represent means ±SE from 3 independent experiments.

Figure 5. Fibroblasts suppress CD4+ T cell proliferation

PBMCs from a healthy donor were stained with CFSE. Labled PBMCs were plated with four fibroblast strains (FB) or our standard BM MSCs (positive control) at different PBMC:MSC/FB ratios. After a 4-day co-culture, cells were collected, stained with anti-CD4, and analyzed by flow cytometry. A non-stimulated (ns) co-culture at 1-0.05 PBMC:MSC/FB ratio served as a negative control as well as to measure the level of stimulation induced by the strain itself. The value of p-m/f indicates the ratio of PBMCs to MSCs/fibroblasts. Bars represent means ±SE.

Figure 6. Immunophenotype of Fibroblast-educated macrophages

CD14+ cells were isolated from peripheral blood of 3 different donors and plated in 6-well plates (1x10⁶/well) for 7 days to allow for differentiation into macrophages. Then fibroblasts or MSCs (2x10⁵/well) were added and co-cultured for 3 days. Macrophages were harvested by scraping the plates. Cells were stained with anti-CD14 and anti-CD206, and CD206 expression was analyzed in CD14-positive cells. Representative histograms are shown. The bar graph represents the average CD206 mean channel fluorescence in units relative to the control (no MSCs or fibroblasts added to CD14+ cells) ±SE from 3 independent experiments. (B) Q-PCR was used to assess gene expression of IL-10 and IL-12 normalized to 3 housekeeping genes (18S rRNA, GAPDH, and beta-actin). Relative mRNA levels (2^-ΔCt) are plotted. Samples were analyzed in triplicate, and bars represent means ±SE. *P<0.05 compared to control.

Figure 7. MSCs express fibronectin and collagen matrices that are indistinguishable from that of fibroblasts

MSCs and fibroblasts were grown on coverslips in 24-well plates to near confluency. For collagen formation, 0.2mM ascorbic acid was added to the media. Samples were probed for fibronectin (A), collagen I (B), or non-specific rabbit serum (C) and analyzed with fluorescent microscopy using the 10x (A) or 20x (B, C) objectives. DNA was counterstained with DAPI (blue). Images are representative micrographs.