Characteristics of human adipose derived stem cells in scleroderma in comparison to sex and age matched normal controls: implications for regenerative medicine

Abstract

Background: Adipose-derived stem cells (ADSCs) are emerging as an alternative stem cell source for cell-based therapies. Recent data suggest that autologous ADSC-enriched micrografting improves the effects of facial involvement in systemic sclerosis (SSc). We have extensively characterised ADSCs from SSc patients and compared their phenotype and function to healthy age- and sex-matched control ADSCs.

Methods: ADSCs were isolated and characterised from a cohort of six SSc patients (ADSC-SSc) and were compared to six healthy age- and sex-matched controls (ADSC-N). Cell surface phenotype lineage commitment was explored by flow cytometric analysis of mesenchymal and hematopoietic markers and by the capacity to differentiate to chondrogenic, osteogenic, and adipogenic lineages. Functional activities of ADSCs were assessed by biochemical and cellular assays for proliferation, metabolism, adhesion, morphology, migration, and invasion.

Results: Upon characterization of ADSC-SSc, we found that there was no alteration in the phenotype or surface antigen expression compared to healthy matched control ADSCs. We found that the differentiation capacity of ADSC-SSc was equivalent to that of ADSC-N, and that ADSC-SSc did not display any morphological or adhesive abnormalities. We found that the proliferation rate and metabolic activity of ADSC-SSc was reduced (p < 0.01). We found that the migration and invasion capacity of ADSC-SSc was reduced (p < 0.01) compared to healthy matched control ADSCs.

Conclusions: This study provides important findings that can differentially characterise ADSCs from SSc patients. Results indicate that the surface phenotype and differentiation capacity of ADSCs from SSc patients are identical to healthy matched ADSCs. While the findings indicate that the proliferation and migration capacity of ADSC-SSc is reduced, ADSC-SSc are capable of ex-vivo culture and expansion. These findings encourage further investigation into the understanding by which ADSCs can impact upon tissue fibrosis.

Keywords: Adipose-derived stem cells; Invasion; Metabolism; Migration; Proliferation; Regenerative medicine; Scleroderma; Systemic sclerosis.

Fig. 1

Flow cytometric analysis of adipose-derived stem cells from systemic sclerosis patients (ADSC-SSc) and from a healthy age- and sex-matched control cohort (ADSC-N). ADSCs from SSc patients are phenotypically identical to healthy control ADSCs. a Example of the flow cytometric dot plot, histogram analysis, and the gating used to obtain percent expression on the surface of ADSC-SSc and ADSC-N at passage 2 (P2). b Summary table displaying the average percentage of antigens for each FACS marker expressed on the surface of the cells. FACS analysis was performed on ADSC-SSc and ADSC-N at P2. FACS analysis was performed for mesenchymal markers CD105, CD90, CD73, and hematopoietic markers CD14, CD45, CD34, CD19, and HLA-DR. SD standard deviation

Fig. 2

Differentiation capacity of ADSCs from SSc patients. ADSCs from SSc patients have comparable differentiation capacity to healthy control ADSCs. a Adipogenic differentiation of adipose-derived stem cells from SSc patients (ADSC-SSc; left image) and ADSCs from healthy controls (ADSC-N; right image) was assessed by Oil Red O staining. Positive Oil Red O staining and fat globules were observed in both ADSC-SSc and ADSC-N at 3 weeks. Quantitative analysis of Oil Red O staining at optical density of 495 nm (graph on right) determined that there was no difference in the adipogenic differentiation capacity of ADSC-SSc compared to ADSC-N (10× magnification). b Chondrogenic differentiation of ADSC-SSc (left image) and ADSC-N (right image) was assessed by Alcian Blue dye. Positive Alcian Blue staining for proteoglycans was observed in both ADSC-SSc and ADSC-N at 3 weeks. Quantitative analysis of Alcian blue staining at optical density of 595 nm (graph on right) determined that there was no difference in the chondrogenic differentiation capacity of ADSC-SSc compared to ADSC-N (10× magnification). c Osteogenic differentiation of ADSC-SSc (left image) and ADSC-N (right image) was assessed by Alizarin Red dye. Positive Alizarin red staining for calcium was observed in both ADSC-SSc and ADSC-N at 3 weeks. Quantitative analysis of Alizarin staining at optical density of 405 nm (graph on right) determined that there was no difference in the osteogenic differentiation capacity of ADSC-SSc compared to ADSC-N (n = 3) (10× magnification)

Fig. 3

Quantitative RT-PCR analysis of osteogenic-, chondrogenic-, and adipogenic-specific genes expressed in ADSCs from SSc patients. Adipose-derived stem cells from systemic sclerosis patients (ADSC-SSc) have a similar gene expression profile following differentiation when compared to healthy control ADSCs (ADSC-N). a Fold-change in expression of the osteogenic genes collagen type 1 (ColI), alkaline phosphatase (ALP), and osteocalcin (OC) in ADSC-SSc and ADSC-N. b Fold-change in gene expression of the chondrogenic genes aggrecan (Agg) and collagen type II (ColII) in ADSC-SSc and ADSC-N. c Fold-change in gene expression of the adipogenic genes fatty acid binding protein 4 (FABP4), peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBBPα), and lipoprotein lipase (LPL) in ADSC-SSc and ADSC-N. Gene expression profiling was carried out at 3 weeks following differentiation. Fold change was normalised to GAPDH (n=3)

Fig. 4

Analysis of the morphology and adhesion properties of ADSCs from SSc patients. There was no alteration in the adhesive properties or the morphology of adipose-derived stem cells from systemic sclerosis patients (ADSC-SSc) compared to healthy control ADSCs (ADSC-N). a DNA quantification assay was carried out to determine the number of cells adhering to tissue culture plastic 24 h after seeding. Adhesion of ADSCs was assessed at passage 1 (P1) to P4. There was no statistical difference in the number of cells that adhered between ADSC-SSc and ADSC-N (n = 6). b Representative bright field images of ADSC-N at passage 2 (top left panel) and SSc-ADSC s at passage 2 (top right panel) (10× magnification). Representative images of Rhodamine Phalloidin staining of F-actin in ADSC-N at passage 2 (bottom left panel) and ADSC-SSc at passage 2 (bottom right panel) (40× magnification). Cells were counter-stained with DAPI to visualize DNA. Scale bars = 50 μm. c Cell area (left graph) and circularity index (right graph) of ADSC-SSc and ADSC-N at P1 to P4 was assessed 24 h after seeding using image J software (n = 20). Statistical significance was calculated using unpaired t test

Fig. 5

Analysis of the proliferative and metabolic properties of ADSCs derived from SSc patients. Proliferation and metabolism of adipose-derived stem cells from systemic sclerosis patients (ADSC-SSc) was decreased over 14 days compared to healthy control ADSCs (ADSC-N). a Cell metabolism was assessed by the Alamar blue assay on days 1, 3, 7, and 14. Cells were assessed at passage 1 (P1) to P4. ADSC-SSc displayed significantly less metabolic activity on days 3, 7, and 14 at all passages assessed (p < 0.01) (n = 6). b Cell proliferation was assessed by DNA quantification on days 1, 3, 7, and 14. Cells were assessed at P1 to P4. ADSC-SSc were significantly less proliferative on days 3, 7, and 14 at all passages assessed (p < 0.01) (n = 6). **p < 0.01

Fig. 6

Analysis of the migration and invasion potential of ADSCs derived from SSc patients. Migration and invasion potential of adipose-derived stem cells from systemic sclerosis patients (ADSC-SSc) was reduced compared to healthy control ADSCs (ADSC-N) at all passages examined. a Cell migration was assessed by QCM 24-Well Colorometric Cell migration Assay. Cells were assessed at passage 1 (P1) to P4. Migration was assessed at 24 h. Migration of ADSC-SSc was significantly reduced at P1, P2, and P4 compared to healthy control ADSCs (p < 0.01) (n = 6). b Cell invasion was assessed by QCM ECMatrix Cell Invasion Assay. Cells were assessed at P1 to P4. Invasion was assessed at 24 h. Invasion of ADSC-SSc was significantly reduced at P1, P2, and P4 compared to healthy control ADSCs (p < 0.015) (n = 6). *p <0.05, **p < 0.01