Iberian pig mesenchymal stem/stromal cells from dermal skin, abdominal and subcutaneous adipose tissues, and peripheral blood: in vitro characterization and migratory properties in inflammation

Abstract

Background: Recently, the capacity of mesenchymal stem/stromal cells (MSCs) to migrate into damaged tissues has been reported. For MSCs to be a promising tool for tissue engineering and cell and gene therapy, it is essential to know their migration ability according to their tissue of origin. However, little is known about the molecular mechanisms regulating porcine MSC chemotaxis. The aim of this study was to examine the migratory properties in an inflammatory environment of porcine MSC lines from different tissue origins: subcutaneous adipose tissue (SCA-MSCs), abdominal adipose tissue (AA-MSCs), dermal skin tissue (DS-MSCs) and peripheral blood (PB-MSCs).

Methods: SCA-MSCs, AA-MSCs, DS-MSCs and PB-MSCs were isolated and analyzed in terms of morphological features, alkaline phosphatase activity, expression of cell surface and intracellular markers of pluripotency, proliferation, in vitro chondrogenic, osteogenic and adipogenic differentiation capacities, as well as their ability to migrate in response to inflammatory cytokines.

Results: SCA-MSCs, AA-MSCs, DS-MSCs and PB-MSCs were isolated and showed plastic adhesion with a fibroblast-like morphology. All MSC lines were positive for CD44, CD105, CD90 and vimentin, characteristic markers of MSCs. The cytokeratin marker was also detected in DS-MSCs. No expression of MHCII or CD34 was detected in any of the four types of MSC. In terms of pluripotency features, all MSC lines expressed POU5F1 and showed alkaline phosphatase activity. SCA-MSCs had a higher growth rate compared to the rest of the cell lines, while the AA-MSC cell line had a longer population doubling time. All MSC lines cultured under adipogenic, chondrogenic and osteogenic conditions showed differentiation capacity to the previously mentioned mesodermal lineages. All MSC lines showed migration ability in an agarose drop assay. DS-MSCs migrated greater distances than the rest of the cell lines both in nonstimulated conditions and in the presence of the inflammatory cytokines TNF-α and IL-1β. SCA-MSCs and DS-MSCs increased their migration capacity in the presence of IL-1β as compared to PBS control.

Conclusions: This study describes the isolation and characterization of porcine cell lines from different tissue origin, with clear MSC properties. We show for the first time a comparative study of the migration capacity induced by inflammatory mediators of porcine MSCs of different tissue origin.

Keywords: Cell migration; Iberian pig; Inflammation; Mesenchymal stem/stromal cells.

Fig. 1

Morphology of MSCs at (a) passage 0 and 8 days of culture and (b) first passage and 13 days of culture. Phase-contrast images acquired with 100× magnification. Bars = 70 μm. (c) Representative P10 metaphase and karyotype. No chromosomal aberrations observed in AA-MSCs after long-term cultivation. AA-MSC abdominal adipose tissue mesenchymal stem/stromal cell, DS-MSC dermal skin tissue mesenchymal stem/stromal cell, PB-MSC peripheral blood mesenchymal stem/stromal cell, SCA-MSC subcutaneous adipose tissue mesenchymal stem/stromal cell

Fig. 2

Analysis by flow cytometry of expression levels of cell surface markers CD34, CD44, CD105, CD90 and MHCII and intracellular markers cytokeratin, vimentin and POU5F1 in AA-MSCs, DS-MSCs, SCA-MSCs and PB-MSCs. Data correspond to mean fluorescence intensity (fold of negative control) for each sample. AA-MSC abdominal adipose tissue mesenchymal stem/stromal cell, DS-MSC dermal skin tissue mesenchymal stem/stromal cell, MHCII major histocompatibility complex II, PB-MSC peripheral blood mesenchymal stem/stromal cell, POU5F1 POU class 5 homeobox 1, SCA-MSC subcutaneous adipose tissue mesenchymal stem/stromal cell

Fig. 3

Analysis of alkaline phosphatase (AP) activity. Bright-field images obtained at 100× (a) or 32× (b) magnification, showing some red-stained cell groups after action of alkaline phosphatase on Fast Red in presence of Napthol AS-mx phosphate. Bars = 70 μm (top panels) and 150 μm (bottom panels). AA-MSC abdominal adipose tissue mesenchymal stem/stromal cell, DS-MSC dermal skin tissue mesenchymal stem/stromal cell, PB-MSC peripheral blood mesenchymal stem/stromal cell, SCA-MSC subcutaneous adipose tissue mesenchymal stem/stromal cell

Fig. 4

In vitro proliferation of MSCs. (a) Absolute number of cells/dish (mean ± SD). (b) Doubling time of each MSC line (mean ± SD). Different lowercase letters indicate significant differences (p < 0.05). AA-MSC abdominal adipose tissue mesenchymal stem/stromal cell, DS-MSC dermal skin tissue mesenchymal stem/stromal cell, MSC mesenchymal stem/stromal cell, PB-MSC peripheral blood mesenchymal stem/stromal cell, SCA-MSC subcutaneous adipose tissue mesenchymal stem/stromal cell

Fig. 5

In vitro differentiation of MSCs to different lineages. Images show Oil red O staining of lipid droplets in cells cultured in basal medium (Control) or in adipogenic differentiation medium (top panel); Alcian blue staining of acidic proteoglycan in cells cultured in basal medium (Control) or in chondrogenic differentiation medium (middle panels); and Alizarin Red S staining of calcium deposits in cells cultured in basal medium (Control) or in osteogenic differentiation medium (bottom panels). Bright-field images acquired with 200× magnification (bars = 70 μm) for top panels and 3× magnification (bars = 150 μm) for middle and bottom panels. AA-MSC abdominal adipose tissue mesenchymal stem/stromal cell, DS-MSC dermal skin tissue mesenchymal stem/stromal cell, PB-MSC peripheral blood mesenchymal stem/stromal cell, SCA-MSC subcutaneous adipose tissue mesenchymal stem/stromal cell

Fig. 6

Representative images of AA-MSC migration assay into PBS, TNF-α or IL-1β-agarose spot after 48 h. Images obtained in a light stereomicroscope at 20× magnification. IL-1β interleukin-1β, PBS phosphate buffered saline, TNF-α tumor necrosis factor alpha

Fig. 7

Migration analysis in agarose spot assay. Distance migrated from border of agarose spot measured in two independent experiments for AA-MSCs, SCA-MSCs, DS-MSCs and PB-MSCs at 48 h (mean ± SD). Different lowercase letters indicate significant differences (p < 0.05 for MSC migration mediated by PBS (a, b, c) and TNF-α (f, g, h); p < 0.005 for MSC migration mediated by IL-1β (j, k, l)). *p < 0.05; **p < 0.005; ***p < 0.0005. AA abdominal adipose tissue, DS dermal skin tissue, IL interleukin, MSC mesenchymal stem/stromal cell, PB peripheral blood, PBS phosphate buffered saline, SCA subcutaneous adipose tissue, TNF tumor necrosis factor