Isolation, Characterization, Differentiation and Immunomodulatory Capacity of Mesenchymal Stromal/Stem Cells from Human Perirenal Adipose Tissue

Abstract

Mesenchymal stromal/stem cells (MSCs) are immature multipotent cells, which represent a rare population in the perivascular niche within nearly all tissues. The most abundant source to isolate MSCs is adipose tissue. Currently, perirenal adipose tissue is rarely described as the source of MSCs. MSCs were isolated from perirenal adipose tissue (prASCs) from patients undergoing tumor nephrectomies, cultured and characterized by flow cytometry and their differentiation potential into adipocytes, chondrocytes, osteoblasts and epithelial cells. Furthermore, prASCs were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or a mixture of cytokines (cytomix). In addition, prASC susceptibility to human cytomegalovirus (HCMV) was investigated. The expression of inflammatory readouts was estimated by qPCR and immunoassay. HCMV infection was analyzed by qPCR and immunostaining. Characterization of cultured prASCs shows the cells meet the criteria of MSCs and prASCs can undergo trilineage differentiation. Cultured prASCs can be induced to differentiate into epithelial cells, shown by cytokeratin 18 expression. Stimulation of prASCs with LPS or cytomix suggests the cells are capable of initiating an inflammation-like response upon stimulation with LPS or cytokines, whereas, LTA did not induce a significant effect on the readouts (ICAM-1, IL-6, TNFα, MCP-1 mRNA and IL-6 protein). HCMV broadly infects prASCs, showing a viral load dependent cytopathological effect (CPE). Our current study summarizes the isolation and culture of prASCs, clearly characterizes the cells, and demonstrates their immunomodulatory potential and high permissiveness for HCMV.

Keywords: adipose tissue; characterization; cytokines; cytomegalovirus; fat; lipopolysaccharide; mesenchymal stromal/stem cells; perirenal; stimulation.

Figure 1

Characterization of human perirenal mesenchymal stromal/stem cells (prASCs) in vitro. (A) Characteristic phase contrast microscopy of prASCs in passage 2 (bar: 100 µm); (B) Immunofluorescence staining of intermediate filament vimentin, nuclei were counterstained with DAPI (bar: 20 µm); (C) Representative flow cytometric overlay histograms of characteristic marker expression (CD73, CD90, CD105, CD29) and of CD45, a pan leukocyte marker which is not expressed on MSCs. Thick black histograms represent isotype controls. A dot plot shows the forward and sideward scatter analysis with the gating strategy to eliminate debris.

Figure 2

Trilineage differentiation of cultured prASCs. Differentiation into adipocytes, chondrocytes and osteoblasts was induced by adipogenic (B), chondrogenic (D) and osteogenic (F) medium for 14 days. Control cells were cultured in standard culture medium for 14 days (A,C,E) After 14 days of incubation in either standard or differentiation medium, cultures were stained with Oil Red O (A,B), Alcian Blue 8GX (C,D) or Alizarin Red S (E,F) (bar: 100 µm).

Figure 3

Induction of epithelial differentiation. (A) Analysis of cytokeratin 18 (CK-18) induction by qPCR. The expression levels in each experiment were normalized using β-actin as a housekeeping gene and are expressed relative to the unstimulated control using the ∆∆CT method (n = 6; * p < 0.05); (B) Characteristic immunofluorescence staining of CK-18 after epithelial differentiation with ATRA (14 days). Nuclei were stained with DAPI (bar: 100 µm); (C) Characteristic Western blots. Expression of CK-18 (45 kDa) and β-actin (42 kDa) of the unstimulated control cells (Co) and after incubation with ATRA (5 µM) for 14 days; (D) Densitometric evaluation of CK-18 induction in relation to undifferentiated controls (= 100%) (n = 5; * p < 0.05).

Figure 4

Effect of lipopolysaccharide (LPS) (10, 100, 1000 ng/mL), LTA (1000 ng/mL) or a cytokine mix (cytomix). (A–D) Assessment at the mRNA level by qPCR for ICAM-1, MCP-1, IL-6, and TNFα after 4 h stimulation. The expression levels in each experiment were normalized to a housekeeping gene (β-actin) and are expressed relative to the control using the ∆∆CT method. (mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 versus control), n = 4.; (E) Constitutive expression of TLR-2- and TLR-4-mRNA of prASCs in vitro. The PCR products were separated by agarose electrophoresis and observed under UV illumination (TLR-2: 96 kb, TLR-4: 211 kb, and β-actin: 169 kb (housekeeper)); (F) Quantification of IL-6 protein in the supernatant after 48 h stimulation. Data from four independent immunoassays (ELISA) are represented as mean ± SD (*** p < 0.0001).

Figure 5

Cell viability after stimulation with LPS, LTA or cytomix for 96 h. A total of 5000 cells were cultured in 96 well plates (n = 3, each in quintuplicate) and stimulated for 96 h. (A) The XTT assay was performed and optical density (OD) was measured in a microplate reader at 492 vs. 650 nm (arbitrary units). (B) The calcein assay was performed and fluorescence was measured in a reader at 485 nm (excitation) and 515 nm (emission). No significant effects of the different stimulations could be detected with both assays (mean ± SD, n = 3, each in quintuplicate).

Figure 6

Infection of prASCs with HCMV Hi 91 for 96h. (A, B) Bright field microscopy of mock-infected prASCs (A) and HCMV-infected cells (B). Compared to the mock-infected controls (A) a strong CPE with very few adherent cells is seen 96 h after infection with HCMV at a MOI of 4 (B). All remaining cells are positive for HCMV late antigen, as shown by immunostaining. (C) Levels of UL83 mRNA were assessed after 96h. The expression levels in each experiment were normalized to β-actin and are calculated relative to the control using the ∆∆CT method (mean ± SD; * p < 0.05 versus control, n = 3).