Characterisation of Mesenchymal Stromal Cells (MSCs) from Human Adult Thymus as a Potential Cell Source for Regenerative Medicine

Abstract

Background: Mesenchymal stem cell-based therapy may be indicated in ischaemic heart disease. The use of autologous adipose-derived mesenchymal stromal cells (AdMSCs) offers regenerative potential due to their paracrine effects. The aim of this study was to expand and characterise adult human thymus-derived MSCs harvested during open heart surgery with respect to their stem cell and paracrine properties. Methods: Enzymatically and non-enzymatically isolated human thymic AdMSCs (ThyAdMSCs) were cultured in xeno-free media containing pooled human platelet lysate (pPL). MSC characterisation was performed. Ex vivo expanded ThyAdMSCs were differentiated into three lineages. Proliferative capacity and immunomodulatory properties were assessed by proliferation assays and mixed lymphocyte reaction, respectively. Gene expression analysis was performed by qPCR. Results: Both isolation methods yielded fibroblast-like cells with plastic adherence and high proliferation. Flow cytometry revealed distinct expression of MSC markers in the absence of haematopoietic cell surface markers. Ex vivo expanded ThyAdMSCs could be differentiated into adipocytes, osteocytes, and chondrocytes. Activated peripheral blood mononuclear cells were significantly reduced when co-cultured with ThyAdMSCs, indicating their ability to inhibit immune cells in vitro. Gene expression analysis showed significantly less IFNγ and TNFα, indicating an alteration of the activated and pro-inflammatory state in the presence of ThyAdMSCs. Conclusions: These results demonstrate an efficient method to generate AdMSCs from human thymus. These MSCs have a strong immunomodulatory capacity and are, therefore, a promising cell source for regenerative medicine. The culture conditions are crucial for cells to proliferate in culture. Further research could explore the use of ThyAdMSCs or their secretome in surgical procedures.

Keywords: adipose tissue; adult thymus; cardiac surgery; cell therapy; mesenchymal stromal cell; platelet lysate.

Figure 1

Isolation of mesenchymal stromal cells from adult thymus adipose tissue. (A) After “cleansing”, the tissue was divided for two different isolation methods: two-thirds of the tissue was used for collagenase I enzyme digestion to obtain the stromal vascular fraction, which was seeded into culture plates. The other third of the tissue was cut into small pieces of ~3–5 mm² to allow the outgrowth of MSCs (explant culture). (B) Morphology of ThyAdMSCs after 4 and 7 days, respectively.

Figure 2

Flow cytometry of ThyAdMSCs cultured in αMEM or DMEM with 10% pPL and pen/strep. (A) Expression of positive stem cell markers CD90, CD73, and CD105 and negative markers Lin (=CD11b, CD19, CD31, CD34, CD45, CD144, and HLA-DR) in “outgrowth” ThyAdMSCs cultured in αMEM and “enzymatic” MSCs grown in DMEM (n = 10). (B) Surface expression of CD105 as a function of medium and culture time (n = 5). (C) Surface markers SSEA4 and CD271 as a function of isolation method and culture medium. The values are given as [%] of viable cells.

Figure 3

ThyAdMSCs proliferation assay using PrestoBlue^®. For cell proliferation analysis at different time points, media was discarded, and cells were incubated in PrestoBlue^TM Cell Viability Reagent (Invitrogen^TM; ThermoFisher) for 2 h according to the manufacturer’s instruction. Photometric measurements were performed at 560 and 620 nm. The values are given in [%] of reduced PrestoBlue (PB). PrestoBlue is a non-toxic, resazurin-based solution that exploits the ability of living cells to convert resazurin to the fluorescent molecule resorufin. The resorufin can be detected photometrically. It is used to measure the amount of PrestoBlue reagent metabolized during incubation and is an indicator of the amount of metabolically active cells present in the culture. The proliferation assays presented in Figure 3 are based on 20,000 cells/well seeded on day 0. The assay was performed in triplicate at each time point (n = 5; * = p < 0.05).

Figure 4

In vitro multilineage differentiation of ThyAdMSCs. Representative images of ThyAdMSCs differentiating into adipocytes (Oil Red O staining), osteocytes (Alizarin Red staining), or chondrocytes (Alcian Blue staining) under specific culture conditions. ThyAdMSCs cultured in DMEM or αMEM were used as controls.

Figure 5

ThyAdMSCs in mixed lymphocyte reaction with activated PBMCs. MLR was carried out with activated PBMCs (PBac) and mitomycin C-treated ThyAdMSCs (MSM) in 96-well plates at a ratio of 1 (MSM): 8 (PBac). The number of PBMCs present in the cell culture was counted on days 4, 6, and 7 using a Neubauer grating haemocytometer and expressed as the mean value per µL (n = 4, ** = p < 0.01.) All measurements were carried out in triplicate.

Figure 6

Gene expression under MLR conditions. For the gene expression studies we used cultured ThyAdMSCs without mitomycin C as the baseline for ThyAdMSCs with mitomycin C (MSM) and ThyAdMSCs with mitomycin co-cultured with activated PBMCs (CoMS). Non-activated PBMCs were used as the control group for activated PBMCs (PBac) or activated PBMCs co-cultured with mitomycin-treated ThyAdMSCs (CoPB). Expression was measured by quantitative RT-PCR and is stated as x-fold change relative to the corresponding control (* = p < 0.05; ** = p < 0.01).

Figure 7

IDO production by PBMCs and ThyAdMSCs during MLR. For IDO detection ELISA, cells were seeded in triplicate in 24-well plates at a ratio of 1 (MSM): 8 (PBac) (n = 3). After culturing, supernatants (containing PBMCs) (A) and adherent ThyAdMSCs (B) were collected separately on days 4 and 7 for quantification of the IDO levels by ELISA. Three freeze–thaw cycles were performed before samples were used for ELISA. The IDO measurement was performed in triplicate according to manufacturer’s instructions.