Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells

Abstract

Background: Congenital abnormalities, cancers as well as injuries can cause irreversible damage to the urinary tract, which eventually requires tissue reconstruction. Smooth muscle cells, endothelial cells, and urothelial cells are the major cell types required for the reconstruction of lower urinary tract. Adult stem cells represent an accessible source of unlimited repertoire of untransformed cells.

Aim: Fetal bovine serum (FBS) is the most vital supplement in the culture media used for cellular proliferation and differentiation. However, due to the increasing interest in manufacturing xeno-free stem cell-based cellular products, optimizing the composition of the culture media and the serum-type used is of paramount importance. In this study, the effects of FBS and pooled human platelet (pHPL) lysate were assessed on the capacity of human adipose-derived stem cells (ADSCs) to differentiate into urothelial-like cells. Also, we aimed to compare the ability of both conditioned media (CM) and unconditioned urothelial cell media (UCM) to induce urothelial differentiation of ADCS in vitro.

Methods: ADSCs were isolated from human lipoaspirates and characterized by flow cytometry for their ability to express the most common mesenchymal stem cell (MSCs) markers. The differentiation potential was also assessed by differentiating them into osteogenic and adipogenic cell lineages. To evaluate the capacity of ADSCs to differentiate towards the urothelial-like lineage, cells were cultured with either CM or UCM, supplemented with either 5% pHPL, 2.5% pHPL or 10% FBS. After 14 days of induction, cells were utilized for gene expression and immunofluorescence analysis.

Results: ADSCs cultured in CM and supplemented with FBS exhibited the highest upregulation levels of the urothelial cell markers; cytokeratin-18 (CK-18), cytokeratin-19 (CK-19), and Uroplakin-2 (UPK-2), with a 6.7, 4.2- and a 2-folds increase in gene expression, respectively. Meanwhile, the use of CM supplemented with either 5% pHPL or 2.5% pHPL, and UCM supplemented with either 5% pHPL or 2.5% pHPL showed low expression levels of CK-18 and CK-19 and no upregulation of UPK-2 level was observed. In contrast, the use of UCM with FBS has increased the levels of CK-18 and CK-19, however to a lesser extent compared to CM. At the cellular level, CK-18 and UPK-2 were only detected in CM/FBS supplemented group. Growth factor analysis revealed an increase in the expression levels of EGF, VEGF and PDGF in all of the differentiated groups.

Conclusion: Efficient ADSCs urothelial differentiation is dependent on the use of conditioned media. The presence of high concentrations of proliferation-inducing growth factors present in the pHPL reduces the efficiency of ADSCs differentiation towards the urothelial lineage. Additionally, the increase in EGF, VEGF and PDGF during the differentiation implicates them in the mechanism of urothelial cell differentiation.

Keywords: Adipose stem cells; Cellular differentiation; Pooled human platelet lysate; Urothelial cells.

Figure 1. Characterization and differentiation potential of ADSCs.

Characterization and differentiation potential of ADSCs. (A) Primary ADSCs morphology after 14 days in culture under the inverted phase contrast microscope. Scale bar = 100 µm. (B–F) Flow cytometry staining of ADSC markers. Cells showed positive staining for mesenchymal stem cells markers CD-44, CD-105, CD-73, CD-90 and negative for CD-34, CD-11b, CD-19, CD-45 and HLA-DR in the negative cocktail.

Figure 2. Multilineage differentiation potential of ADSCs.

(A–C) Osteogenic mineral deposition was observed after 21 days of osteogenic induction and positively stained with Alizarin Red S stain (ARS). (B–D) Uninduced ADSCs were used as a negative control and stained negatively with ARZ. (E–G) Lipid droplets were observed after 14 days of adipogenic differentiation of ADSCs and positively stained with Oil Red O. (F–H) ADSCs with normal culture media stained negatively for oil red O. Scale bar = 100 µm. All differentiation experiments were repeated at least three times.

Figure 3. Relative gene expression of urothelial markers.

Real time-PCR performed on urothelial-like cells differentiated from ADSCs, cultivated in either conditioned medium (CM) or unconditioned urothelial cell media (UCM) in the presence of either 5% pHPL, 2.5% pHPL or 10% FBS. Uninduced ADSCs were used as the calibrator sample. (A) Relative expression of cytokeratin-18, (B) cytokeratin-19 and (C) uroplakin-2. * P < 0.05, ** P < 0.01. All experiments were repeated at least three times.

Figure 4. Immunofluorescence staining of urothelial markers at the cellular level.

(A) ADSCs cultured in CM and different concentrations of pHPL (5% & 2.5%) or with FBS, were assessed for the expression of cytokeratin-18 (FITC, green) and uroplakin- 2 (Cy3, red). SV-HUC cells were used as a positive control, meanwhile ADSCs were utilized as a negative control. Scale bar = 50 µm. (B & C) Semi-quantitative analysis of immunoflouresence representing percentage of positive cells relative to negative control. All experiments were repeated at least three times.

Figure 5. Growth factor levels assessment in induced ADSCs.

Enzyme linked immunosorbent assay (ELISA) was preformed on conditioned media collected after 24 h from cells induced for 14 days with urothelial cell derived conditioned medium (CM) or unconditioned urothelial cell media (UCM) with 5% pHPL, 2.5%pHPL or FBS. (A) Measurement of epidermal growth factor levels (EGF). (B) Platelet derived growth factor-BB (PDGF-BB). (C) Vascular endothelial growth factor (VEGF). * P < 0.05, ** P < 0.01. All experiments were repeated at least three times.