Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells
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...
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PeerJ Inc.
2021-03-01
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| author | Ban Al- kurdi Nidaa A. Ababneh Nizar Abuharfeil Saddam Al Demour Abdalla S. Awidi |
| author_facet | Ban Al- kurdi Nidaa A. Ababneh Nizar Abuharfeil Saddam Al Demour Abdalla S. Awidi |
| author_sort | Ban Al- kurdi |
| collection | DOAJ |
| description | 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. |
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| spelling | doaj.art-ba2732f1274b44e1b55dbd8050332b062023-12-02T21:54:55ZengPeerJ Inc.PeerJ2167-83592021-03-019e1089010.7717/peerj.10890Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cellsBan Al- kurdi0Nidaa A. Ababneh1Nizar Abuharfeil2Saddam Al Demour3Abdalla S. Awidi4Cell Therapy Center, University of Jordan, Amman, JordanCell Therapy Center, University of Jordan, Amman, JordanDepartment of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, Irbid, JordanDepartment of Urology, School of medicine, University of Jordan, Amman, Jordan, University of Jordan, Amman, JordanCell Therapy Center, University of Jordan, Amman, JordanBackground 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.https://peerj.com/articles/10890.pdfAdipose stem cellsUrothelial cellsPooled human platelet lysateCellular differentiation |
| spellingShingle | Ban Al- kurdi Nidaa A. Ababneh Nizar Abuharfeil Saddam Al Demour Abdalla S. Awidi Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells PeerJ Adipose stem cells Urothelial cells Pooled human platelet lysate Cellular differentiation |
| title | Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells |
| title_full | Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells |
| title_fullStr | Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells |
| title_full_unstemmed | Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells |
| title_short | Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells |
| title_sort | use of conditioned media cm and xeno free serum substitute on human adipose derived stem cells adscs differentiation into urothelial like cells |
| topic | Adipose stem cells Urothelial cells Pooled human platelet lysate Cellular differentiation |
| url | https://peerj.com/articles/10890.pdf |
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