Dataset Information

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells.

ABSTRACT: Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin-agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results showed that the biofabrication methods used here succeeded in generating a tubular structure consisting of a stromal substitute with a stratified epithelial-like layer on top, especially using a medium containing epithelial growth and differentiation factors (EM), although differentiation was not complete. At the functional level, UM substitutes were able to synthesize collagen fibers, proteoglycans and glycosaminoglycans, although the levels of control UM were not reached ex vivo. Epithelial differentiation was partially achieved, especially with EM after 14 days of development, with expression of keratins 7, 8, and 13 and pancytokeratin, desmoplakin, tight-junction protein-1, and uroplakin 2, although at lower levels than controls. These results confirm the partial urothelial differentiative potential of HWJSC and suggest that the biofabrication methods explored here were able to generate a potential substitute of the human UM for future clinical use.

SUBMITTER: Garzon I

PROVIDER: S-EPMC8153323 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells.

Garzón Ingrid I Jaimes-Parra Boris Damián BD Pascual-Geler Manrique M Cózar José Manuel JM Sánchez-Quevedo María Del Carmen MDC Mosquera-Pacheco María Auxiliadora MA Sánchez-Montesinos Indalecio I Fernández-Valadés Ricardo R Campos Fernando F Alaminos Miguel M

Polymers 20210513 10

Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin-agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results sh ...[more]

PMID: 34068343

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Project description:Purpose: Obtaining sufficient numbers of cells in a short time is a major goal of cell culturing in cell therapy and tissue engineering. However, current bidimensional (2D) culture methods are associated to several limitations, including low efficiency and the loss of key cell differentiation markers on cultured cells. Methods: In the present work, we have designed a novel biofabrication method based on a three-dimensional (3D) culture system (FIBRIAGAR-3D). Human Wharton's jelly mesenchymal stromal cells (HWJSC) were cultured in 3D using 100%, 75%, 50%, and 25% concentrations of fibrin-agarose biomaterials (FA100, FA75, FA50 and FA25 group) and compared with control cells cultured using classical 2D systems (CTR-2D). Results: Our results showed a significant increase in the number of cells generated after 7 days of culture, with cells displaying numerous expansions towards the biomaterial, and a significant overexpression of the cell proliferation marker KI67 was found for the FA75 and FA100 groups. TUNEL and qRT-PCR analyses demonstrated that the use of FIBRIAGAR-3D was not associated with an induction of apoptosis by cultured cells. Instead, the 3D system retained the expression of typical phenotypic markers of HWJSC, including CD73, CD90, CD105, NANOG and OCT4, and biosynthesis markers such as types-I and IV collagens, with significant increase of some of these markers, especially in the FA100 group. Finally, our analysis of 8 cell signaling molecules revealed a significant decrease of GM-CSF, IFN-g, IL2, IL4, IL6, IL8, and TNFα, suggesting that the 3D culture system did not induce the expression of pro-inflammatory molecules. Conclusion: These results confirm the usefulness of FIBRIAGAR-3D culture systems to increase cell proliferation without altering cell phenotype of immunogenicity and opens the door to the possibility of using this novel biofabrication method in cell therapy and tissue engineering of the human cornea, oral mucosa, skin, urethra, among other structures.

Project description:Purpose: Iron oxide based magnetic nanoparticles (MNP) are versatile tools in biology and medicine. Adipose derived mesenchymal stem cells (ADSC) and Wharton Jelly mesenchymal stem cells (WJMSC) are currently tested in different strategies for regenerative regenerative medicine (RM) purposes. Their superiority compared to other mesenchymal stem cell consists in larger availability, and superior proliferative and differentiation potential. Magnetic field (MF) exposure of MNP-loaded ADSC has been proposed as a method to deliver mechanical stimulation for increasing conversion to musculoskeletal lineages. In this study, we investigated comparatively chondrogenic conversion of ADSC-MNP and WJMSC with or without MF exposure in order to identify the most appropriate cell source and differentiation protocol for future cartilage engineering strategies. Methods: Human primary ADSC and WJMSC from various donors were loaded with proprietary uncoated MNP. The in vitro effect on proliferation and cellular senescence (beta galactosidase assay) in long term culture was assessed. In vitro chondrogenic differentiation in pellet culture system, with or without MF exposure, was assessed using pellet histology (Safranin O staining) as well as quantitative evaluation of glycosaminoglycan (GAG) deposition per cell. Results: ADSC-MNP complexes displayed superior proliferative capability and decreased senescence after long term (28 days) culture in vitro compared to non-loaded ADSC and to WJMSC-MNP. Significant increase in chondrogenesis conversion in terms of GAG/cell ratio could be observed in ADSC-MNP. MF exposure increased glycosaminoglycan deposition in MNP-loaded ADSC, but not in WJMSC. Conclusion: ADSC-MNP display decreased cellular senescence and superior chondrogenic capability in vitro compared to non-loaded cells as well as to WJMSC-MNP. MF exposure further increases ADSC-MNP chondrogenesis in ADSC, but not in WJMSC. Loading ADSC with MNP can derive a successful procedure for obtaining improved chondrogenesis in ADSC. Further in vivo studies are needed to confirm the utility of ADSC-MNP complexes for cartilage engineering.

Project description:BackgroundWharton's jelly derived stem cells (WJMSCs) are gaining attention as a possible clinical alternative to bone marrow derived mesenchymal stem cells (BMMSCs) owing to better accessibility, higher expansion potential and low immunogenicity. Usage of allogenic mesenchymal stem cells (MSC) could be permissible in vivo only if they retain their immune properties in an inflammatory setting. Thus the focus of this study is to understand and compare the immune properties of BMMSCs and WJMSCs primed with key pro-inflammatory cytokines, Interferon-gamma (IFNgamma) and Tumor Necrosis Factor-alpha (TNFalpha).Methodology/principal findingsInitially the effect of priming on MSC mediated suppression of alloantigen and mitogen induced lymphoproliferation was evaluated in vitro. Treatment with IFNgamma or TNFalpha, did not ablate the immune-suppression caused by both the MSCs. Extent of immune-suppression was more with WJMSCs than BMMSCs in both the cases. Surprisingly, priming BMMSCs enhanced suppression of mitogen driven lymphoproliferation only; whereas IFNgamma primed WJMSCs were better suppressors of MLRs. Further, kinetic analysis of cytokine profiles in co-cultures of primed/unprimed MSCs and Phytohematoagglutinin (PHA) activated lymphocytes was evaluated. Results indicated a decrease in levels of pro-inflammatory cytokines. Interestingly, a change in kinetics and thresholds of Interleukin-2 (IL-2) secretion was observed only with BMMSCs. Analysis of activation markers on PHA-stimulated lymphocytes indicated different expression patterns in co-cultures of primed/unprimed WJMSCs and BMMSCs. Strikingly, co-culture with WJMSCs resulted in an early activation of a negative co-stimulatory molecule, CTLA4, which was not evident with BMMSCs. A screen for immune suppressive factors in primed/unprimed WJMSCs and BMMSCs indicated inherent differences in IFNgamma inducible Indoleamine 2, 3-dioxygenase (IDO) activity, Hepatocyte growth factor (HGF) and Prostaglandin E-2 (PGE2) levels which could possibly influence the mechanism of immune-modulation.Conclusion/significanceThis study demonstrates that inflammation affects the immune properties of MSCs distinctly. Importantly different tissue derived MSCs could utilize unique mechanisms of immune-modulation.

Dataset Information

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells.

Publications

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells.

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