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.

Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds

“…To promote attachment, cells were resuspended in a minimal amount of medium (50 µL) and DLs were immobilized using agarose casts, as previously reported [38]. In order to induce epithelial differentiation of both cell types seeded on the RL, these tissues were cultured for 21 days in EM epithelial differentiation medium containing epithelial growth and differentiation factors, as previously described [39]. The EM medium consisted of a mixture of 150 mL of HAM-F12, 300 mL of DMEM, 50 mL of fetal bovine serum, 1% antibiotics/antimycotics, 24 µg/mL of adenine, 5 µg/mL of insulin, 1.3 ng/mL of triiodothyronine, 0.4 µg/mL of hydrocortisone, and 10 ng/mL of EGF (epidermal growth factor) (all of them, from Merck).…”

“…For the immunohistochemical analyses, results were semiquantitatively categorized as strongly positive signal (+++), positive signal (++), slightly positive signal (+), or negative signal (−), as previously reported [39].…”

Development of stromal differentiation patterns in heterotypical models of artificial corneas generated by tissue engineering

“…In this regard, our research group previously demonstrated the potential usefulness of HWJSC to generate epithelial-like cell layers in several models of bioengineered human tissues, including the urothelial mucosa ( Garzón et al, 2021 ), the skin epidermis ( Martin-Piedra et al, 2019 ), oral mucosa epithelium ( Alfonso-Rodríguez et al, 2015 ) and also the corneal epithelium ( Garzón et al, 2014 ). In the case of the human cornea, the use of HWJSC were able to stratify and differentiate very efficiently on a bioartificial corneal stroma, especially when the bioengineered corneas were grafted in vivo ( Garzón et al, 2020 ).…”

Development of secretome-based strategies to improve cell culture protocols in tissue engineering

“…To generate primary cell cultures of human MSC, we obtained samples of human adipose tissue, dental pulp, and umbilical cord according to previously described protocols 19 – 21 . Samples corresponding to three different donors were used (n = 3 for each tissue).…”

“…hADSC and hDPSC were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich/Merck, Burlington, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics and antimycotics (100 U/mL penicillin G, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B) (Sigma-Aldrich/Merck). hWJSC were cultured in Amniomax-C100 culture medium (Gibco-Thermo Fisher Scientific) 21 – 23 . In all cases, cells were kept under standard cell culture conditions at 37 °C in a humidified incubator (Esco Lifesciences Group, Singapore, Singapore) with 5% CO 2 .…”