Urine-Derived Stem Cells: Applications in Regenerative and Predictive Medicine

Despite being a biological waste, human urine contains a small population of cells with self-renewal capacity and differentiation potential into several cell types. Being derived from the convoluted tubules of nephron, renal pelvis, ureters, bladder and urethra, urine-derived stem cells (UDSC) have a similar phenotype to mesenchymal stroma cells (MSC) and can be reprogrammed into iPSC (induced pluripotent stem cells). Having simple, safer, low-cost and noninvasive collection procedures, the interest in UDSC has been growing in the last decade. With great potential in regenerative medicine applications, UDSC can also be used as biological models for pharmacology and toxicology tests. This review describes UDSC biological characteristics and differentiation potential and their possible use, including the potential of UDSC-derived iPSC to be used in drug discovery and toxicology, as well as in regenerative medicine. Being a new cellular platform amenable to noninvasive collection for disease stratification and personalized therapy could be a future application for UDSC.

The search for optimal UDSC culture medium was described by Zhou et al. The authors cultivated primary cells in a medium that contained 10% (vol/vol) FBS to increase the initial cell adherence and survival. Later, for cellular expansion, authors cultivated cells in two distinct media: (a) RE (renal epithelial) proliferation medium [6] and (b) mixture of RE/MC (renal epithelial/mesenchymal cell) proliferation medium in a ratio 1:1. According to the results, UDSC grew in both media, but the proliferation rate was higher in the latter [7].
Urothelial cells grow in serum-free medium, which is a different feature from UDSC and BMSC [3,8].
Another experimental condition that distinguishes urothelial cells from UDSC and MSC is the need for collagen matrices and salt concentration. To improve cellular growth and the formation of stratified epithelium, it is required to cultivate urothelial cells in a medium with high concentrations of calcium chloride [9,10] on flasks covered by collagen [11,12]. Moreover, UDSC long-term cultivation was more efficient by cultivating cells on porous collagen matrix in a cell medium supplemented with 5% FBS, hormones and calcium [10,13]. Instead of collagen matrices, some researchers seeded cells on feeder cells (embryonic mesenchymal-derived (Swiss 3T3)) [11], at least for the primary culture [14,15]. In comparison to urothelial cells, UDSC do not require such specific conditions. Whenever urothelial differentiation is required, UDSC can be cultivated on plates, covered by collagen, in a serum-free keratinocyte medium [16] or in a mixture of medium containing equal amounts of KSFM and EFM with 2% FBS [17]. EGF is a growth factor required for urothelial differentiation [3,17]. Urothelial differentiation of cells is characterized by specific cobblestone morphology [18]; the presence of tight junction genes and protein markers (ZO-1 and E-cadherin) and the expression of uroplakin Ia and III, AE1/AE3 and CK-7 [3]; pancytokeratins and cytokeratin20 [16]. Stratification of confluent UDSC monolayer cultures can be induced in a culture medium containing 1.5 mM calcium chloride [19].
Addition of EGF may also induce endothelial differentiation of UDSC, as it was demonstrated by Dong et al. [5]. In comparison to the previous urothelial differentiation, cells were cultivated in a mixture of DMEM 10% FBS with KSFM at a 1:4 ratio and 30 ng/mL EGF. A more specific medium content was later used by Bharadwaj S. et al. [17]. In this work, cells were cultured on fibronectin-coated plates in an endothelial basal medium (EBM2, Lonza) supplemented with 50 ng/mL VEGF. Such medium was used also for the induction of MSC endothelial differentiation [20]. This differentiation was demonstrated through measuring endothelial cell-specific gene transcripts (vWF and CD31); protein markers (CD31 [5], vWF, KDR, FLT1 and eNOS) and a tight endothelial junction marker (VE-cadherin) [17]. Formation of vessel-like structures was also visualized during cultivation on Matrigel [17].
For the induction of UDSC neuronal differentiation, Bhadawaraj et al. described a specific protocol. Cells were initially grown in pre-induction medium containing DMEM with 20% FBS and 10 ng/mL basic fibroblast growth factor (bFGF). Then, 24 hours later, the medium was replaced with a nerve induction medium for a further 48 hours. As a result, UDSC exhibited several neurogenic extensions and processes; approximately 40% of cells expressed the neuronal specific protein markers nestin, S100, NF200 and glial fibrillary acidic protein [17]. In other studies, the neural differentiation medium consisted of DMEM/Hamm's F12 medium supplemented with 20 ng/mL EGF, 40 ng/mL bFGF, 2% B27, 1% nonessential amino acid, 1% l-glutamine and 1% insulin-transferrin-selenite. As a result of the neurogenic differentiation of UDSC, an increased number of Sox2-and Nestin-positive cells was confirmed by the significantly increased gene expression of neuronal progenitor cell markers Nestin and Sox2, while mature neuron marker β-III-tubulin was not upregulated [21]. Alternatively, neurogenic differentiation of UDSC was also induced by cultivation in a commercial NeuroCult NS-A differentiation kit (StemCell) that lead to neuronspecific morphological changes and β-III-tubulin gene expression [18].
Confirming the initial ideas that UDSC have mesodermal origin, these cells can be differentiated into derivatives of mesoderm-osteogenic, chondrogenic, myogenic and adipogenic differentiation lineages [22].
For osteogenic differentiation, most publications reported the use of osteogenic induction media from commercial sources [18,21]. During cultivation, 70%-80% of UDSCs appeared to produce mineralized tissue that was confirmed by von Kossa, alkaline phosphatase and Alizarin Red S staining for calcium deposition. Adipogenic commercial medium is widely used to demonstrate adipogenic differentiation of UDSC [18,21]. After adipogenic induction, cells were positive for oil red-O staining and 30%-40% expressed adipocyte gene markers-peroxisome proliferator-activated receptor, acetyl-CoA synthase, adiponectin, CCAAT/enhancer-binding protein a, fatty acid binding protein 4 and lipoprotein lipase [17,21,23]. For chondrogenic differentiation, UDSC can be cultivated with commercial chondrogenic induction medium, [18,21] and cells can be stained with Alcian blue, toluidine blue, and safranin-O for labeling sulfated glycosaminoglycan proteins to check the efficiency of differentiation. According to the authors' results obtained, about 60% of the induced UDSCs expressed the chondrogenic lineage markers Sox9, collagen-II and aggrecan [21]. However, in comparison to osteo-and myogenic differentiation, chondrogenic differentiation of UDSC was less efficient [2,17].
UDSC may also be differentiated into skeletal muscle cells. Such differentiation may be induced by two methods: (a) DMEM with an addition of 50 µM hydrocortisone, 0.1 µM dexamethasone, 10% FBS and 5% horse serum [17] and (b) conditioned medium from skeletal muscle cell cultures for 12 hours [18,24]. After one month of cultivation, UDSC displayed an elongated spindle-shaped morphology and expression of skeletal muscle-related transcripts (MyoD and myogenin). Moreover, 50%-60% of cells stained for MyoD formed myotube-like structures [17]. It was demonstrated that this myogenic differentiation properties of UDSC and ADCS were comparable and that the expression of MyoD in UDSC cultures was higher. Thus, UDSC seem to be more prone to a skeletal muscle differentiation lineage commitment [18].
Therefore, UDSC medium content and the presence of different supplements play a crucial role in cell viability, growth and phenotype. The culture medium used may contribute to distinguishing various cell types and to induce differentiation into multiple cell lineages, allowing for a high versality of this type of cell for multiple purposes.