Functional characterization of the immunomodulatory properties of human urine-derived stem cells

BackgroundUrine-derived stem cells (USCs) have been widely researched as a novel cell source for stem cell therapy, but their immunomodulatory characteristics remain to be investigated. This study aimed to characterize the immunomodulatory properties of human USCs.MethodsHuman USCs were isolated from fresh voiding urine samples from healthy male donors and expanded. Their cell surface markers were characterized by flow cytometry analysis and the telomerase activities for several USCs clones were determined. The immunosuppressive potential of USCs was evaluated by the performing the mixed lymphocyte reaction (MLR) [co-culture with peripheral blood mononuclear cells (PBMNCs)] and natural killer cells (NK) cytotoxicity assay. USCs cytokines release profile was determined by using human cytokine proteome array.ResultsUSCs exhibited high cell surface expression of embryonic/mesenchymal stem cells (MSCs) markers CD29, CD44, CD54, CD73, CD90, CD146, and CD166, while lacked expression of hematopoietic stem cell markers CD11, CD14, CD19, CD31, CD34, CD45, B cell marker CD79, and co-stimulatory factors CD80 and CD86, thus, exhibiting the phenotype of MSCs. MLR indicated that USCs significantly inhibited the proliferation of PBMNCs, as compared to that of the human smooth muscle cells (SMCs). In cell cytotoxicity assays, NK cells displayed less cytotoxicity against USCs than against bone marrow mesenchymal stem cells (BMSCs) and SMCs. Furthermore, upon PBMNCs stimulation, USCs secreted higher levels of immunomodulatory cytokines, including IL-6, IL-8, MCP-1, RANTES, GROα, and GM-CSF, compared to those of BMSCs, especially when directly contact mix-culture with PBMNCs.ConclusionsUSCs secreted immunoregulatory cytokines and possessed immunomodulatory properties, comparable to those of BMSCs.     

Advanced Properties of Urine Derived Stem Cells Compared to Adipose Tissue Derived Stem Cells in Terms of Cell Proliferation,Immune Modulation and Multi Differentiation

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However, ADSCs require invasive procedures, and has potential complications. Recently, urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study, we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization, and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation, colony formation, cell surface markers, immune modulation, chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3, 5, and 7. USCs showed high cell proliferation rate, enhanced colony forming ability, strong positive for stem cell markers expression, high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3, 5, and 7. In chromosome stability analysis, both cells showed normal karyotype through all passages. In analysis of multi-lineage capability, USCs showed higher myogenic, neurogenic, and endogenic differentiation rate, and lower osteogenic, adipogenic, and chondrogenic differentiation rate compared to ADSCs. Therefore, we expect that USC can be an alternative autologous stem cell source for muscle, neuron and endothelial tissue reconstruction instead of ADSCs.     

The effect of urine-derived stem cells expressing VEGF loaded in collagen hydrogels on myogenesis and innervation following after subcutaneous implantation in nude mice

Impairment of sphincter muscles or their neural and vascular support leads to stress urinary incontinence. The aim of this study was to determine the role of urine-derived stem cells (USCs) over-expressing vascular endothelial growth factor (VEGF) in collagen-I gel on angiogenesis, cell survival, cell growth, myogenic phenotype differentiation of the implanted cells and innervations following implantation in vivo. USCs were infected with adenovirus containing the human VEGF₁₆₅ and green fluorescent protein genes. A total of 5 × 10⁶ cells, USCs alone, or plus endothelial cells or human skeletal myoblasts (as control) suspended in collagen-I gel were subcutaneously implanted into nude mice. Extensive vascularization and more implanted cells was noted in VEGF-expressing USCs groups compared to the non-VEGF groups in vivo. Numbers of the cells displaying endothelial markers (CD 31 and von Willebrand's factor) and myogenic markers (myf-5, MyoD and desmin), and regenerated nerve fibers displaying neural markers (S-100, GFAP and neurofilament) significantly increased in the grafts of VEGF-expressing USCs. Improved angiogenesis by VEGF-expressing USCs enhanced grafted cell survival, recruited the resident cells and promoted myogenic phenotype differentiation of USCs and innervation. This approach has important clinical implications for the development of cell therapies for the correction of stress urinary incontinence.