In vitro differentiation potential of human urine-derived stem cells into neuron-like cells and their protective effects against spinal cord injury in rats / 中国组织工程研究

BACKGROUND: Human urine-derived stem cells are newly discovered adult stem cells, characterized by rich sources, simple extraction, good proliferative ability and multi-directional differentiation potential. In recent years, human urine-derived stem cells have been used for the repair of neurological functions in urinary diseases, such as stress urinary incontinence and vesicoureteral reflux. OBJECTIVE: To explore the biological characteristics of human urine-derived stem cells and to study their repairing effect in a rat model of spinal cord injury. METHODS: Cell phenotypes of human urine-derived stem cells were detected using flow cytometry, and the immunohistochemical staining was used to identify neuron-like cells differentiated from human urine-derived mesenchymal stem cells. Then, an animal model of spinal cord injury at T9 segment was made by Allen method, and after modeling 24 Sprague-Dawley rats were assigned into spinal cord injury group or cell treatment group (n=12/group). In the cell treatment group, the model rats were injected 2 μL of 1.0×1011/L human urine-derived stem cells, while in the spinal cord injury group, the rats were administered the same volume of L-DMEM containing 10% fetal bovine serum. Basso, Beattie and Bresnahan scores were valued at 1, 10, 20, and 30 days after modeling. Spinal cord samples from all the rats were taken out at 30 days after modeling, and Luxol Fast Blue staining, microglia/macrophages staining and glial fibrillary acidic protein staining were used to value the injured area of the spinal cord and the fluorescence intensity of glial fibrillary acidic protein. RESULTS AND CONCLUSION: (1) Flow cytometry showed high expression on CD29 and CD90, and low expression on CD45 in human urine-derived mesenchymal stem cells. Moreover, human urine-derived mesenchymal stem cells could be induced to differentiating into neuron-like cells in vitro. (2) Basso, Beattie and Bresnahan scores showed no significant difference between the two groups at 1 and 10 days after modeling (P > 0.05), while, at 20 and 30 days after modeling, the scores in the cell treatment group were significantly higher than those in the spinal cord injury group (P < 0.05). (3) Luxol Fast Blue staining showed that the injured area of the spinal cord in the cell treatment group was markedly less than that in the spinal cord injury group (P < 0.05), and the glial fibrillary acidic protein showed lower fluorescence intensity in the cell treatment group than the spinal cord injury group (P < 0.05). To conclude, human urine-derived stem cells can differentiate into neuron-like cells and have therapeutic effects in the rat model of spinal cord injury.

Urine-derived stem cells differentiate into urothelial cells and smooth muscle cells in vitro / 中国组织工程研究

BACKGROUND: Adult stem cells are pluripotent stem cells that exist in differentiated tissues. Urine-derived stem cells are newly discovered adult stem cells. They have attracted increasing attentions in the tissue engineering, due to its advantages of convenient sampling, highly proliferative ability and multidirectional differentiation potential. OBJECTIVE: To investigate the separation and extraction method of urine-derived stem cells and to investigate the feasibility of differentiation into urothelial cells and smooth muscle cells in vitro. METHODS: Urine specimens were collected from healthy adults, and urine-derived stem cells were obtained by isolation and culture in vitro. Cell counting kit-8 assay was used to detect cell proliferation and plot cell growth curve. Cell phenotype was detected by flow cytometry. The differentiation into urothelial and smooth muscle cells was induced by special medium respectively in vitro. The cell differentiation was detected by quantitative PCR, immunohistochemical staining, immunofluorescence cell staining and western blot assay. RESULTS AND CONCLUSION: Urine-derived stem cells were successfully isolated from the urine specimens of healthy adults. Urine-derived stem cells possessed high proliferation rate and the cell growth curve exhibited an S-shape. Urine-derived stem cells exhibited high expression of CD29 (98.11%) and CD90 (95.74%), both of which are mesenchymal stem cell surface markers. After 14 days of induction in vitro, urine-derived stem cells were able to express urothelial cell specific genes Cytokeratin 7, Cytokeratin 20, Uroplakin II and smooth muscle cell specific genes α-SMA and SM22. These results suggest that urine-derived stem cells can differentiate into urothelial cells and smooth muscle cells after induction in vitro and can be used as ideal seed cells for urinary tract tissue engineering.

Effect of astragalus polysaccharide combined with urine stem cell transplantation on type 2 diabetes in rats / 国际生物医学工程杂志

Objective To study the effect of astragalus polysaccharides combined with hUSCs transplantation on type 2 diabetic rats. Methods Twenty-five SD rats were randomly selected into the normal control group, and the remaining 105 SD rats were used to establish type 2 diabetes model. The 100 rats successfully modeled were randomly divided into the diabetes group, astragalus polysaccharide treatment group, hUSCs treatment group, and astragalus polysaccharide+hUSCs treatment group, with 25 rats in each group. After 2 weeks of treatment, the FBG concentration, insulin and C-peptide concentrations, and body weight changes were measured in each group. The distribution and survival of PKH-26-labeled hUSCs in rat pancreatic tissue were observed by fluorescence microscopy. TUNEL method was used to detect the apoptosis of rat islet cells. Real-time quantitative PCR and Western Blot were used to detect the expression of TGF-β/Smad signaling pathway-related genes in rat pancreatic tissue. Results The FBG concentration of rats in the astragalus polysaccharide treatment group, hUSCs treatment group and astragalus polysaccharide +hUSCs treatment group were significantly decreased, and that in the combination treatment group was significantly lower those in the astragalus polysaccharide group and hUSCs group, and the differences were statistically significant ( all P<0 . 05 ) . Compared with the diabetic group , the insulin concentration, C-peptide concentration and body weight in the astragalus polysaccharide treatment group, hUSCs treatment group and combination treatment group rats were significantly increased, and those in the combination treatment group was significantly higher than those in the astragalus polysaccharide treatment group and in the hUSCs treatment group, the differences were statistically significant( all P<0.05). The results of fluorescence microscopy showed that the number of PKH-26 positive hUSCs in the combined treatment group was 74.64 ±9.75 in each high power field, which was significantly higher than that in the hUSCs treatment group (43.64±5.83), the difference was statistically significant (P<0.05). Compared with the diabetic group, the apoptotic rates of islet cells in the astragalus polysaccharide treatment group and the hUSCs treatment group were reduced, and the relative expressions levels of mRNA and protein of TGF-β1, Smad3, and Smad7 in the pancreatic tissue were also significantly reduced(all P<0.05). The reduction was more significant in the combination treatment group, and the differences were statistically significant (all P<0.05). Conclusions Astragalus polysaccharide combined with hUSCs transplantation can effectively reduce the FBG concentration, increase the concentration of insulin, C-peptide and body weight, reduce the apoptosis of pancreatic islet tissue, which may be related to the reduction of TGF-β/Smad in pancreatic tissue. Signaling pathways are involved in suppressing the inflammatory response.

Laminin and Platelet-Derived Growth Factor-BB Promote Neuronal Differentiation of Human Urine-Derived Stem Cells

Urine-derived stem cells (USCs) are considered as a promising cell source capable of neuronal differentiation. In addition, specific growth factors and extracellular matrix are essential for enhancing their neuronal differentiation efficiency. In this study, we investigated the possibility of neuronal differentiation of USCs and the role of laminin and platelet-derived growth factor BB (PDGF-BB) as promoting factors. USCs were isolated from fresh urine of healthy donors. Cultured USCs were adherent to the plate and their morphology was similar to the cobblestone. In addition, they showed chromosome stability, rapid proliferation rate, colony forming capacity, and mesenchymal stem cell characteristics. For inducing the neuronal differentiation, USCs were cultured for 14 days in neuronal differentiation media supplemented with/without laminin and/or PDGF-BB. To identify the expression of neuronal markers, RT-PCR, flow cytometry analysis and immunocytochemistry were used. After neuronal induction, the cells showed neuron-like morphological change and high expression level of neuronal markers. In addition, laminin and PDGF-BB respectively promoted the neuronal differentiation of USCs and the combination of laminin and PDGF-BB showed a synergistic effect for the neuronal differentiation of USCs. In conclusion, USCs are noteworthy cell source in the field of neuronal regeneration and laminin and PDGF-BB promote their neuronal differentiation efficiency.

The application of urine derived stem cells in regeneration of musculoskeletal system / 中国修复重建外科杂志

Objective: To review the application of urine derived stem cells (USCs) in regeneration of musculoskeletal system. Methods: The original literature about USCs in the regeneration of musculoskeletal system was extensively reviewed and analyzed. Results: The source of USCs is noninvasive and extensive. USCs express MSCs surface markers with stable proliferative and multi-directional differentiation capabilities, and are widely used in bone, skin, nerve, and other skeletal and muscle system regeneration fields and show a certain repair capacity. Conclusion: USCs from non-invasive sources have a wide application prospect in the regeneration of musculoskeletal system, but the definite biological mechanism of its repair needs further study.

Potency of Human Urine-Derived Stem Cells for Renal Lineage Differentiation

Kidney is one of the most difficult organs for regeneration. Several attempts have been performed to regenerate renal tissue using stem cells, the results were not satisfactory. Urine is major product of kidney and contains cells from renal components. Moreover, urine-derived stem cells (USCs) can be easily obtained without any health risks throughout a patient's entire life. Here, we evaluated the utility of USCs for renal tissue regeneration. In this study, the ability of USCs to differentiate into renal lineage cells was compared with that of adipose tissue-derived stem cells (ADSCs) and amniotic fluid-derived stem cells (AFSCs), with respect to surface antigen expression, morphology, immunocytochemistry, renal lineage gene expression, secreted factors, immunomodulatory marker expression, in vivo safety, and renal differentiation potency. Undifferentiated USCs were positive for CD44 and CD73, negative for CD34 and CD45, and formed aggregates after 3 weeks of renal differentiation. Undifferentiated USCs showed high SSEA4 expression, while renal-differentiated cells expressed PAX2, WT1, and CADHERIN 6. In the stem/renal lineageassociated gene analysis, OCT4, SSEA4, and CD117 were significantly downregulated over time, while PAX2, LIM1, PDGFRA, E-CADHERIN, CD24, ACTB, AQP1, OCLN, and NPHS1 were gradually upregulated. In the in vivo safety evaluation, renal-differentiated USCs did not show abnormal histology. These findings demonstrated that USCs have a similar MSC potency, renal lineage-differentiation ability, immunomodulatory effects, and in vivo safety as ADSCs and AFSCs, and showed higher levels of growth factor secretion for paracrine effects. Therefore, urine and USCs can be one of good cell sources for kidney regeneration.

The Efficacy and Safety of Collagen-I and Hypoxic Conditions in Urine-Derived Stem Cell Ex Vivo Culture

Upper urinary tract-derived urine stem cells (USCs) are considered a valuable mesenchymal stem cell source for autologous cell therapy. However, the reported culture condition for USCs is not appropriate for large-quantity production, because cells can show limited replicativity, senescence, and undesirable differentiation during cultivation. These drawbacks led us to reconstitute a culture condition that mimics the natural stem cell niche. We selected extracellular matrix protein and oxygen tension to optimize the ex vivo expansion of USCs, and compared cell adhesion, proliferation, gene expression, chromosomal stability, differentiation capacity, immunity and safety. Culture on collagen type I (ColI) supported highly enhanced USC proliferation and retention of stem cell properties. In the oxygen tension analysis (with ColI), 5% O₂ hypoxia showed a higher cell proliferation rate, a greater proportion of cells in the S phase of the cell cycle, and normal stem cell properties compared to those observed in cells cultured under 20% O₂ normoxia. The established reconstituted condition (ColI/hypoxia, USCs(recon)) was compared to the control condition. The expanded USCs(recon) showed highly increased cell proliferation and colony forming ability, maintained transcription factors, chromosomal stability, and multi-lineage differentiation capacity (neuron, osteoblast, and adipocyte) compared to the control. In addition, USCs(recon) retained their immune-privileged potential and non-tumorigenicity with in vivo testing at week 8. Therefore, reconstituted condition allows for expanded uUSC cell preparations that are safe and useful for application in stem cell therapy.

Human Urine-derived Stem Cells Seeded Surface Modified Composite Scaffold Grafts for Bladder Reconstruction in a Rat Model

We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold(heparin-bFGF) group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 microL/cm H2O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 microL/cm H2O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 microL/cm H2O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 microL/cm H2O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 microL/cm H2O for the scaffold(heparin-bFGF) group, respectively). In histological and immunohistochemical analysis, the USC-scaffold(heparin-bFGF) group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold(heparin-bFGF) exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.