We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages.When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction.

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

uUSC possess expansion and differentiation (urothelial and myogenic) capabilities, and can potentially be used as an alternative cell source in bladder tissue engineering for patients needing cystoplasty.

Despite successful approaches to preserve organs, tissues, and isolated cells, the maintenance of stem cell viability and function in body fluids during storage for cell distribution and transportation remains unexplored. The aim of this study was to characterize urine-derived stem cells (USCs) after optimal preservation of urine specimens for up to 24 hours. A total of 415 urine specimens were collected from 12 healthy men (age range 20–54 years old). About 6×104 cells shed off from the urinary tract system in 24 hours. At least 100 USC clones were obtained from the stored urine specimens after 24 hours and maintained similar biological features to fresh USCs. The stored USCs had a “rice grain” shape in primary culture, and expressed mesenchymal stem cell surface markers, high telomerase activity, and normal karyotypes. Importantly, the preserved cells retained bipotent differentiation capacity. Differentiated USCs expressed myogenic specific proteins and contractile function when exposed to myogenic differentiation medium, and they expressed urothelial cell-specific markers and barrier function when exposed to urothelial differentiation medium. These data demonstrated that up to 75% of fresh USCs can be safely persevered in urine for 24 hours and that these cells stored in urine retain their original stem cell properties, indicating that preserved USCs could be available for potential use in cell-based therapy or clinical diagnosis.

To provide site-specific delivery and targeted release of growth factors to implanted urine-derived stem cells (USCs), we prepared microbeads of alginate containing growth factors. The growth factors included VEGF, IGF-1, FGF-1, PDGF, HGF and NGF. Radiolabeled growth factors were loaded separately and used to access the in vitro release from the microbeads with a gamma counter over 4 weeks. In vitro endothelial differentiation of USCs by the released VEGF from the microbeads in a separate experiment confirmed that the released growth factors from the microbeads were bioactive. USCs and microbeads were mixed with the collagen gel type 1 (2 mg/ml) and used for in vivo studies through subcutaneous injection into nude mice. Four weeks after subcutaneous injection, we found that grafted cell survival was improved and more cells expressed myogenic and endothelial cell transcripts and markers compared to controls. More vessel formation and innervations were observed in USCs combined with six growth factors cocktail incorporated in microbeads compared to controls. In conclusion, a combination of growth factors released locally from the alginate microbeads induced USCs to differentiate into a myogenic lineage, enhanced revascularization and innervation, and stimulated resident cell growth in vivo. This approach could potentially be used for cell therapy in the treatment of stress urinary incontinence.

As a chronic metabolic disease, diabetes mellitus (DM) is broadly characterized by elevated levels of blood glucose. Novel epidemiological studies demonstrate that some diabetic patients have an increased risk of developing dementia compared with healthy individuals. Alzheimer’s disease (AD) is the most frequent cause of dementia and leads to major progressive deficits in memory and cognitive function. Multiple studies have identified an increased risk for AD in some diabetic populations, but it is still unclear which diabetic patients will develop dementia and which biological characteristics can predict cognitive decline. Although few mechanistic metabolic studies have shown clear pathophysiological links between DM and AD, there are several plausible ways this may occur. Since AD has many characteristics in common with impaired insulin signaling pathways, AD can be regarded as a metabolic disease. We conclude from the published literature that the body’s diabetic status under certain circumstances such as metabolic abnormalities can increase the incidence of AD by affecting glucose transport to the brain and reducing glucose metabolism. Furthermore, due to its plentiful lipid content and high energy requirement, the brain’s metabolism places great demands on mitochondria. Thus, the brain may be more susceptible to oxidative damage than the rest of the body. Emerging evidence suggests that both oxidative stress and mitochondrial dysfunction are related to amyloid-β (Aβ) pathology. Protein changes in the unfolded protein response or endoplasmic reticulum stress can regulate Aβ production and are closely associated with tau protein pathology. Altogether, metabolic disorders including glucose/lipid metabolism, oxidative stress, mitochondrial dysfunction, and protein changes caused by DM are associated with an impaired insulin signal pathway. These metabolic factors could increase the prevalence of AD in diabetic patients via the promotion of Aβ pathology.

Senescence restricts the development of applications involving mesenchymal stem cells (MSCs) in research fields, such as tissue engineering, and stem cell therapeutic strategies. Understanding the mechanisms underlying natural aging processes may contribute to the development of novel approaches to preventing age-related diseases or slowing individual aging processes. Nampt is a rate-limiting NAD biosynthetic enzyme that plays critical roles in energy metabolism, cell senescence and maintaining life spans. However, it remains unknown whether Nampt influences stem cell senescence. In this study, the function of Nampt was investigated using a rat model of natural aging. Our data show that Nampt expression was significantly lower in MSCs obtained from aged rats than in those obtained from young rats during physiological aging. Reducing the level of Nampt in aged MSCs resulted in lower intracellular concentrations of NAD+ and downregulated Sirt1 expression and activity. After the Nampt inhibitor FK866 was added, young MSCs were induced to become aged cells. The enhanced senescence was correlated with NAD+ depletion and Sirt1 activity attenuation. In addition, Nampt overexpression attenuated cell senescence in aged MSCs. Our findings provide a new explanation for the mechanisms underlying stem cell senescence and a novel target for delaying stem cell senescence and preventing and treating age-related diseases.

In vitro replicative senescence affects MSC characteristics and functionality, thus severely restricting their application in regenerative medicine and MSC-based therapies. Previously, we found that MSC natural senescence is accompanied by altered intracellular nicotinamide adenine dinucleotide (NAD + ) metabolism, in which Nampt plays a key role. However, whether Nampt influences MSC replicative senescence is still unclear. Our study showed that Nampt expression is down-regulated during MSC replicative senescence. Nampt depletion via a specific Nampt inhibitor FK866 or Nampt knockdown in early passage MSCs led to enhanced senescence as indicated by senescence-like morphology, reduced proliferation, and adipogenic and osteogenic differentiation, and increased senescence-associated-β-galactosidase activity and the expression of the senescence-associated factor p16 INK4a . Conversely, Nampt overexpression ameliorated senescence-associated phenotypic features in late passage MSCs. Further, Nampt inhibition resulted in reduced intracellular NAD + content, NAD + /NADH ratio, and Sirt1 activity, whereas overexpression had the opposite effects. Exogenous intermediates involved in NAD + biosynthesis not only rescued replicative senescent MSCs but also alleviated FK866-induced MSC senescence. Thus, Nampt suppresses MSC senescence via mediating NAD + -Sirt1 signaling. This study provides novel mechanistic insights into MSC replicative senescence and a promising strategy for the severe shortage of cells for MSC-based therapies.