Table 1.
Preservation solutions and methods to store the urine samples for 24 hours period.
Table 2.
Numbers of USC clones in different preservation solutions of urine.
Figure 1.
Gravity of different solution and urine samples.
The gravity of nine urine samples from three different donors and five different preservation solutions were determined at room temperature and 24 hours storage in 4 degree. The specific gravity of UW solutions was the highest as 1.052 in room temperature and increased to 1.060 after 24 hours storage in 4 degree which was higher than the urine samples (1.0066 and 1.0080 in room temperature and after stored 24 hours in 4 degree respectively), and the USC culture medium without FBS's specific gravity was the lowest one (1.0055 and 1.0076 in room temperature and after stored 24 hours in 4 degree respectively).
Figure 2.
The cell morphology and cell growth curve of USCs from fresh urine and 24 hr preservation.
(A) Cell morphology of the USCs preserved in culture media in different percentage of serum was similar to that of fresh USCs. (B) Cell proliferation curve showed cell growth pattern of 24-hr preserved USCs that was similar to that of fresh USCs. Images at 100×.
Figure 3.
Cell surface marker expression of fresh USCs and 24-hr preserved USCs assessed by flow cytometry.
Both fresh USCs and 24-hr preserved USC cells at passage 2 were strongly positive for the mesenchymal stem cell markers, such as CD44, CD73, CD90, CD 146 and CD105, and negative for the hematopoietic stem cell markers such as CD31, CD34 and CD45.
Table 3.
Comparison of growth characteristics of urine-derived cells from fresh urine and 24-hours storage urine sample.
Figure 4.
Smooth muscle differentiation of USCs.
(A) Myogenically differentiated USCs from fresh urine and preserved urine expressed smooth muscle markers (such as desmin, myosin smoothelin, calponin and α-smooth muscle actin) that were similar to the ureter SMCs, while few cells displayed the same specific staining in un-induced USCs assessed by immunofluorescent staining. Scale bar = 50 uM. SMCs = ureter smooth muscle cells; USCs = urine derived stem cells. (B) Preserved USCs and fresh USCs 14 days after myogenic differentiation expressed about one-half to two thirds the amount of smooth muscle-specific protein (i.e. smoothelin, desmin and myosin ) compared to control (ureter SMCs) assessed by Western blot analysis. (C) Preserved USCs and fresh USCs displayed a strong contraction to calcium-ionophore A21387 (10−5 M) (61.4% and 60.2%, respectively) similar to the response of ureter SMC (68.9%). Non-induced USCs from fresh or preserved urine specimens lacked responses to calcium-ionophore in contractility analysis.
Figure 5.
Urothelial differentiation of USCs.
(A) Preserved USCs and fresh USCs 14 days after urothelial differentiation expressed urothelial cell markers (uroplakin Ia/III, CK7, CK13 and CK20) that were similar to the urothelial cells, while few uninduced USCs expressed these markers assessed by immunofluorescent staining. Scale bar = 50 uM. (B) Preserved USCs and fresh USCs expressed almost same amount of urothelial-specific proteins (uroplakin Ia/III, CK7, E-cadherin and cingulin) assessed with Western blotting. (C) Preserved USCs and fresh USCs displayed tight junction markers (cingulin and E-cadherin) on cell membrane boundaries (arrow) between cells detected by immunofluorescent staining. Scale bar = 20 uM. (D) Preserved USCs and fresh USCs exhibited tight junction-desmosomes (arrows) between two adjacent cells, whereas non-induced fresh USCs and preserved USCs showed desmosomes when examined by transmission electron microscopy (TEM). (E) A barrier function assay performed by using a fluorescent tracer on confluent cells cultured on inserts. Both preserved USCs and fresh USCs showed similar leakage protection that was significantly better than uninduced USCs (p<0.05).
Figure 6.
Telomerase activity in fresh USCs and 24-hr preserved USCs.
Four of 10 fresh USCs, 5 of 10 preserved USCs (both preserved media with 0.5% and 10% FBS), respectively, showed significant telomerase activity (red bars). TS 8 were the positive control.