Self-Renewal and Differentiation Capacity of Urine-
Derived Stem Cells after Urine Preservation for 24 Hours
Ren Lang1,2., Guihua Liu2., Yingai Shi2, Shantaram Bharadwaj2, Xiaoyan Leng3, Xiaobo Zhou4,
Hong Liu5, Anthony Atala2, Yuanyuan Zhang2*
1Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, People’s Republic of China, 2Wake Forest Institute for Regenerative
Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 3Department of Biostatistical Sciences, Wake Forest School of
Medicine, Winston-Salem, North Carolina, United States of America, 4Radiology/Translational Biology Department, The Methodist Hospital Research Institute, Houston,
Texas, United States of America, 5Center for Bioengineering and School of Electrical and Computer Engineering, University of Oklahoma, Norman, Oklahoma, United
States of America
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 66104cells 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.
Citation: Lang R, Liu G, Shi Y, Bharadwaj S, Leng X, et al. (2013) Self-Renewal and Differentiation Capacity of Urine-Derived Stem Cells after Urine Preservation for
24 Hours. PLoS ONE 8(1): e53980. doi:10.1371/journal.pone.0053980
Editor: Irina Kerkis, Instituto Butantan, Brazil
Received October 16, 2012; Accepted December 4, 2012; Published January 18, 2013
Copyright: ? 2013 Lang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Although preservation of various organs and tissues in
protective solutions at low temperature (4uC) for 24 hours and
cryopreservation of cells in liquid nitrogen have been successfully
established [1,2], the storage of stem cells in body fluid has not yet
been explored. We have recently found stem cells in urine (termed
urine-derived stem cells, or USCs) that possess a high capability for
expansion and multi-potent differentiation properties toward
osteocyte, chrondocyte, adipocyte, myocyte, endothelial and
urothelial cells [3,4,5,6,7]. To develop a reliable method of
preservation of body fluid-derived stem cells, such as USCs,
preservation of stem cells in urine would enable a maximum
amount of high-quality donor cells in a short period of time and
alleviate damage by storing the cells in urine. USCs at earlier
passages have more potential for self-renewal and differentiation;
thus, it would be an advantage to generate more of these cells at
early passages (such as p2 or p3) within a short period of time (7–
10 days). Patients’ urine samples could be transferred from home
to the hospital. This method would also improve cell transporta-
tion or distribution from sites where cell isolation is not
immediately available, to places where cell isolation and cell
culture can be done. USCs can be obtained via a simple, safe, non-
invasive, reliable and low-cost approach, and their use has great
potential for clinical application. USCs might be a viable cell
source for cell therapy and tissue engineering in urology, such as
cell therapy for the treatment of stress urinary incontinence [7,8],
vesicoureteral reflux, or bladder and urethra tissue engineering
[4,9], and in other fields as well.
The purpose of this study was to determine whether USCs still
possess stem cell features and functions after being stored in urine
at 4uC for 24 hours. We determined the total number of cells shed
off from entire urinary tract system into the urine within 24 hours;
we also optimized preservation methods to retain the maximum
number of high-quality USCs. We then characterized the
preserved stem cells 24 hours after urine storage, and compared
them to fresh USCs with regard to cell morphology, cell growth
patterns, population doubling, stem cell surface marker expression,
telomerase activity, karyotypes, myogenic protein marker expres-
sion, contractility of myogenic differentiated USCs, urothelial
protein marker expression, tightness of junctions, and barrier
function of urothelial differentiated USCs.
PLOS ONE | www.plosone.org1January 2013 | Volume 8 | Issue 1 | e53980
Materials and Methods
This study was approved by the Wake Forest University
Institutional Review Board (IRB00014033). Written informed
consents have been obtained and were approved by Wake Forest
University institutional review board.
Collection of Urine Samples
A total of 415 urine samples were collected from 12 healthy
adult men (age range 20–54 years old). Two types of cells were
investigated in this study: i.e. urine derived cells (total numbers of
cells in the urine) and USCs. To determine total numbers of cells
shed into the urine (urine derived cells) in 24 hours, 166 urine
specimens were used on 3 consecutive days (24 h63 d). The cells
were stained with trypan blue and counted. A total of 189 urine
samples were preserved in seven different preservation solutions at
4uC for 24 hours. Group 8 specimens (total of 9) were only
preserved for 12 hours (Table 1). To evaluate the effect of
preservation solution on cell survival and function of USCs, several
solutions were tested: 1) histidine-tryptophan-ketoglutarate (HTK)
solution; 2) University of Wisconsin (UW) solution; 3) culture
media (a mixture of keratinocyte-serum free medium [KSFM] and
embryonic fibroblast medium [EFM]) with a final concentration of
0.5% fetal bovine serum (FBS) for USCs ; 4) culture media with
10% FBS; 5) culture media without FBS; 6) 0.5% FBS alone and
7) culture media with 0.5% FBS for a 12 hour. All preservation
solutions were used at 10% (v/v), e.g. 25 ml solution in 225 ml
voided urine. Urine samples were also stored without any
preservation solution as a negative control. Cells from fresh urine
samples were cultured as a positive control. All donors had no
urinary tract infection.
Specific Gravity of Urine Samples and Preservation
Nine urine specimens from three different donors and the five
different preservation solutions were used to detect specific gravity
at room temperature and after storage at 4uC for 24 hours.
Specific gravity was measured by a Mettler Toledo Densito 30PX
device (Schwerzenbach, Switzerland) following the manufacturer’s
Culture of Urine-Derived Cells
For isolation of cells from fresh urine samples and preserved
urine samples after storage at 4uC for 24 hrs, the urine samples
were centrifuged at 5006g for 5 min at room temperature. The
cell pellets were re-suspended and then plated into 24-well tissue
culture plates (Becton Dickinson, Franklin Lakes, NJ). The
medium used was composed of keratinocyte-serum free medium
(KSFM, Invitrogen, Carlsbad, CA) and embryonic fibroblast
medium (EFM) at a 1:1 ratio with 5% FBS . Urine-derived cells
were isolated and characterized as previously described [3,5].
Numbers of cell clones were counted from each urine sample after
they were cultured for 3 weeks. Cell colonies from fresh urine
samples often appeared as a cluster of 5–12 cells within 5–7 days
after plating on the cell culture wells. However, cell colonies from
24-hr preserved urine samples appeared 7–8 days after being
initially plated. The cells were allowed to grow to a confluence of
50–70% before subculture. A single cell clone per well was used for
further experimentation; further colonies in one well were
discarded. Cell morphology at each passage, population doublings
(PD), cell growth pattern, expression of mesenchymal stem cell
surface markers, relative telomerase activity, and bi-potential
differentiation capability from fresh and 24-hr preserved urine
samples were assayed when USCs were at passage 2.
USCs from fresh and 24 hr preserved culture urine samples (P2)
were seeded in 24-well plates at a density of 1,250 cells/cm2. The
culture medium was changed every other day. Cell proliferation
was measured on days 1, 3, 5 and 7 using an MTS assay
(Promega). Briefly, the MTS reagent was incubated with the cells
in the dark for 1 hour at 37uC. Following the incubation, 100 ml of
the supernatant was transferred to a 96 well micro-plate and the
absorbance was measured at 490 nm using a spectrophotometer
(Molecular Devices Inc, Sunnyvale, CA). Repeated measurements
(n=5) were carried out for each time point. To calculate PD and
doubling time (DT), cell numbers and culture time were counted
at each passage from p0 onwards in fresh and 24 hr preserved
samples. PD and DT were calculated using the following formula:
PD=ln(Nf/Ni)/ln(2); DT=Ct/PD (Nf: Final number of cells,
Ni: Initial number of cells, Ct: Culture time)
Table 1. Preservation solutions and methods to store the urine samples for 24 hours period.
Preservation solutionsStorage Procedures
G1: Fresh urine sample (as control) 10% USC culture media in urine sample with no storage. Cells were harvested and
G2. HTK solution 10% HTK solution in voided urine sample at 4uC for 24 hours (25 ml HTK in 225 ml
G3: UW solution10% UW solution in urine sample at 4uC for 24 hours.
G4: USC culture medium (0.5% serum) 10% USC with 5% FBS culture media in urine sample (0.5% serum) at 4uC for
G5: USC culture media (10% FBS) 10% USC culture media and add extra 9.5% FBS in urine sample (final FBS
concentration is 10%) at 4uC for 24 hours.
G6: USC culture medium (serum free) 10% USC culture media in urine sample (no serum) at 4uC for 24 hours.
G7: 0.5% FBS0.5% serum in urine sample at 4uC for 24 hours.
G8. USC culture medium (0.5% FBS) 10% USC with 5% FBS culture media in urine sample (0.5% serum) at 4uC for
G9: No preservation solution (as control)No preservation solution was used with the urine sample at 4uC for 24 hours.
Characteristic of USCs after Urine Preserved 24 H
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Fresh USCs and 24-hr cultured USCs undergoing exponential
growth at passage 2 were stained with specific anti-human
antibodies labeled for CD45-FITC, CD31-FITC, CD73-PE,
FITC and CD146-PE. Briefly, cells were trypsinized and
5.06105cells were washed and re-suspended in ice-cold PBS
containing 1% bovine serum albumin (BSA). Fluorochrome-
conjugated antibodies (Table S1) were added to cells in 50 ml PBS
containing 3% BSA and incubated on ice for 30 min in the dark.
IgG1-PE, IgG1-FITC, IgG2b-FITC and IgG1- PerCP-CyTM5.5
conjugated isotype control antibodies were used to determine
background fluorescence. Cells were then washed twice in wash
buffer, passed through a 70 mm filter, and analyzed by flow
cytometry (FACS Calibur BD Biosciences, Franklin Lakes, NJ).
A total of 30 USC clones collected from fresh and 24-hr
preserved urine samples (preserved in USC media containing
0.5% serum and 10% serum) were used for this study. Each group
consisted of 10 samples. Whole cell lysates from 26105USCs
(passage 2) were assayed for telomerase activity using the Telo
TAAGG ELISA kit (Roche Applied Sciences, Upper Bavaria,
Germany), according to the manufacturer’s instructions. Telo-
meric repeats (TS) 8 provided in the kit was used as a positive
control. Samples were considered to be positive for telomerase
activity when the difference in absorbance was at least twice that of
the negative control.
Both USC clones from fresh urine samples and 24-hr preserved
samples were analyzed for chromosomal stability at passage 4.
Briefly, the cultured cells were treated with a hypotonic solution
and fixed using methanolacetic acid solution. The metaphase
spread on glass slides were digested using trypsin and followed by
Giemsa staining to generate G bands along each chromosome.
Standard cytogenetic analysis was carried out on the captured
images and karyotyping performed using CytoVision software
(Leica, Buffalo Grove, IL).
Urothelial and Smooth Muscle Differentiation of USCs
To confirm that one single USC clone gave rise to two bladder
cell lineages, smooth muscle cells (SMC, mesoderm) and urothelial
cells (UCs, endoderm), fresh USCs and 24-hr preserved USCs
were used from initially-generated single clones. USCs (p2) were
plated at a density of 1000 cells/cm2and then induced to
differentiation into SMCs and UCs by culture in specific induction
media for 14 days. For myogenic differentiation, equal volumes of
DMEM and EFM containing 2.5 ng/ml transforming growth
factor (TGF-b1) and 5 ng/ml of platelet-derived growth factor
(PDGF-BB) was used . For urothelial differentiation, DMEM
and KSFM mixed at a 4:1 ratio containing 30 ng/ml epidermal
growth factor (EGF) were used . Final serum concentrations
were maintained at 10% and 8% in the myogenic and urothelial
differentiation medium, respectively. The differentiation medium
was replaced every third day. All growth factors were purchased
from R&D Systems (Minneapolis, MN). Cell morphology was
recorded before and after growth factor addition for up to 14 days.
The fresh USCs and 24 hr preserved USCs were induced to
differentiate into UCs or SMCs on 8-well chamber slides (Thermo
Scientific, Waltham, MA) for 14 days and then immunofluorescent
staining with uroepithelial cell markers (Uroplakin Ia and III,
cytokeratin (CK-7, CK-13 and CK-20) or smooth muscle cell
markers (desmin, myosin, a-SM actin, smoothelin, and calponin)
was done (Table S1). The slides were fixed with freshly prepared
4% paraformaldehyde for 15–20 min at room temperature. After
removing excess fixative with PBS washes, the cells were
permeabilized with 0.1% Triton-X100 in PBS for 10 min and
blocked with serum-free block solution (Dako, Denmark) for
15 min. Lineage-specific primary antibodies were diluted appro-
priately in the antibody diluent solution (Dako) and incubated
overnight at 4uC. Unbound primary antibody was removed by
using three times PBS washes. Appropriate secondary antibody
conjugated to fluorescein-isothiocyanate (FITC, Vector Labora-
tories, Burlingame, CA) were used to visualize hybridization
(green) to primary antibody and incubated at room temperature
for 30–45 min in the dark. The slides were mounted using antifade
mounting media (Vector Laboratories) containing propidium
iodide (PI), and images were captured using a Leica upright
microscope (DM 4000B, Germany).
Various cultured cells (non-induced USCs, urothelially differ-
entiated USCs, myogenically differentiated USCs, normal human
ureter urothelial or smooth muscle cells as control) were harvested
from each 10 cm culture dish, respectively, and the proteins were
extracted using RIPA lysis buffer (Thermo) containing proteinase
inhibitor cocktail (Complete mini; Roche Applied Sciences).
Protein extracts (15–20 mg) were run on 8–10% sodium dodecyl
sulfate-polyacrylamide gels to separate the proteins. A pre-stained
protein ladder (Benchmark, Invitrogen) was used to monitor
resolution of protein bands. After electrophoresis, the separated
proteins were transferred to a PVDF membrane (Millipore,
Billerica, MA) by semi-dry transfer. Following transfer, the
membrane was incubated in blocking solution (5% nonfat dry
milk in PBS) for 1 h at room temperature. For protein signal
analysis, the membrane was incubated overnight at 4uC with
primary antibodies (Table S1) in 1% milk in PBS, subsequently
washed with 0.05% Tween and then incubated with the
appropriate diluted HRP-conjugated secondary antibody for
1 hour at room temperature. The blots were visualized with an
enhanced chemiluminescence detection system by an enhanced
chemiluminescence assay (Supersignal West Femto, Thermo
Scientific, Rockford, IL).
Cell-collagen lattice contractility
To measure cell contractile function of smooth muscle
differential USCs in vitro, non-induced USCs, myogenically
differentiated USCs,and normal
(36105cells/mL) were mixed with soluble collagen I (1 mg/mL)
neutralized by NaOH, F-12 and NaHCO3(Sigma) to create a
USC-collagen I solution. An aliquot (250 mL) of the cell-collagen I
solution was placed onto a 35-mm tissue culture plate [5,10],
allowed to set and maintained for 7 days with or without myogenic
growth factors. The cell-collagen lattice was then mechanically
released from the underlying plastic. The diameters of the cell-
collagen lattices were measured before and after releasing
(10 min), and the relative change in diameter calculated. Each
experiment was performed in triplicate. SMCs-lattices released in
the presence of serum (10% FBS) and agonist (10 uM calcium-
ionophore, Sigma), served as positive controls; lattices released
under serum-free conditions served as negative controls. For the
negative controls, cell-collagen lattices were washed twice with
serum-free medium to remove any residual serum and incubated
for an additional 5 minutes before release. The percentage of
human SMCs (control)
Characteristic of USCs after Urine Preserved 24 H
PLOS ONE | www.plosone.org3January 2013 | Volume 8 | Issue 1 | e53980
contraction was calculated as follows: (Du-Dr)/Du X100 (Du and
Dr are the diameters of unreleased lattice and released lattice,
Barrier function of urothelially differentiated USC
To assess the barrier function of urothelially differentiated
USCs, cells were cultured on 0.4 mm trans-well inserts (Becton
Dickinson) and placed in 6-well dishes as reported earlier  with
minor modifications. Briefly, the inserts were coated with collagen-
IV (3 mg/cm2), air dried in a laminar hood and sterilized by a 70%
ethanol rinse. The ethanol was allowed to evaporate completely
before the inserts were used. After USCs (16105/cm2) were plated
in the 6-well plate inserts (top chamber) and maintained for 7 days
with or without epidermal growth factors (EGF). The 0.5 ml of
tracer-containing medium (1 mg/ml, FITC-dextran-FD4, Sigma)
was changed in the insert (top chamber) and 3 ml tracer-free
medium was added in the bottom well. Phenol-free medium was
used to avoid interference of the indicator in the assay. A day
before the tracer was added, the media was supplemented with
2 mM CaCl2solution to promote multilayer formation by the
differentiated USCs. Media aliquots (150 ml) were removed from
the bottom chamber for fluorescence measurements (excitation at
490 nm and emission at 520 nm) after 3 hours. Diffusion of these
dyes through a nude insert membrane served as an internal
control. Non-induced USCs and normal human urothelial cells
were used as negative and positive controls, respectively.
Transmission electron microscopy (TEM)
TEM studies were used to visualize the presence of tight
junctions after induction of USCs to urothelial-like cells. The cells
were cultured and induced as described above on 6-well plates
trans-well inserts, fixed, and sectioned according to standard TEM
protocols . Briefly, the cultured cells were fixed in 2.5%
glutaraldehyde, postfixed with 1% osmium tetroxide, dehydrated
in graded alcohols, embedded in Spurr’s resin (Polysciences,
Warrington, PA), and cut into 80-nm sections with a Reichert
Ultracut E ultramicrotome. The specimens were viewed and
photographed with a Tecnai Spirit BioTwin transmission electron
microscope (FEI, Hillsboro, OR) equipped with an AMT 12
megapixel 2 Vu camera (Woburn, MA).
Values are expressed as mean 6 standard deviation (SD). One-
way ANOVA followed by a Student-Newman-Keuls post hoc test
for multiple comparisons were used when appropriate. Pearson
correlation analyses were used to study the relationship between
total/living cell numbers and age, height. SPSS16.0 software was
used for analyses. P#0.05 was considered as statistically signifi-
The total number of cells and ratio of living cells after 24-
hour preservation in urine
No bacterial contamination was found in any urine samples.
The average total number of cells derived from 166 urine samples
from 12 healthy adult donors was 58,560627,980 cells (range
25,226 to 107,543 cells) in 1,6806846 ml urine in a 24-hr period.
The average total number of living cells in that period was
8,07664,784 (range 3,070 to 15,820 cells), as measured by trypan
blue exclusion. The ratio of live cells to total cells was
13.6564.96% (range 5.96%,22.53%) (Table S2). Pearson
correlation analyses showed that number of living cells in the
urine appeared to be correlated with age (r=0.58, p=0.06). Live
cell/total cell ratio also appeared to be correlated with height
(r=0.56, p=0.07). The boundary statistically significant p values
might be due to the small sample size. No other trend of
association was found.
Preserved USCs at 4uC
The preserved USCs initially appeared as a single cell with a
rice grain-like appearance after the first 3–5 days. They then
formed cell clones with a cluster of 7–8 cell clones, compared to
fresh USCs at 2–3 days for single cells and 5–7 days for the clones.
The average number of USC clones in every 100 ml urine sample
is shown in Table 2. No clones were formed when urine was
preserved in UW preservation solution and preservation-free urine
sample groups. Among all preservation methods, the USC culture
medium with 0.5% serum or 10% serum had the best results
(Table 2). USC clones were able to be obtained from each
individual. The gross success rate for generating USCs was highest
(83%) from fresh samples, and the success rate from urine
specimens stored for 24 hr was 70–73%. Although there were
differentiating cells in each of above groups, the differentiating
cells stopped proliferation, died and were washed away from
culture dishes after 1–2 passages.
Specific gravity of urine samples and preservation
To further study the mechanisms of preservation, the specific
gravity of urine samples and preservation solutions were measured
at room temperature and after being stored 24 hours at 4uC. The
specific gravity of UW solutions was the highest; it was 1.052 at
room temperature and increased to 1.060 after 24-hours storage in
4uC, higher than the urine samples (1.0066 and 1.0080 in room
temperature and after being stored for 24 hours at 4uC,
respectively). The USC culture medium’s specific gravity was the
lowest (1.0055 and 1.0076 at room temperature and after being
stored 24 hours at 4uC, respectively). The higher gravity of UW
solutions might be a reason why no cells survived because of cell
membrane injuries (Fig. 1).
Cell morphology, growth patterns, and cell surface
The cell morphology and growth pattern of the 24- hours
preserved USC clones were similar to those obtained from fresh
USCs (Fig. 2A, B). The cell clones all displayed ‘‘rice grain’’-like
morphology in fresh and 24-hours preserved urine samples. They
had a bright, uniform and compact appearance, although USC
clones appeared 1–2 days later in primary culture from 24-hours
preserved samples than the cells from fresh urine. USCs from fresh
and 24-hours preserved samples all could extensively expand in
vitro. The average PD from passage 0 forward to passage 8 of fresh
USCs was 49.567.2. On the other hand, 24-hours preserved
USCs displayed an average PD of 47.565.8 with 0.5% serum and
46.966.7 with 10% serum to passage 8, respectively. The average
DT was 32.2613.51 hrs in 24-hours preserved samples with 10%
serum, 28.368.55 hrs in 24-hours preserved samples with 0.5%
serum, and 25.666.32 hrs in fresh sample (Table 3). There was no
significant difference between these three groups. When immu-
nophenotyping for surface antigens by flow cytometry was
performed, fresh and 24-hours preserved clonal lines showed
similar results. Both fresh and 24-hours preserved USCs were
strongly positive for the mesenchymal stem cell markers CD44,
CD73, CD90; weakly positive for CD105; and negative for the
hematopoietic stem cell markers CD31, CD34, and CD45.
Characteristic of USCs after Urine Preserved 24 H
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Furthermore, these cells also expressed pericyte membrane marker
CD 146 (Fig. 3).
Bipotent differentiation of preserved USCs
To test whether 24-hr preserved USCs displayed at least
bipotential differentiation capability, single clones of fresh and
preserved USCs (p2) were subjected to myogenic as well as
uroepithelial differentiation conditions for 14 days. A distinct
morphologic change was evident by day 14 in each culture in both
types of cells. The long, spindle-shaped morphology of stromal
cells was evident in the fresh and 24-hours preserved USCs in
myogenic differentiation medium, and a cuboidal phenotype all
appeared in fresh and 24-hours preserved USCs in the uroep-
ithelial differentiation medium. The bipotential differentiation
capacity of fresh and 24-hours preservation USCs was also
confirmed by the expression of specific proteins visualized by
immunofluorescent staining for desmin, myosin, ASMA, calponin,
and smoothelin for myogenic differentiation (Fig. 4A) and
uroplakin-Ia (Up-Ia), Up-III, CK7, and CK13 for urothelial
differentiation (Fig. 5A) and by analysis of the expression of
lineage-specific proteins with Western blotting (Fig. 4B and 5B).
We used cell-collagen contractility analysis to measure the cell
functionality of smooth muscle differentiated USCs (Fig. 4C). After
mechanically releasing the cell-collagen gel lattices, contractile
action was assayed by measuring the diameter before and after
release. The gels mixed with smooth muscle differentiated from
Table 2. Numbers of USC clones in different preservation solutions of urine.
Gross success rate of
generating USC clone (%)
Numbers of cell clones/100 ml urine
Total clones/100 ml
USC clones Differentiating cells
G1. Fresh urine (control)(24 hours storage, n=30) (83%) 25/30 urine samples 6.2964.6 1.3961.47 7.6265.86
G2. HTK solution(24 hours storage, n=30)2.0761.42 0.8560.54 2.6861.93
G3. UW solution(24 hours storage, n=30)000
G4. USC culture medium (0.5% FBS)
(24 hours storage, n=30)
(70%) 21/30 urine samples3.1661.95 0.9860.723.9162.50
G5. USC culture medium (10% FBS)
(24 hours storage, n=30)
(73%) 22/30 urine samples3.3761.290.7360.324.0461.55
G6. USC culture medium (serum free)
(24 hours storage, n=30)
G7. 0.5% FBS(24 hours storage, n=30)1.1960.360.6960.29 1.8660.57
G8. USC culture medium (0.5% FBS)
(12 hours storage, n=9)
G9. No preservation (control)000
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).
Characteristic of USCs after Urine Preserved 24 H
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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
Characteristic of USCs after Urine Preserved 24 H
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fresh and preserved USCs and ureter SMCs showed an average
baseline contraction of 61.4%, 60.2%, and 68.9% in DMEM with
10% fetal bovine serum (FBS) separately, whereas gels from non-
induced fresh and preserved USCs only showed about 8.9% and
16.1% contraction to serum. On the other hand, smooth muscle
differentiated USCs showed a strong contraction to calcium-
ionophore A21387 (1025M)  similar to the response of ureter
SMC (Fig. 4C). Non-induced USCs lacked responses to calcium-
ionophore. Calcium-ionophore A21387 is considered to be a
unique smooth muscle agonist, as it causes a rapid contraction of
smooth muscle cells but not fibroblasts .
To assess barrier function of UC-differentiated USCs, specificity
of tight junction marker expression (E-cadherin and cingulin) was
confirmed by Western blotting (Fig. 5B) and immunofluorescence
staining (Fig. 5C). All tight junction markers stained the cell
membrane boundaries between cells, and staining was even more
pronounced upon induction (Fig. 5C). Using a fluorescent tracer
on cells cultured on inserts revealed at least 50% decrease in
leakage or protection after 3 h in vitro (Fig. 5D). To further
determine the tight junction of urothelial differentiated USC
clones from fresh urine samples and 24-hr mixed USC media
preserved samples, TEM analysis was performed. The desmo-
somes were observed in both UC and induced USC clones and did
not show significant differences, whereas non-induced samples
fresh and preserved USCs did not show desmosomes (Fig. 5E).
Telomerase activity of preserved USC
Relative telomerase activity was measured in 30 single USC
clones from fresh and 24-hours preserved urine samples from one
individual at passage 2, compared to TS8 as positive control. Four
of 10 clones in fresh samples (40%), five of 10 clones in 24-hours
preserved samples with 0.5% serum (50%), and five of 10 clones in
24-hours preserved samples with 10% serum (50%) showed
significant higher telomerase activity, defined as more than twice
the background value (Fig. 6). There were no significant
differences between fresh and 24-hours preserved USCs in relative
telomerase activity assays.
Karyotyping of preserved USCs
Karyotype analysis was performed to test the chromosomal
stability of USC clones from fresh urine samples and 24-hours
preserved samples after serial cultures. Both USC lines all
displayed a karyotype of 1 X and 1 Y chromosome, as expected
for a male donor, and a normal diploid (2n=46) complement of
autosomes. No multiploidy or obvious chromosomal rearrange-
ments at metaphase were detected by Giemsa bandings at passage
4 of both USC clones.
Cell turnover in physiological conditions indicates that differ-
entiated or aged cells are naturally reduced by apoptosis and
replaced by the division progeny of adult stem cells. The rate of
cell turnover depends on each type of tissue or organ . For
example, intestinal epithelium is restored every 3–5 days in
mammals , whereas stratum corneum replacement takes about
14 days and 28 days for replacement of the entire epidermis .
Urothelial cells display a much slower turnover rate of many
weeks, and are basically quiescent until injury. Our hypothesis is
that the more urine derived cells, including urothelial cells, renal
tubule epithelial cells and others can be shed off from the urinary
tract system into urine in the taller individuals due to the body size.
We found a turnover rate of approximately 66104cells in the
urinary tract system over 24 hours, depending on the individual’s
age and height. Cells from older and taller donors shed off more
cells into their urine samples. Although a large of experiment
samples were not performed in the present study, the preliminary
data showed a strong correlation between numbers of shed cells
and individual height and age. Among these total numbers of cells,
the living cells were about 46103cells (13.7% of the total cells in
urine). We used urine from healthy men, since voided urine from
women is more likely to be contaminated with epithelial cells or
vaginal bacteria. In addition, more cells may be present in urine
collected by urethral catheter due to catheter induced injury of the
bladder and urethral mucosa. Therefore, it is easier to collect the
of 24-hr preserved USCs that was similar to that of fresh USCs. Images at 1006.
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.
Characteristic of USCs after Urine Preserved 24 H
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Table 3. Comparison of growth characteristics of urine-derived cells from fresh urine and 24-hours storage urine sample.
Population Doubling with Passage (p)/Doubling Time (DT) in hours
PD/Average DT (hour)
P1/DT P2/DTP3/DTP4/DT P5/DTP6/DTP7/DT P8/DTP9/DT
Fresh Urine 12.2/23.45.7/25.14.2/23.6 4.7/19.84.8/19.34.4/21.6 4.4/26.94.5/31.5 4.2/38.949.567.24/25.666.32
24 hours preserved
12.8/27.9 5.0/28.64.4/15.8 3.5/19.74.6/21.74.7/30.54.7/29.1 3.5/41.1 4.0/40.347.565.86/28.368.55
24 hours preserved
12.2/23.55.1/28.1 4.7/15.84.7/19.9 4.1/23.64.5/31.13.2/50.3 4.0/46.94.1/50.346.966.75/32.2613.51
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 a-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 (1025M) (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.
Characteristic of USCs after Urine Preserved 24 H
PLOS ONE | www.plosone.org8 January 2013 | Volume 8 | Issue 1 | e53980
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.
Characteristic of USCs after Urine Preserved 24 H
PLOS ONE | www.plosone.org9 January 2013 | Volume 8 | Issue 1 | e53980
urine sample and derive more representative cells when cells are
collected from voided urine than via a urethral catheter.
There are various sources of autologous mesenchymal stem cells
[17,18,19], such as bone marrow and adipose tissue. However, our
purpose was to identify a cell source with high self-renewal and
multi-potent differentiation capacities that can be obtained via a
simple and non-invasive approach. We recently found that a
subpopulation of cells isolated from voided urine or urine from
upper urinary tract  possess stem cells features, i.e. are highly
expandable and have multi-lineage differentiation capability
[3,4,5,7,9]. These urine-derived stem cells are capable of
multipotent differentiation to mesoderm lineages (i.e. SMCs
[4,5,7,9] and endothelial cells ) and endoderm lineages such
as urothelial cells [4,5,7,9].
Our previous studies showed that USCs can survive for only a
few hours in urine without any preservation, and eventually all the
cells died within 24 hours after the urine was discharged from the
body. Urine itself is unfavorable for cell survival due the lack of
nutrients, metabolic waste material that might be toxic, osmotic
pressure, and a non-physiological pH value. This toxic environ-
ment impairs cellular membrane and causes cellular lysis. Two
strategies can be used to preserve cells and improve the results of
their necessary storage. One is cryopreservation, to spin down the
cells from urine and freeze them in dimethyl sulfoxide (DMSO) in
liquid nitrogen immediately, which requires the appropriate
facilities to isolate and store cells. The other strategy is to add
the preservation medium in urine and store whole urine samples at
4uC to retain cell membrane stability, slow down metabolic
processes, and prevent cell lysis. The advantage of this approach is
that no special equipment or processes are needed to isolate and
freeze cells, which could be more convenient for patients.
An effective and simple preservation method is greatly
advantageous for accumulating more USCs for necessary storage
or for USC distribution within a restricted time period for
potential clinical application. When large amounts of USCs at
early stages are needed rapidly for cell-based therapy, collecting
more urine samples to preserve living USCs is necessary. MSC at
earlier passages possess greater plasticity and higher growth
potential . It would be ideal for cell therapy to obtain USCs at
as early passage as possible, since cells often lose differentiation
function and proliferation capacity with number of passage in
cultures. Our previous studies demonstrated that 5–10 USC
colonies can be formed per fresh 100 ml urine. Each single USC
clone can generate four million cells at passage 3 within 2 weeks
, and the USCs at early passage can be more efficiently
differentiated into urothelial and smooth muscle cells [4,5,7,9].
It would be beneficial and convenient for patients to collect their
urine samples, refrigerate them at home, and then bring the
samples to the hospital rather than keeping them as inpatients. In
addition, using urine samples stored for 24 hours allows batching
of processing, which minimizes costs and decreases the possibility
of contamination in each step of the cell isolation process. The
ability to preserve cells in urine permits transportation of the cells
if necessary for cell isolation, characterization and culture of USCs
for research or future clinical use.
In this study, we optimized preservation techniques to maintain
the maximum amount of cell viability and optimal stem cell
properties, i.e. self-renewal and multi-potency during cell preser-
vation in urine. In each 100 ml of urine samples, 3–4 USC clones
existed in 24-hours preserved urine, 4–5 clones in 12-hours
preserved urine, and about 6–7 USC clones in fresh urine.
Overall, in about 100 USC clones, nearly 50–80% of fresh USCs
can be preserved from adult urine during 24-hours storage by
these preservation methods. USCs can be obtained from each
donor. Although USCs cannot be obtained from each urine
sample, they can be obtained from about 70% of preserved urine
specimens of each individual. Importantly, the quality of stored
USCs was the same as fresh USCs in each area tested, consistent
with our previous study [6,21]. Like fresh USCs, the preserved
USC clones initially grew a single rice-grain-like cell and then
formed a cluster of 6–9 cells in the initial primary culture. They
underwent more than 47 population doublings compared to fresh
USCs, with nearly 50 population doublings. The preserved cells
expressed mesenchymal stem cell [22,23] and perictye markers
, such as CD44, CD 73, CD90, and CD105, CD146, but not
hematopoietic stem cell markers [25,26] such as CD31, CD34,
and CD45. Half (5 of 10) preserved cell clones had high telomerase
activity and 4 of 10 fresh USCs had telomerase activity; all had
normal karyotypes. Prominently, the preserved USCs maintained
bi-potent differentiation capacity. After storage, differentiated
USCs expressed myogenic-specific genes and proteins [27,28]
such as desmin and myosin contractile function when exposed to
myogenic differentiation medium. They also expressed urothelial
genes and proteins [29,30] such as uroplakin I and III a, and tight
junction genes, proteins such as E-cadherin and cingulin when
exposed to urothelial differentiation medium. Furthermore, the
urothelially differentiated cells possessed tight junction ultra-
microstructure and barrier function.
To optimize preservation conditions, six different solutions were
tested, and USC culture medium with serum was the best
environment for USCs during simple cold storage, compared to
serum alone, culture medium alone or organ preservation
solutions. The numbers of the cells declined markedly using other
solutions. As the densities of cells and preserved medium (.1) are
both slightly higher than urine, the cells sank on the bottle of
containers with medium at 4uC during storage due to gravity.
Most cells were immersed in the culture medium. This might be
why a small amount of preserved medium (about 10–15% of urine
volume) can retain the USC clones.
Maintaining stability of the cellular membrane is critical in
preservation for stem cells in urine, given its limited content of
nutrients and oxygen. Although it is unknown how culture
medium with serum provides cell protection, multiple factors
might be involved in stabilization of the cell membrane. For
example, the culture medium may offer a favorable osmotic
concentration, or the presence of serum and supplements in the
medium (e.g. hydrocortisone and insulin) may help to maintain
cell membrane stability, prevent edema and chemical toxicity .
Therefore, the combination of culture medium and serum
provided better conditions to preserve USC clones. In addition,
the lack of glucose in the medium may prevent cellular
metabolism. Furthermore, hypothermic storage at 4uC reduces
the rate of cell metabolism and oxygen consumption, and thus
reduces cellular impairment.
Compared to organ solutions, culture medium provided better
conditions for cell preservation in these experiments. Although
both HTK Solution [32,33] and University of Wisconsin solution
[33,34] have been successfully used in preservation of donor
kidneys, livers, pancreas, and hearts, in our studies, neither
solution retained USCs better than the USC culture medium. In
addition, both media are very expensive and might not be
recommended to use in preservation of cells in body fluid.
In summary, human USCs are a potential cell source for cell
therapy in the urinary tract system. It would be beneficial to
preserve the USCs or cells in urine and also retain cell quality and
function of USCs during necessary storage and transportation. In
this study, we demonstrated that USCs can be obtained from each
individual’s preserved urine specimens. The cell quality and
Characteristic of USCs after Urine Preserved 24 H
PLOS ONE | www.plosone.org10 January 2013 | Volume 8 | Issue 1 | e53980
function of preserved USCs are the nearly same as the fresh USCs Download full-text
in cell morphology, cell growth patterns, expression of stem cell
surface makers, self-renewal capacity, differentiation capacity,
expression of telomerase activity, and chromosome stability.
Culture media with a minimum of serum added into the urine
significantly increased cell viability and maintained membrane
integrity of USCs compared with cells in organ preservation
solutions. This preservation approach is simple, effective and low
cost, which makes it possible to transport living USCs in the urine,
or possibly or to preserve cells contained in other body fluids for
transportation or for short-term storage.
Details of antibodies used in this study.
urine samples on a series of 3 days. The average total number of
cells derived from 166 urine samples from 12 healthy adult donors
The average number of cells in 24 hour storage of
was 58,560627,980 cells (range 25,226 to 107,543 cells) in
1,6806846 ml urine in a 24-hr period. The average living cells in
that period was 8,07664,784 (range 3,070 to 15,820 cells), as
measured by trypan blue exclusion. The ratio of live cells to total
cells was 13.6564.96% (range 5.96%,22.53%).
The authors would like to thank Karl-Erik Andersson for his valuable
comments and Ms. Karen Klein (Research Support Core, Wake Forest
School of Medicine) for her editorial assistance with this manuscript.
Administrative support: AA. Editorial help: AA. Conceived and designed
the experiments: YYZ. Performed the experiments: RL GL YS SB.
Analyzed the data: RL GL YS SB XL XZ HL YYZ. Contributed
reagents/materials/analysis tools: AA. Wrote the paper: RL GL YYZ.
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