TISSUE-SPECIFIC STEM CELLS

Multipotential Differentiation of Human Urine-Derived Stem Cells:

Potential for Therapeutic Applications in Urology
SHANTARAM BHARADWAJ,a GUIHUA LIU,a YINGAI SHI,a RONGPEI WU,a,b BIN YANG,a,c TONGCHUAN HE,d
YUXIN FAN,e XINYAN LU,f XIAOBO ZHOU,g HONG LIU,h ANTHONY ATALA,a JAN ROHOZINSKI,a,i YUANYUAN ZHANGa
a
Wake Forest Institute of Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North
Carolina, USA; bDepartment of Urology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou,
GuangDong, People’s Republic of China; cDepartment of Urology, Shanghai Tenth People’s Hospital, Tongji
University School of Medicine, Shanghai, People’s Republic of China; dMolecular Oncology Laboratory,
Department of Surgery, The University of Chicago Medical Center, Chicago, Illinois, USA; eJohn Welsh
Cardiovascular Diagnostic Laboratory at Department of Pediatric, Baylor College of Medicine, Houston, Texas,
USA; fClinical Cytogenetics Laboratory, Section of Hematology-Oncology, Department of Pediatrics, Baylor
College of Medicine, Houston, Texas, USA; gRadiology/Translational Biology Department, The Methodist
Hospital Research Institute, Houston, Texas, USA; hCenter for Bioengineering and School of Electrical and
Computer Engineering, University of Oklahoma, Norman, Oklahoma, USA; iDepartment of Obstetrics and
Gynecology, Baylor College of Medicine, Houston, Texas, USA

Key Words. Urine • Bladder • Stem cells • Telomerase • Differentiation • Smooth muscle • Urothelial cells

ABSTRACT
We sought to biologically characterize and identify a
subpopulation of urine-derived stem cells (USCs) with
the capacity for multipotent differentiation. We demon-
strated that single USCs can expand to a large popula-
tion with 60–70 population doublings. Nine of 15
individual USC clones expressed detectable levels of telo-
merase and have long telomeres. These cells expressed
pericyte and mesenchymal stem cell markers. Upon
induction with appropriate media in vitro, USCs differ-
entiated into bladder-associated cell types, including
functional urothelial and smooth muscle cell lineages.
Disclosure of potential conflicts of interest is found at the end of this article.
When the differentiated USCs were seeded onto a scaf-
fold 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, chon-
drogenic, 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. STEM CELLS
2013;31:1840–1856

differentiation [3]. In the clinical setting, the use of autolo-

INTRODUCTION
The ability of stem cells to differentiate into various tissue-
specific lineages is the basis for exploring their use in cell-
based therapies and tissue regeneration. Tissue-specific stem
cells and/or other progenitor cells exist in almost every tissue
and organ of the body [1]. These cells differ from somatic
cells by their ability to differentiate into the many types of
cells required for tissue maintenance, repair, and regeneration
[2]. Once isolated, these stem cells can expand in culture, and
under appropriate experimental conditions, give rise to a
variety of cell types in vitro via induction of lineage-specific
gous stem cells is advantageous, as they do not induce
immune responses and/or rejection. Because tissue-specific
stem cells are a very small subpopulation of cells, they are
difficult to isolate from most differentiated somatic cells pres-
ent in the tissues and organs [4]. In this article, we demon-
strate that a population of stem cells can be easily isolated
from human urine; we term these urine-derived stem cells
(USCs). Single clones of USCs can expand to yield a large
population, and they have the capacity for multipotent differ-
entiation. These cells express a combination of pericyte and
mesenchymal stem cell markers. USCs express detectable lev-
els of telomerase and have long telomeres but did not form

Author contributions: S.B. and G.L.: collection and assembly of data, data analysis and interpretation, and manuscript writing; Y.S.,
R.W., B.Y., T.H., and Y.F.: collection and assembly of data and data analysis and interpretation; X.L., X.Z., H.L., and J.R.: data
analysis and interpretation; A.A.: financial support, administrative support, and provision of study material; Y.Z.: conception and design,
data analysis and interpretation, and manuscript writing; S.B., G.L., Y.S., R.W., B.Y., T.H, Y.F., X.L., X.Z., H.L., J.R., A.A., and Y.Z.:
final approval of manuscript. S.B. and G.L. contributed equally to this article.
Correspondence: Yuanyuan Zhang, M.D. Ph.D., Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine,
Medical Center Boulevard, Winston-Salem, NC 27157, USA. Telephone: 336-713-1189; Fax: 336-713-7290; e-mail: yzhang@
wakehealth.edu Received December 11, 2012; accepted for publication April 21, 2013; first published online in STEM CELLS EXPRESS
May 10, 2013. V
C AlphaMed Press 1066-5099/2013/$30.00/0 doi: 10.1002/stem.1424

STEM CELLS 2013;31:1840–1856 www.StemCells.com

Bharadwaj, Liu, Shi et al.
teratomas in vivo during the 1-month study. In this study, we
have characterized USCs comprehensively and identified multi-
lineage differentiation potential of USCs at gene and protein
expression levels, cellular function, and tissue formation. We
have extended our previously published data to further confirm
the plasticity of USCs in vivo and induced these cells to poten-
tially give rise to different tissue types, systematically analyzed
the function of differentiated cells, particularly urological tis-
sues such as urothelium, smooth muscle, and microvessel.
Obtained via a simple and noninvasive method [5–9], USCs
could potentially provide a virtually limitless supply of autolo-
gous cells for regenerative medicine and tissue engineering
applications, in urology [10–12] and other fields as well.
MATERIALS AND METHODS
Isolation of Human USCs
Approval for this study was obtained from the Wake Forest Uni-
versity Health Sciences Institutional Review Board. To determine
whether USCs can be obtained for cell differentiation from
healthy individuals at different ages, 59 fresh urine samples were
collected from 17 healthy people ranging from 5 to 75 years old.
USCs were isolated and characterized as previously described [6,
8, 9]. Briefly, USCs were grown and expanded in initial/culture
media composed of keratinocyte serum-free medium (KSFM) and
embryonic fibroblast medium (EFM) mixed at a ratio of 1:1 with
5% fetal bovine serum (FBS) [9]. Only wells in 24 plates that
contained single cells were scored and cultured. To keep results
more consistent, cell clones were selected from six donors (3–7
clones per individual) between 28 and 41 years old for further
assessments (Supporting Information Table S1).
Cell Morphology and Proliferation
Cell morphology and proliferation capacity were assessed for
each USC clone from 17 donors. For cell population doubling
(PD) studies, 1,720 cells per centimeter square were plated and
manual counts were performed 3 or 4 days later (70%–85% con-
fluence). The PD and doubling time were calculated. For cell
morphology images, the cells were stained with 0.05% crystal
violet dye.
Fluorescence Activated Cell Sorting Analysis,
Immunofluorescence Analysis, RNA Analysis,
Immunoblotting
Fluorescence activated cell sorting (FACS), immunofluorescence
staining, RNA analysis, immunblotting were used to characterize
the USCs and differentiated USCs according the Supporting In-
formation methods.
Determination of Telomerase Activity
USCs isolated from 28 urine samples, collected from 15 healthy
individuals, were used. Whole cell lysates from 2  104 USCs
(p3) were assayed using a Telo TAAGG ELISA kit (Roche
Applied Sciences, Upper Bavaria, Germany, http://www.roche-
applied-science.com), according to the manufacturer’s
instructions.
Creation of Adenoviruses Expressing BMP2, BMP9,
and Green Fluorescent Protein
The recombinant adenovirus vectors were generated by using the
AdEasy system [13–17]. Briefly, the coding regions of human
BMP2 and BMP9 were polymerase chain reaction (PCR)
amplified and subcloned into the shuttle vector pAdTrack-TO4.
After sequencing verification, the shuttle vectors were used to
generate adenoviral recombinants. Adenoviruses were produced
and amplified in HEK-293 cells. AdBMP viruses also express
www.StemCells.com
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green fluorescent protein (GFP) as a marker for monitoring infec-
tion efficiency. An analogous adenovirus only expressing GFP
(AdGFP) was used as a control [14, 15, 17-24].
Multipotent Differentiation Potential of USCs
In Vitro
In order to characterize the differentiation potential of USC
clones, cells were subjected to the following regimen described
below, and their changes in morphology and/or histochemical
staining for specific components were recorded. The multipoten-
tial capability of independent USC clones was evaluated for vary-
ing levels of response (þ and ) as follows. þþþþ, 80%–100%
differentiated; þþþ, 60%–80% differentiated; þþ, 40%–60%
differentiated; þ, 20%–40% differentiated; 6, 20% or less differ-
entiated; , no differentiation/change in morphology.
Smooth Muscle Cell Induction. USCs were plated at 2,000
cells per centimeter square in smooth muscle differentiation media
containing equal amounts of Dulbecco’s modified Eagle’s medium
(DMEM) (high glucose) and EFM with 10% FBS and the growth
factors transforming growth factor beta 1 (TGF-b1) and platelet
derived growth factor-BB (PDGF-BB) (R&D Systems, Minneapo-
lis, MN, http://www.rndsystems.com) at concentrations of 2.5 and
5 ng/mL, respectively[5]. Cell morphology was recorded before
and after growth factor additions for up to 14 days.
Uroepithelial Induction. USCs were plated at 3,000 cells per
centimeter square in a mixture of medium containing equal
amounts of KSFM and EFM with 2% serum and the growth fac-
tor, epidermal growth factor (EGF) (R&D Systems, Minneapolis,
MN) at 30 ng/mL for 14 days [5].
Endothelial Induction. USCs were plated at a density of
5,000 cells per centimeter square on fibronectin-coated dishes
(2 lg/cm2) and allowed to grow for 2 days to reach subconfluent
levels. Endothelial basal medium (EBM2, Lonza) supplemented
with 50 ng/mL vascular endothelial growth factor (VEGF) was
used to culture USCs for 9 days for induction of endothelial-line-
age differentiation as reported earlier [25].
Neuronal Induction. USCs were seeded in culture dishes
(6,000 cells per centimeter square) and grown in preinduction
medium (PIM) containing DMEM supplemented with 20% FBS
and 10 ng/mL basic fibroblast growth factor (Peprotech, Rocky
Hill, NJ, http://www.peprotech.com). After 24 hours of culture in
PIM, the medium was substituted with nerve induction medium
for a further 48 hours.
Osteogenic Induction. USCs were seeded at a density of
4,000 cells per centimeter square and cultured in serum contain-
ing DMEM low-glucose medium with osteogenic supplements.
Chondrogenic Induction. For high-density cell pellet cultures,
3  105 cells were centrifuged at 600g for 5 minutes in a sterile
15 mL conical polypropylene tube and incubated overnight to
form an aggregate. The culture medium was replaced with 0.5
mL of chondrogenic medium for a total of 28 days. USCs were
also cultured at 5,000 cells per centimeter square on regular tis-
sue culture dishes using chondrogenic medium for 28 days.
Adipogenic Induction. USCs were seeded at a density of
4,000 cells per centimeter square and cultured in serum contain-
ing adipogenic medium.
Skeletal Myogenic Induction. USCs were induced by two
methods for 1 month, respectively: (a) using skeletal muscle sup-
plements and DMEM containing 50 lM hydrocortisone, 0.1 lM
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Figure 1. Schematic isolation of urine-derived stem cell (USCs) and morphology of USC clone with passage. (A): USCs were derived from
the urine by three major steps: isolation, selection, and propagation. (B): A single USC (inset) is followed through different passages (p0–p12).
The cells were expanded to a colony and were cultured in keratinocyte serum-free medium and embryonic fibroblast medium with 5% serum and
images recorded with passage. Images shown at 100.
dexamethasone, 10% FBS, and 5% horse serum was used to cul-
ture USCs; (b) conditioned medium was collected from skeletal
muscle cell culture for 12 hours.
Cell-Collagen Lattice Contractility Assay
To measure cell contractile function in vitro, smooth muscle dif-
ferentiated USCs, noninduced USCs, and human ureter smooth
muscle cells (SMCs) were assessed as reported earlier [26] with
minor modifications (Supporting Information Methods).
Permeability Studies
To assess barrier function, urothelial-differentiated USCs, nonin-
duced USCs, SMCs, and ureter urothelial cells (UCs) were tested.
Cells were cultured on 0.4 lm transwell inserts (Becton Dickin-
son, Franklin Lakes, NJ, http://www.bd.com) placed in six-well
dishes as reported earlier [27] with minor modifications (Support-
ing Information Methods).
In Vitro Tube Formation Assay
Endothelial-differentiated USCs (5,000 cells in 0.1 mL medium)
were seeded on Matrigel and the plates left undisturbed for 18–
20 hours. Bright-field images were then captured for analysis of
network formation. Nontreated USCs served as negative or back-
ground controls with human umbilical vein endothelial cells as a
positive control.
In Vivo Study
Institutional Animal Care and Use Committee approvals for this
project were obtained for animal surgery. Small intestinal submu-
cosa (SIS) as a scaffold was harvested from porcine intestine and
processed as previously described [ 7, 28]. In vitro cell-seeded
scaffolds either with urothelially differentiated USCs or smooth
muscle differentiated USCs were seeded on the mucosal side of
SIS, as previously described [7, 28, 29]. The cells were main-
tained on scaffolds for 14 days and then cell-scaffold constructs
were implanted in 22 immunodeficient female mice for 1 month.
The constructs were harvested and assessed by histology and
immunohistochemistry.
Multipotential of Human Urine Derived Stem Cells
To study the feasibility of adenoviral infection and expression
of transgenes in USCs, the GFP expressing transgene-BMP2
(Group 1), BMP9 (Group 2), or GFP alone (Group 3, control)
were implanted in 15 female athymic nude (nu/nu) mice (4–6
weeks old; Harlan Sprague Dawley, five mice per group). Briefly,
the USCs were seeded at subconfluence and infected with adeno-
viruses expressing GFP-BMP2, GFP-BMP9, and GFP at an multi-
plicity of infection (MOI) of 10. For experiments involving bone
morphogenic protein (BMP) infection, the cells were implanted
as described [15, 16, 23, 24, 30-33]. Briefly, subconfluent USCs
were infected with adenoviruses. At 16-hour postinfection, cells
were harvested and resuspended in phosphate buffered saline
(PBS) for subcutaneous injection (2  106 per injection) into the
flanks of the mice. At 4 weeks after implantation, animals were
sacrificed, and the implantation sites were retrieved for histologic
evaluation.
Histochemical Analysis
For Bone. The implanted grafts with induced USCs were har-
vested after 28 days and fixed in 95% ethanol prior to histochem-
ical staining as described in Supporting Information methods.
For Cartilage. The grafts with induced USCs were histochemi-
cally stained at the end of 21 days after fixing cells with 10% for-
malin. Sulfated glycosaminoglycan (GAG) synthesis was visual-
ized by Alcian blue staining as described in Supporting
Information Methods.
For Fat. The grafted samples were fixed in 4% formaldehyde
solution for 20 minutes at room temperature and incubated after
being stained with 0.5% oil red-O in propylene glycol for
50 minutes. Accumulation of lipid globules was visualized after
extensive water washes under a light microscope.
For the in vivo cell-seeded SIS grafts and collagen scaffolds,
immunohistochemical triple staining was performed by incubating
specific antibodies (Supporting Information Table S3) along with
human nuclear antibody for detection of implanted human cells.
For the BMP-infected USCs, serial sections of the paraffin-em-
bedded samples were deparaffinized, then rehydrated in a
Figure 2. Determination of stem cell surface marker expression in USCs by flow cytometry. (A): USCs at passage three were labeled with
mesenchymal stem cell markers (CD29, CD44, CD54, CD73, CD90, CD105, CD166 and STRO-1). (B): USCs (p3) were labeled with hematopoi-
etic stem cell markers (CD11b, CD14, CD19, CD31, CD34, CD45, and MHC-I and -II). No visible positive labeled cells were observed except
for the MHC-class-I protein. (C): Analysis of USCs for pericyte markers; (i) by fluorescence activated cell sorting (FACS) (CD146, CD140b, and
NG2), (ii) semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the three pericyte markers on different passages of cul-
tured USCs (p2-7). (D): Embryonic stem cell marker detection by (i) FACS analysis (Tra-1-60, Tra-1-81, SSEA4); (ii) real-time PCR (sox2,
oct4, c-myc, and klf4), and (iii) immunofluorescence staining (sox2, oct4, c-myc, and klf4). Representative positive cells stained green at their
membranes (SSEA4) or yellow in their nucleus are shown by arrows. Nuclei were counterstained with PI (red). Inset shows positive control
NCCIT cells. Scale ¼ 50 lm. Abbreviations: BMSC, bone marrow mesenchymal stromal cell; ES cell, embryonic stem cell; GAPDH, glyceralde-
hyde 3-phosphate dehydrogenase; MHC, major histocompatibility complex; NG2, NG2 chondroitin sulfate proteoglycan; NCCIT, embryonic car-
cinoma cell line; PDGF-rb, platelet-derived growth factor receptor beta; PI, propidium iodide; SSEA-1, stage specific embryonic antigen-1; TRA,
tumor related antigen; USC, urine-derived stem cell.
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Figure 3. Telomerase activity and length in USCs. (A): Nine out of the 15 samples tested showed significant telomerase activity (red bars)
over the background. Positive represents lysates prepared from HEK-293 cells; heat-denatured HEK-293 lysate served as the negative control.
(B): DNA extracted from USCs at different passages analyzed for telomere length. Lanes 1 and 2, USCs at passages 2 and 7, respectively; lanes
3 and 4, low and high telomere length controls; lane 5, DNA marker. Abbreviations: BMSCs, bone marrow mesenchymal stromal cells; SMCs,
human bladder smooth muscle cells; USCs, urine-derived stem cells.
graduated fashion, and stained with hematoxylin and eosin,
Alcian blue, and Masson’s trichrome [15, 16, 23, 24, 30-33]. Oil-
red O staining was carried out on the decalcified and frozen sec-
tion samples, as previously described [15, 16, 23, 24, 30-33].
Additionally, to determine the origin of the USCs in urine,
human renal cortex, medullar, renal pelvic and ureter tissues were
assessed with immunofluorescent staining of the pericyte marker
(i.e., CD 146 antibody).
Fluorescence In Situ Hybridization and Amelogenin
Gene PCR Analysis
To determine the origin of the USCs in urine, study of cells with
y-chromosome from sex-mismatched kidneys following renal
transplantation (i.e., male donor-to-female recipient kidney trans-
plant combination) by fluorescence in situ hybridization (FISH)
and amelogenin gene PCR analysis was performed. FISH using
alpha satellite probe for chromosomes X and Yq12 region from
Abbott Molecular (Abbott Park, Illinois, U.S.A. http://www.
abbottmolecular.com) was performed according to the manufac-
turer’s protocol [34] as described in Supporting Information
Methods.
Statistical Analysis
Student’s t test and paired-sample t test were used for statistical
analysis. p values of .05 or less were considered significant. One-
way or two-way ANOVA followed by a Student-Newman-Keuls
post hoc test for multiple comparisons were used when
appropriate.
RESULTS
Characteristics of USCs
Isolation of USCs from urine involves minimal processing
(Fig. 1A). Single, small, compact ‘‘rice-grain’’ like cells was
observed 2–3 days after initial seeding. These cells further
formed clones within an additional 4–6 days (Fig. 1B). FACS
analysis showed that USCs consistently expressed mesenchy-
mal stem cells (MSCs)-like cell surface markers (CD29,
CD44, CD54, CD73, CD90, CD105, CD166 and STRO-1)
(Fig. 2A), but not hematopoietic stem cell markers (CD11b,
CD14, CD19, CD31, CD34, CD45, and MHC-II) except for
MHC-1, endothelial cell markers (CD31), or human leukocyte
antigen(locus)DR (HLA-DR) (Fig. 2B; Supporting Informa-
Multipotential of Human Urine Derived Stem Cells
tion Table S5). Additionally, more than 90% of cultured
USCs and a few cells isolated from fresh urine from a prior
culture expressed pericyte markers (CD146, Fig. 2C).
However, only a few USCs expressed NG2 and PDGF-rb,
although we found the cells consistently displayed the NG2
and PDGF-rb genes in serial passages from p2 to p7 (Fig.2C;
Supporting Information Table S5). USCs expressed the em-
bryonic stem cell markers TRA 1-60, TRA 1-81, and SSEA4,
as well as Sox2, Oct3/4, c-Myc, and klf4. Gene expression
profiles and nuclear protein levels for these markers are
shown in Figure 2D. Importantly, 9 of 15 clones tested
showed telomerase activities more than twice those of the
background levels (0.2 units, p < .005, Student’s t test) (Fig.
3A). An assay of terminal restriction fragments [35] showed
that USCs had long telomeres; the average telomere length
remained constant between passages 2 and 7 (Fig. 3B). How-
ever, both bone marrow mesenchymal stromal cells (BMSCs)
and SMCs displayed telomerase levels below the background
control levels.
Multipotent Differentiation of USCs In Vitro
USCs derived from one single clone (p4) were able to differ-
entiate into smooth muscle lineage (mesodermal origin) and
urothelial lineage (endodermal origin) (Fig. 4A). The differen-
tiation capacity of USCs was repeatable in each USC clone
(six clones per donor) from six donors. (Supporting Informa-
tion Table S1). Myogenic differentiated USCs became elon-
gated and spindle-shaped (Fig. 4B) and expressed at least a
twofold increase in SMC-specific gene transcripts (desmin,
myosin, smoothelin, and a-SMA) upon induction (Fig. 4C,
4D). Up to 80% of induced cells expressed smoothelin, a
smooth muscle-specific protein marker (Fig. 4E–4G).
Although noninduced USCs expressed detectable levels of
a-SMA, the fluorescent intensity of a-SMA increased in a
time-dependent manner upon induction (Fig. 4F, 4G). In cell-
collagen contractility analysis, myogenic-induced USCs and
SMC demonstrated rapid lattice contraction of 24.4% 6 3.5%
and 20.8% 6 1.9%, respectively, which was significantly
higher than the relative contraction occurred in the
noninduced USCs (p < .05) (Fig. 4H).
Urothelial differentiated cells developed a cobblestone-
like morphology (Fig. 4I) and expressed urothelial-specific
transcripts and proteins of uroplakin-III, uroplakin-Ia, and
generic epithelial cell markers CK7 and AE1/AE3 (Fig. 4I–K).
Figure 4. Differentiation of single USC clone into UCs and SMCs. (A): USCs (p3) were used to differentiate into two distinct lineages. Culture
in SMCs-lineage differentiation (2.5 ng/mL TGF-b1 and 5 ng/mL PDGF-BB) and UCs-lineage differentiation (30 ng/mL EGF) medium was used
for 14 days. (B): Morphology of USCs after induction with SMC induction media for 7 and 14 days, respectively. Human ureter SMCs were pos-
itive controls. Scale bar ¼ 100 lm. (C): Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and (D) quantitative real-
time PCR was performed on total RNA using smooth muscle-specific primers (desmin, myosin, aSMA, and smoothelin). Ind-USCs, USCs
induced to myogenic lineage. SMCs RNA was used as a positive control and H2O: negative control. (E): Detection of smooth muscle-specific
proteins by immunoblotting. Specific signals (bands) were seen in lanes with proteins from induced USCs and positive control. (F): Immunofluo-
rescence staining of myogenic induced USCs with time. Ind-USCs-7d and -14d, USCs induced with myogenic medium for 7 and 14 days, respec-
tively. Scale bar ¼ 50 lm. (G): Quantitation of immunofluorescent images for the different markers shown in (F). Differentiation to SMC
lineage significantly increased expression of smooth muscle-specific markers when compared to nontreated USCs as controls. *, p  .05; **, p <
.005. (H): Contractility after myogenic differentiation of USCs. Serum and calcium ionophore promoted significant lattice contraction in SMC
(cross-hatched bars) and induced USCs-collagen lattices (open bars) but not in noninduced USCs-collagen lattice (patterned bars). (I): Morphol-
ogy of USCs after induction with UC induction media following 7 and 14 days. The cell morphology changed from a ‘‘rice-grain-like’’ to a cu-
boidal morphology, which was more pronounced with time. (J): Quantitative real-time PCR (i, ii) and (K) semiquantitative RT-PCR (iii) was
performed on total RNA using urothelial-specific primers (uroplakins-Ia and -III, CK7). Ind-USCs, USCs induced to urothelial lineage. UCs were
used as a positive control and H2O as negative control. (L): Detection of urothelial-specific proteins (uroplakin-Ia, -III, AE1/AE3 and CK7) by
immunoblotting. Specific signals (bands) were seen in lanes with proteins from induced USCs and positive control. (M): Immunofluorescent
staining of urothelial-induced USCs with time. Ind-USC-7d and -14d, USCs induced with urothelial medium for 7 and 14 days, respectively. Spe-
cific marker staining (green) and nuclear staining by propidium iodide (red) are seen. Scale bar ¼ 50 lm. (N): Quantitation of immunofluorescent
images for the different markers shown in (E). Differentiation to an urothelial lineage significantly increased expression of urothelial-specific
markers when compared to nontreated USC controls. *, p  .05; **, p < .005. Functional analysis of urothelial-differentiated USC: (O): Tran-
script analysis by real-time and (P) semiquantitative RT-PCR for functional tight junctions (ZO-1 and E-cadherin). (Q): Immunoblot analysis for
the same tight junction proteins on day 14. (R): Membrane junctions of UC-induced USCs were intensely stained with tight junction antibodies
(ZO-1, occludin, E-cadherin, and cingulin). Inset shows overall staining at a lower magnification. Scale bar ¼ 50 lm. (S): Transmission electron
microscopic analysis for tight junctions in UC differentiated USCs. Arrows indicate position of tight junction; arrow head indicates desmosomes.
(T): Barrier function analysis was performed on USC differentiated to UC-like cells for 3 days. Fluorescein isothiocyanate (FITC)-dextran was
incubated with cells grown on inset; media in the bottom chamber was analyzed after 3 hours. Results were plotted as a percentage of the ‘‘no
cell’’ control (**, p < .05). Abbreviations: ASMA, alpha-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epi-
dermal growth factor; Non-USC, noninduced; PI, propidium iodide; SMCs, authentic human ureter smooth muscle cells; UCs, human ureter uro-
thelial cells as control; USCs Con, noninduced USCs control; Ind-USCs, USCs induced to myogenic lineage, urothelial lineage; Ind-USCs-7d and
-14d, USCs induced with myogenic medium, urothelial medium for 7 and 14 days.
1846
Figure 4.
In addition, more than 90% of USCs expressed urothelial
markers after 14 days of culture, compared to 55%–70% of
USCs that expressed urothelial markers after 7 days of culture
(Fig. 4M, 4N). USCs that expressed urothelial protein markers
increased significantly when the cells were cultured in medium
containing 30 ng/mL EGF (60%–70%), compared to expansion
medium containing 7.5 ng/mL EGF (30%–40%) [9]. In an
assay of cellular barrier function, urothelial differentiated USCs
expressed specific tight junction gene and protein markers (ZO-
1 and E-cadherin) (Fig. 4O–4R). Transmission electron micros-
copy demonstrated the ultrastructure of tight junction formation
between neighboring cells (Fig. 4S). In barrier function assays,
urothelial differentiated USCs showed at least a 60% decrease
in leakage of fluorescent tracer through the insert in vitro, com-
pared to nondifferentiated USCs (Fig. 4T).
Endothelial differentiated USCs exhibited in vitro
‘‘vessel’’ formation when they were plated in culture wells
Multipotential of Human Urine Derived Stem Cells
(Continued)
containing solidified Matrigel (Fig. 5A). In addition, the
differentiated cells expressed endothelial cell-specific gene
transcripts (vWF and CD31), protein markers (CD31, vWF,
KDR, FLT1, and eNOS), and a tight endothelial junction
marker (VE-cadherin) (Fig. 5B, 5C).
Neurogenic differentiated USCs exhibited several neuro-
genic extensions and processes (arrows) (Fig. 5D) and
expressed neuronal transcripts (Fig. 5E). Approximately 40%
of induced USCs expressed the neuronal specific protein
markers nestin, S100, NF200, and GFAP (Fig. 5F).
Skeletal myogenic differentiated USCs displayed an elon-
gated spindle-shaped morphology (Fig. 5G). This change was
accompanied by expression of skeletal muscle-related tran-
scripts (Fig. 5H). Formation of myotube-like structures was
seen in 50%–60% of cells stained for MyoD (Fig. 5I, arrows).
Nuclear staining with antibodies to myogenin revealed
differentiated populations (Fig. 5I).
Bharadwaj, Liu, Shi et al.
Figure 4.
Finally, USCs were also induced to differentiate into
osteogenic, adipogenic, and chondrogenic lineages using pub-
lished differentiation protocols [36]. Adipogenic-differentiated
USCs were positive for oil red-O staining (Fig. 5J-i) and
30%–40% of adpogenically induced USCs expressed adipo-
cyte gene markers including peroxisome proliferator-activated
receptor (PPARc), acetyl-CoA synthase, adiponectin,
CCAAT/enhancer-binding protein a; fatty acid binding protein
4, and lipoprotein lipase (Fig. 5K-i). Osteogenic-differentiated
USCs appeared to produce mineralized tissue as shown via
von Kossa, alkaline phosphatase, and Alizarin Red staining
for calcium deposition (Fig. 5J-ii,iii,iv). Induction of the
osteogenic markers osteocalcin, runt-related transcription
factor-2, and alkaline phosphatase (Fig. 5K-ii) occurred in
70%–80% of osteogenically induced USCs. Chondrogenic-dif-
ferentiated USCs were stained with Alcian blue, toluidine
www.StemCells.com
1847
(Continued)
blue, and safranin-O for GAGs and all were positive (Fig. 5J-
v,vi,vii,viii). About 60% of the induced USCs that gave rise
to the chondrogenic lineage markers expressed Sox9, collagen
II, and aggrecan (Fig. 5K-iii). It appeared that USCs can
highly efficiently differentiate into almost all of the above
cell lineages except chondrogenic differentiation (Supporting
Information Table S1).
This supposition was confirmed with a study performed on
USC clones from three different donor samples (Fig. 5L; Sup-
porting Information Table S1). One of the clones could differ-
entiate to multiple lineages (osteogenic, chondrogenic, adipo-
genic myogenic, urothelial, and endothelial) whereas another
was highly restricted to a few lineages (osteogenic, myogenic,
urothelial, and endothelial). The other clones showed a mixed
response in vivo. This result confirms the presence of cells at
varying stages of differentiation in human urine.
1848
Figure 4.
Possible Renal Origin of USCs
USCs from female recipients of male kidneys showed the
presence of the Y-chromosome when assessed by FISH and
amelogenin gene PCR analysis (Fig. 6A), indicating that
USCs originated from either the upper urinary tract, including
kidney, renal pelvic, and/or upper segment of the transplanted
ureters. To further reveal whether USCs were from kidney or
ureters, we tested for kidney-specific gene expression. The
normal renal cell genes and proteins, CD224, CD13, NR3C2,
Multipotential of Human Urine Derived Stem Cells
(Continued)
CD146, pax2, and pax8, were expressed in cultured USCs from
different individuals (Fig. 6B, 6C). Importantly, USCs
expressed podocytes and parietal cell gene and protein markers
(i.e., synaptopodin and podocin) (data not shown). However,
these genes were not expressed on cultured ureter UCs. Impor-
tantly, using immunofluorescent staining, we found that parietal
cells and podocytes in glomerular tissues and vessels from
human kidney cortex expressed CD146 (Fig. 6D), with no
staining in renal tubule epithelial cells or ureter mucosa.
Figure 5. USCs undergo multipotential differentiation in vitro. (A–C): Endothelial differentiation of USCs. USCs (p3) were induced to endothelial
lineage by culture in endothelial basal medium containing vascular endothelial growth factor (VEGF) 50 ng/mL for 14 days. (A): In vitro vessel forma-
tion. Endothelial differentiated USCs were cultured on Matrigel for 18 hours to form branched networks (angiogenesis) and tubular structures. Scale
bar ¼ 100 lm. (B): Expression analysis of endothelial-specific transcripts by reverse transcriptase polymerase chain reaction (RT-PCR). (C): Immuno-
fluorescence staining using endothelial-specific markers revealed enhanced staining of the markers with differentiation (middle row) compared to the
nontreated control (top row). Scale bar ¼ 50 lm. (D–F): Induction of USCs to neuronal lineage. (D): Cell morphology changed significantly, with cell
extensions present after cells were cultured in neuronal induction medium (arrows). Scale bar ¼ 50 lm. (E): Real-time analysis of neuronal transcript
performed on induced USCs for 3 days. A several fold increase in nestin and NF200 transcripts was observed upon induction. (F): Immunofluorescent
staining using lineage-specific neural antibodies (nestin, S-100, NF200, and GFAP) were used for confirming neuronal induction of USCs. Specific
staining is seen in green and nuclei in red (PI). Scale bar ¼ 50 lm. (G–I): Skeletal myogenic induction of USCs. (G): Cells cultured with skeletal-mus-
cle induction medium caused the cells to change to a spindle shape by day 14. Scale bar ¼ 100 lm. (H): Semiquantitative RT-PCR analysis revealed
that cells expressed specific transcripts after induction. (I): Immunofluorescent staining for skeletal muscle markers (myoD and myogenin) showed
increased specific staining (arrows) in induced cells. Scale bar ¼ 50 lm. (J, K): USCs were induced to multiple lineages using specific media cock-
tails. (J): USCs cultured in different induction media were assessed for differentiation to multiple lineages. (i): Adipogenic differentiation-day 28: oil
red O stain for lipid droplets (enlarged image shown in inset 400). (ii–iv): Osteogenic differentiation-day 28: (ii) von Kossa staining for bone miner-
als; (iii) Alizarin Red staining for calcium deposition; (iv) alkaline phosphatase. (v–ix): Chondrogenic differentiation-day 28: (v) toluidine blue stain-
ing; (vi) Alcian blue staining for GAGs in monolayer (vi) and pellet culture (vii). Safranin-O staining of pellet culture (viii) and in monolayer culture
(ix). Scale bar ¼ 100 lm. (K): Semiquantitative RT-PCR analysis for lineage-specific transcripts: (i) adipogenic; (ii) osteogenic, and (iii) chondro-
genic differentiation. (L): Scoring for multipotential capability of independent USC clones. þþþþ, 80%–100% differentiated; þþ, 60%–80% differ-
entiated; þþ, 40%–60% differentiated; þ, 20%–40% differentiated; 6, 20% or less differentiated; , No differentiation/change in morphology.
Abbreviations: BMSC, bone marrow mesenchymal stromal cells; C/EBP, CCAAT/enhancer-binding protein; FABP, fatty acid binding protein; FLT-
1, fms-like tyrosine kinase-1; eNOS, endothelial nitric oxide synthase; GFAP, glial fibrillary acidic protein; GAPDH, glyceraldehyde 3-phosphate de-
hydrogenase (housekeeping gene); HUVEC, human umbilical vein endothelial cell; KDR, kinase insert domain receptor; LpL, lipoprotein lipase; NF,
neurofilament; PPAR, peroxisome proliferator-activated receptor; runx2, runt-related transcription factor-2; sox-9-SRY, sex determining region Y-
box 9; SkM, skeleton muscle; USC, urine derived stem cell; vWF, von willebrand factor; VE, vascular endothelial.
1850
Figure 5.
Characterization of Differentiated USCs In Vivo
Myogenic differentiated USCs formed multiple layers of
SMCs beneath UC layers in vivo, and the SMCs stained posi-
tively for a-SMA, desmin, and myosin. Scaffolds containing
urothelial differentiated USCs generated stratified layers in
vivo and stained for the uroplakin-Ia and uroplakin-III urothe-
lial markers (Fig. 7A, 7B).
USCs expressing BMP2 and BMP9 formed bony masses,
which were examined using micro CT imaging 4 weeks post-
implantation (Fig. 7C-i). No masses were found in the non-
BMP-infected USC control animals. The isosurface and den-
sity heat maps for the retrieved masses are shown in Figure
7C-ii. Masses were excised from the animals, decalcified, em-
bedded in paraffin, and sectioned for hematoxylin and eosin
staining (Fig. 7C-iii). Sections of these bony masses were also
Multipotential of Human Urine Derived Stem Cells
(Continued)
subjected to Masson’s trichrome (Fig. 7C-iv, top), Alcian blue
(Fig. 7C-iv, middle), and oil-red O staining (Fig. 7C-iv, bot-
tom). Trichrome staining revealed that BMPs induced USCs
to form a highly mature and mineralized osteoid matrix. For-
mation of cartilage-like matrix and chondrocytes was detected
by Alcian blue staining. This staining was more pronounced
in BMP expressing USCs compared to the non-BMP-infected
USC controls (data not shown).
DISCUSSION
A heterogeneous cell population—including differentiated,
differentiating, progenitor, and stem cells—exists in human
Bharadwaj, Liu, Shi et al.
Figure 5.
voided urine. However, of these, only USCs tend to attach to
culture plastic and continuously expand in culture [9]. These
possess MSC-like features such as clonogenicity, self-renewal,
and multipotent differentiation capacity. In this study, we dem-
onstrated that these cells express a whole panel of pericyte/
MSC surface markers, possess telomerase activity, and have
long telomeres. Importantly, USCs from one clone can simulta-
neously differentiate into functional SMCs (mesoderm) and
UCs (endoderm) in a time-dependent fashion and given the
presence of an optimal amount of growth factors. Additionally,
USCs can give rise to osteogenic, chondrogenic, adipogenic,
myogenic, endothelial (mesoderm), and neurogenic (ectoderm)
lineages in vitro. BMP-2 and -9 transduced USCs can form
bone, cartilage, and fat tissue in vivo. Together with our previ-
ous studies [5–9] describing urine-derived progenitor cells, our
data indicate the existence of a stem cell population in urine.
The ability to undergo numerous cycles of cell division
while maintaining an undifferentiated state is associated with
the expression of telomerase in stem cells [37]. Telomerase
www.StemCells.com
1851
(Continued)
activity and telomere maintenance are typical features of
embryonic stem cells, germ cells, some adult stem cells
such as hair follicle stem cells and intestinal stem cells.
Telomerase activity is associated with immortality in cancer
cells and correlates with longer lifespan and extended repli-
cative capacity in cell lines [38]. In this study, telomerase
was detected in 9 of 15 independent USC samples. To mea-
sure telomere length of USCs expressing telomerase activity,
a single USC clone at initial culture generated a large
amount of cells with telomerase activity expression; even
these cells reach passages 7–8. The USCs with the highest
telomerase activity maintained telomere length. In addition,
the USCs retained a complete diploid set of chromosomes
with normal karyotype after several passages. Importantly,
USCs obtained from six individuals did not form any terato-
mas in vivo after being subcutaneously implanted in nude
mice [5-8, 39]. Compared to BMSCs, which usually stop to
grow before passage 10 and reach a PD rate of 30 [40], a
single USC at initial culture can proliferate for up to 20
1852 Multipotential of Human Urine Derived Stem Cells

Figure 6. Determination of USC source. Several clones of USCs (p3) were cultured and analyzed for expression of kidney-lineage markers.
(A): Fluorescence in situ hybridization (left) and amelogenin gene polymerase chain reaction (PCR) analysis (right) analysis of USCs isolated
from urine obtained from a male donor-to-female recipient kidney transplant for presence of Y-chromosome. (B): Real-time PCR analysis using
specific primers (pax8, NR3C2, and L1-CAM) also showed transcript levels similar to those of the kidney cortex tissue and cultured kidney cells.
(C): Immunofluorescent staining using kidney-specific antibodies (CD224, CD13, NR3C2, and pax8) showed specific staining versus the primary
kidney cortex cells. Scale bar ¼ 50 lm. (D): Histology of human kidney cortex. Immunofluorescent staining of podocytes in glomeruli and ves-
sels showed cells positive for CD146 (green). Nuclei were counter-stained with PI (red). Hematoxylin and eosin staining of a serial section
included as a control. Scale bar ¼ 50 lm. Abbreviations: A4, USCs from male donor-to-female recipient urine sample; F, female control; L,
DNA ladder; M, male control; N, negative control; USC, urine derived stem cell.
Bharadwaj, Liu, Shi et al. 1853

Figure 7. In vivo studies. (A): Myogenic differentiation—nontreated and myogenic-differentiated USCs (p3) were analyzed after 4 weeks in
nude mice for smooth muscle-specific markers (desmin, aSMA, and myosin). Lineage-specific staining is seen in green, human nuclei staining in
red, and nuclear staining in blue (DAPI). (B): Urothelial differentiation—nontreated and urothelial-differentiated USCs (p3) were analyzed after
4 weeks in nude mice for urothelial cell markers (Up-Ia and III). Lineage-specific staining is seen in green, human cells nucleus in red, and all
nucleus (mice and human) stained in blue (DAPI). (C): USCs were infected with bone morphogenetic protein 9 (BMP9) or control green fluores-
cent protein and were injected subcutaneously into nude mice. (i): Bony masses were only observed in mice implanted with BMP-transduced
USCs at week 4. (ii): The harvested bony masses were subjected to microCT imaging revealing the isosurface (left) and density heat maps
(right). (iii): The retrieved masses were also sectioned and stained with hematoxylin and eosin. (iv): Mesodermal lineage differentiation of USCs
in vivo. The retrieved bony masses were stained with Masson’s trichrome (top panel), Alcian blue (middle panel), and oil red O (bottom panel).
Magnification, 400. AC, CM, and OM are indicated by arrows. Abbreviations: ASMA, alpha-smooth muscle actin; AC, adipocytes; CM, chon-
droid matrix; DAPI, 40 ,6-diamidino-2-phenylindole; OM, osteoid matrix; USC, urine derived stem cell.

passages and reach a PD rate of 60–70. This indicates that
USCs possess the capacity to generate a sufficient quantity
of cells for potentially use in cell-based therapies. However,
further study is needed to determine whether there is a
difference in numbers of USCs expressing telomerase in
24-hour urine among different age groups.
USC clones can be a unique tool to assess whether a sin-
gle adult stem cell is able to generate a genetically identical
www.StemCells.com
line of stem cells with biological plasticity. Our study demon-
strated that one single USC possesses multipotent differentia-
tion capability; one USC clone with telomerase activity differ-
entiated into endodermal, mesodermal, and ectodermal
lineages. One challenge in urological tissue regeneration is
generating urothelial cells from MSC-derived cells. Only 5%
of BMSCs can give rise to the cells expressing urothelial
markers [41]. In contrast, using the same inductive medium
1854
Figure 7.
as in the BMSC study [6], 60%–70% of the USCs in this
study could not only differentiate into cells expressing uroepi-
thelial cell-specific genes (uroplakin-Ia/III) and protein
markers but also possessed urothelial barrier function and
tight junction ultrastructures. In addition, urothelial differenti-
ated USCs expressed the genes and proteins ZO-1, E-cad-
herin, and cingulin that are associated with tight junctions in
a dose-dependent and time-dependent manner. Although bar-
rier function of the induced USCs did not reach the mature
function of UCs isolated from bladder tissue, it was signifi-
cantly higher than that observed in noninduced USCs, indicat-
ing that USCs possessed stem cell plasticity.
Multipotential of Human Urine Derived Stem Cells
(Continued)
Continuing from the findings of previous studies [9], this
study showed that USCs can efficiently give rise to functional
SMC and urothelial lineage cells. Smooth muscle differenti-
ated USCs expressed smooth muscle markers at both the gene
and protein levels. These markers include: a-SM actin and
calponin (early differentiation markers, but not SMC-specific
markers); desmin and myosin (only displayed in contractile
SMCs); and smoothelin (SMC-specific marker). The mRNA
and protein levels of the induced markers increased
significantly with time in differentiation media. Functional
studies demonstrated that these SMCs have contractile proper-
ties in vitro.
Bharadwaj, Liu, Shi et al.
USCs can differentiate toward mesodermal cell lineages
using two methods: one is through the use of a commercial
inductive media that includes growth factors, and the second
is via gene transfection with BMPs. Several signaling path-
ways, including those activated by the BMPs, regulate lineage
commitment and terminal differentiation of MSCs. Previously,
we conducted a comprehensive analysis of 14 types of BMPs
and compared their abilities to regulate multilineage-specific
differentiation of MSCs[13, 16, 42]. Most BMPs exhibited
distinct abilities to regulate the expression of Runx2, Sox9,
MyoD, and PPARc2 [15]. Further analysis indicated that
BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively
induced both adipogenic and osteogenic differentiation in
vitro and in vivo [15]. In an ongoing study, we are assessing
the relationship between differentiation capacity and expres-
sion of telomerase in USCs.
Identification of the origin of USCs will lead to a better
understanding of the biological role of this multipotent MSC
population within the urinary tract system. Several types of
MSCs could potentially be shed from the kidney or the
urothelial mucosa into the urine. These include BMSCs,
urothelial basal cells, renal tubule epithelial cells, parietal
cells, and pericytes that originate from capillaries of glomeruli
in kidney. Our serial studies involving morphology and/or
growth patterns provided no evidence to suggest that USCs
are actually BMSCs. BSMCs are spindle-shaped and required
culture medium containing 10% serum, whereas USCs do not
proliferate well in this media and require 2%–5% serum. The
maximum number of PDs for BMSCs is about 30, while for
USCs, it can reach up to 60–70. USCs are also not urothelial
basal cells, because USCs cannot grow in serum-free medium
and initially do not express UC markers [5]. USCs isolated
from urine sourced from the upper urinary tract are similar to
voided USCs in morphology, cell phenotypes, growth pattern,
and bipotent differentiation capacity [5]. Importantly, this
study presents strong evidence that the voided USCs originate
from kidney, because cells obtained from female patients who
received a sex-mismatched kidney transplant contained the Y-
chromosome and expressed normal kidney cell gene and pro-
tein markers (CD224, CD13, NR3C2, pax2, and pax8 [43]).
They also expressed the marker CD146, synaptopodin, and
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In conclusion, USCs can be accessed via a simple, non-inva-
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We thank Ms. Karen Klein (Office of Research, Wake Forest
School of Medicine) for her editorial assistance with this
manuscript.
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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