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Nature Reviews Urology
nature reviews urology https://doi.org/10.1038/s41585-023-00787-2
Perspective Check for updates
Body fluid-derived stem cells —
an untapped stem cell source
in genitourinary regeneration
Ru-Lin Huang 1, Qingfeng Li 1, Jian-Xing Ma 2, Anthony Atala 3 & Yuanyuan Zhang 3
Abstract
Somatic stem cells have been obtained from solid organs and tissues,
including the bone marrow, placenta, corneal stroma, periosteum,
adipose tissue, dental pulp and skeletal muscle. These solid tissue-
derived stem cells are often used for tissue repair, disease modelling
and new drug development. In the past two decades, stem cells have
also been identied in various body uids, including urine, peripheral
blood, umbilical cord blood, amniotic uid, synovial uid, breastmilk
and menstrual blood. These body uid-derived stem cells (BFSCs) have
stemne ss properties comparable to those of other adult stem cells and,
similarly to tissue-derived stem cells, show cell surface markers, multi-
dierentiation potential and immunomodulatory eects. However,
BFSCs are more easily accessible through non-invasive or minimally
invasive approaches than solid tissue-derived stem cells and can be
isolated without enzymatic tissue digestion. Additionally, BFSCs
have shown good versatility in repairing genitourinary abnormalities
in preclinical models through direct dierentiation or paracrine
mechanisms such as pro-angiogenic, anti-apoptotic, antibrotic,
anti-oxidant and anti-inammatory eects. However, optimization of
protocols is needed to improve the ecacy and safety of BFSC therapy
before therapeutic translation.
Sections
Introduction
Discovery of stem cells from
body luids
Urine-derived stem cells
Amniotic luid-derived
stem cells
UCB-derived stem cells
Other sources of BFSCs
Mechanisms of BFSC action
Application of BFSCs to treat
genitourinary conditions
Future perspectives and
challenges
Conclusions
1Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong
University School of Medicine, Shanghai, China. 2Department of Biochemistry, Wake Forest School of Medicine,
Winston-Salem, NC, USA. 3Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem,
NC, USA. e-mail: yzhang@wakehealth.edu
Nature Reviews Urology
Perspective
for these cells. For example, implantation of skeletal muscle-derived
stem cells considerably improved quality of life in 16 women with
SUI at a 4-year follow-uptime
24
. Additionally, intra-urethral injection
with adipose tissue-derived stem cells (ADSCs) was shown to be a safe
and feasible procedure, and led to an improvement in the pad test in
3 of 8 men with urinary incontinence after radical prostatectomy, and in 5
of 10 women with SUI
25
. ASCs are usually multipotent but can be also
oligopotent, bipotent or even unipotent. For example, very small
embryonic-like stem cells (VSELs) found in umbilical cord blood (UCB) and
bone marrow express some pluripotency markers such as OCT4, Nanog
and SSEA26. MSCs and HSCs are two unique populations of ASCs. The Inter-
national Society of Cell Therapy defined MSCs as plastic-adherent cells,
isolated from bone marrow and other tissues, that express CD90, CD73 and
CD105 but do not express CD11b, CD14, CD19, CD34, CD45 or HLA-DR
and can differentiate into adipocytes, osteoblasts and chondroblasts
in vitro27. MSCs are gaining popularity owing to the ability of these cells
to repair tissue damage and restore organ function in vivo. For example,
intravenous infusion of allogeneic umbilical cord-derived MSCs showed
promising results in treating patients with heart failure in a phase I/II ran
-
domized controlled trial28. HSCs are lymphoid and myeloid precursors
from bone marrow that can develop into all mature blood cell types
29
.
Following differentiation, HSCs become oligopotent cells and exert
systemic effects by differentiating into mature blood cells after entering
blood circulation30.
Discovery of stem cells from body luids
Stem cells have been identified in various solid tissues (Fig.1a). These
cells are embedded tightly within the extracellular matrix, which
provides a particular microenvironment that enables stem cells to
be maintained in an undifferentiated state
31
. The first step towards
using stem cells for therapeutic purposes is the release of these cells
from the extracellular matrix, which frequently occurs through enzy-
matic digestion
32
. Solid tissue-derived stem cells have been extensively
explored in various laboratory studies and clinical trials, but insur-
mountable limitations related to stem cell isolation from solid tissues
limit the widespread application of these cells. For example, harvesting
fat tissue for ADSCs requires invasive and potentially morbid surgery.
Moreover, collagenase digestion, which is frequently used to detach
ADSCs from extracellular matrix networks, is a double-edged sword
because long-term digestion might affect cell viability but short-term
digestion might be insufficient and lead to waste of precious donor tis-
sues. After digestion, harvested cells form a heterogeneous cell popula-
tion called the stromal vascular fraction, only a tiny portion of which is
ADSCs, and further purification and expansion are usually needed
33
.
Direct use of the stromal vascular fraction has been reported in clini-
cal studies, forexample for knee osteoarthritis
34
, wound healing
35
and
systemic sclerosis36, but concerns regarding efficiency and the presence
of residual enzymes hamper the clinical translation of this tool37.
In addition to solid tissues, stem cells are also present in a series
of ‘liquid tissues’, including urine38, peripheral blood39, menstrual
blood
40
, UCB
41
, synovial fluid
42
, pericardial fluid
43
, amniotic fluid
44
and breastmilk45. These biological body fluids differ in origin, resident
conditions and cellular composition, which have crucial roles in the
phenotypes and biological properties of resident stem cells (Fig.1b
and Table1). We termed these stem cells ‘body fluid-derived stem
cells’(BFSCs). BFSCs have shown superiority over solid tissue-derived
stem cells in terms of accessibility, early isolation, and origin-specific
characteristics and have been used as a stem cell source in animal mod-
els of regenerative diseases
46–48
. First, harvesting body fluid samples
Introduction
The reconstruction of genitourinary organ anomalies caused by con-
genital malformations, diseases and injuries, such as kidney impair-
ment, bladder dysfunction, hypospadias, erectile dysfunction (ED)
and stress urinary incontinence (SUI), is still a challenge. Current treat
-
ments for genitourinary anomalies are insufficient. For example, trans-
plantation of autologous tissues, such as intestinal bowel, remains the
gold-standard therapy for genitourinary tract reconstruction
1–3
. How-
ever, this technique does not enable functional reconstruction owing to
differences in function and structure between the implanted bowel and
the damaged or abnormal bladder tissues1,4. Additionally, allogeneic
organ transplantation, including kidney
5
and uterus transplantation
6
,
has been successfully used to replace failed genitourinary organs in
patients; however, the risk of rejection, the severe shortage of tissue
and organ donors, and the high medical costs limit the widespread
application of this technique
7,8
. Moreover, potential adverse effects and
donor site morbidities might negatively affect patient quality of life8.
Advancements in stem cell research have led to the development
of additional therapeutic options for genitourinary abnormalities.
Stem cells have unique capabilities of primitive self-renewal, infinite
proliferation and multi-differentiation potential towards a variety of
terminal cell types9. Furthermore, stem cells have unique immunomod-
ulatory properties, including anti-inflammatory, immunoregulatory
and immunosuppressive capacity
9
, and are therefore an attractive
therapeutic alternative in genitourinary regeneration. Innovative
stem cell therapies have been extensively explored in the treatment
of various genitourinary diseases and have shown promising results in
preclinical models
9
. However, the clinical translation of stem cell-based
therapies from bench to bedside in genitourinary system regeneration
has lagged, as additional research is needed to optimize the use of
these therapies and to fully understand long-term safety and efficacy
in patients. Thus, a thorough understanding of the essential proper-
ties and curative potential of different stem cells is essential for future
research in regenerative medicine.
Stem cells are categorized into four types based on their differ-
entiation capacity. Totipotent stem cells, originating from the zygote,
have the highest differentiation capacity and can form all the cell
types in an organism
10
. Pluripotent stem (PS) cells, such as embryonic
stem (ES) cells and induced PS (iPS) cells, can differentiate into all three
germ lineages but not into extra-embryonic structures
11,12
. ES cells are
present in the inner cell mass of embryos and are able to generate all
tissues in the body except the placenta12; thus, these cells have the greatest
therapeutic potential. iPS cells are genetically reprogrammed somatic
cells that return to the embryonic-like pluripotent state through forced
overexpression of specific genes
13
. These cells function similarly to ES
cells and might enable researchers to overcome problems associated
with using embryos. Nevertheless, clinical application of ES cells and
iPS cells is challenging owing to bioethical issues, immunological rejec-
tion and teratoma formation
12,14,15
. Multipotent stem cells, such as mes-
enchymal stem cells (MSCs) and haematopoietic stem cells (HSCs), are
found in the developing germ layers or in the respective adult organs.
These cells can self-renew and differentiate into limited populations of
organ-specific cells
16
. Progenitor cells, such as endothelial progenitor
cells (EPCs), are unipotent stem cells with limited self-renewal and the
narrowest differentiation capacity12.
Adult stem cells (ASCs) are located within all organs and tissues,
including adipose tissue17, synovial membrane18, endometrium19, amni-
otic membrane20, umbilical cord21, skeletal muscle22 and bone marrow23
(Fig.1a), and evidence of successful clinical application has been shown
Nature Reviews Urology
Perspective
is quite simple. Urine, amniotic fluid, menstrual blood and UCB are
usually considered clinical waste and can be collected routinely with-
out pain. Other BFSCs residing in blood vessels or joint spaces can be
collected through a minimally invasive puncture. Additionally, body
fluids are relatively abundant. Urine, menstrual blood and breastmilk
(during the lactation period) are regularly discharged by the human
body and can be continually collected. Moreover, BFSCs are easily
separated by centrifugation alone. Characterizing BFSCs immediately
after isolation is important to understand the phenotypic properties
of these cells. Stem cell markers (identified, for example, through
immunofluorescence) are commonly used to distinguish BFSCs from
other cell types in the body fluid. However, a big challenge is the lim-
ited amount of BFSCs available for testing, which can complicate the
conduct of additional parameter assays, such as PCR or western blot-
ting, for rare or low-abundance populations of BFSCs. BFSCs naturally
reside in specific tissues and retain origin-specific differentiation and
immunomodulatory properties, which make these cells particularly
advantageous to repair tissues of the same origin. Indeed, in relation
to other ASCs, BFSCs are more effective in differentiating towards the
tissue of origin than towards other tissues49–51.
In this Perspective, we summarize the origins, phenotypic charac-
teristics, differentiation capabilities and immunomodulatory proper-
ties of different BFSCs. We mainly focus on urine-derived stem cells
(USCs), amniotic fluid-derived stem cells (AFSCs) and UCB-derived
stem cells, highlighting studies in which the therapeutic potential of
these cells was assessed in kidney diseases, ED, bladder dysfunction,
SUI and genitourinary tract tissue engineering. Ongoing challenges
in the clinical translation of BFSCs in genitourinary regeneration are
also discussed.
Urine-derived stem cells
USCs were first identified in human voided urine in 2008 (ref. 38).
Subsequently, USCs were also shown to be present in urine samples
from different animals, including rabbits52,53 and dogs54. The exact
origin of USCs remains controversial. USCs from female recipients
of kidneys from male donors contained the Y chromosome and
expressed kidney cell genes and proteins, indicating that USCs
originated from the donated kidneys and upper segment of the
ureters55. Consistentlywith these observations, USCs expressed
parietal cells and podocyte markers such as CD24, CD44, CD124,
CD133, CD146, synaptopodin and podocin; however, these cells
did not express urothelial cell markers, suggesting that USCs might
not originate from transitional cells located at the mucosa of the
whole urinary tract55. Why renal progenitor cells are present in
the urine of healthy individuals is still unclear. A possible explanation
is that, in a healthy state, parietal epithelial cells can detach from
the interface of the glomerulus to normally replace ageing podo-
cytes and renal tubular epithelial cells56,57. If no podocytes need to
be replaced, the detached parietal epithelial cells shortly persist in
urine in the Bowman space and then exit the kidney with urine as
USCs (Fig.2).
Human adult kidneys produce ~2 l of urine daily, which ensures
large urine samples for USC isolation. Donor age, health status and
sex as well as storage conditions of urine samples affect the quality
and quantity of USCs58. Generally, sterile cryopreserved midstream and
last-stream urine collected from healthy young donors are the best
samples for USC extraction59. However, three urine samples per
donor
38
and fresh urinary samples collected by catheterization are
recommended for isolation of a large number of USCs. For patients
with chronic bladder diseases or cancer, urine samples harvested
from the upper urinary tract are an alternative stem cell source for
bladder tissue regeneration
60
. USCs collected from young individu-
als presented higher cell viability, clonogenicity and proliferation
but lower senescence than cells collected from older donors
61
. Fur-
thermore, the efficiency of USC collection in male donors was higher
than in female donors
62
. The culture contamination rate is higher in
a Tissue-derived stem cells b Body fluid-derived stem cells
ASCs
Multipotent stem cells
Unipotent stem cells
Mesenchymal stem cells from fat, skeletal
muscle, bone marrow, synovial membrane,
amniotic membrane and endometrium
Neural stem cells
Epithelial stem cells from skin, urinary tract
and intestine
Endothelial progenitor cells
Pancreatic progenitor cells Synovial luid
Pluripotent or multipotent stem cells
Urine
Amniotic luid
Umbilical cord blood
Peripheral blood
Menstrual blood
Breastmilk
Fig. 1 | Stem cell types and sources. Classification of different stem cell types
according to tissue origin. Adult stem cells (ASCs) are undifferentiated cells
present in the body throughout most postnatal life. ASCs are self-renewing and
clonogenic but differentiate into only limited mature cell types. According to the
tissue of origin, ASCs are further classified into solid tissue-derived stem cells
(part a), such as bone marrow-derived stem cells, adipose tissue-derived stem
cells and amniotic membrane-derived stem cells, and into body fluid-derived
stem cells (part b) such as urine-derived stem cells, amniotic fluid-derived stem
cells and umbilical cord blood-derived stem cells. Currently, stem cells have been
identified in various body fluids, including urine38, peripheral blood39,
menstrual blood40, umbilical cord blood41, synovial fluid42, pericardial fluid43,
amniotic fluid44 and breastmilk45. Compared with solid tissue-derived stem
cells, body fluid-derived stem cells have shown unique characteristics in
sample harvesting, stem cell isolation, origin-specific differentiation and
immunomodulatory properties and, therefore,have already been recognized
as an alternative stem cell source for the production of extracellular vesicles
and for stem cell therapy and tissue engineering.
Nature Reviews Urology
Perspective
urine samples collected from females than those from males, but the
risk of contamination can be decreased by collecting urine in sterile
conditions63.
USC isolation procedures are simple, quick and reproducible.
USCs can be easily harvested by centrifugation alone and expanded
in plastic plates with a growth medium supplemented with epidermal
growth factor and fetal bovine serum
38
. Several other types of differen-
tiated cells, such as urothelial, endothelial and stromal cells, are present
in urine, but these cells do not have the same self-renewal capacity as
stem cells and cannot survive or proliferate in culture. Conversely,
USCs with self-renewal potential can proliferate in a mixed medium
culture system consisting of keratinocyte serum-free medium and
progenitor cell medium, which is designed to provide the necessary
nutrients and growth factors to maintain stem cell self-renewal capac-
ity. Most USCs can grow in vitro for at least eight passages without
karyotype abnormalities
38,64
but the percentage of stem or progenitor
cells decreases after each passage.
USCs express parietal epithelial cell markers (CD24, CD106, CD133,
CD44, CD146, SIX2, CITED1 and WT1)54,65,66 as well as a repertoire of
surface markers that are similar to those of MSCs. Typically, USCs
express mesenchymal markers (CD73, CD90, CD105)
67,68
and adhe-
sion molecules (CD29 and CD44) but are negative for haematopoietic
markers54,65 and the endothelial cell marker CD31 (refs. 52,65). Addition-
ally, USCs express pluripotency-associated markers, such as TRA-1-60,
TRA-1-81, KIT
69
, SOX2 (ref. 55), MYC
55
, KLF4 (ref. 55), OCT4 (refs. 61,70)
and SSEA4 (refs. 70,71), as reported from several research teams with
different donors and analysis methods. Without induction, USCs do
not express podocyte or renal tubule cell markers, suggesting that
USCs are not fully differentiated renal cells.
After in vitro induction under appropriate conditions, USCs can be
differentiated into all three germ lineages. USCs have shown the capac-
ity to generate cells from multiple mesenchymal derivatives, including
osteogenic64,72, chondrogenic73, adipogenic54,68, endothelial74, skeletal
and smooth muscle cells
75,76
. Ectodermal neural lineages have been
obtained by culturing USCs in neural induction medium
77
. Endodermal
lineages, such as hepatocytes
78
, insulin-producing cells
79
and urothelial
cells52,64,80, have also been successfully obtained by culturing USCs in
specific induction media.
Table 1 | Origin, biological characteristics and applications of BFSCs in genitourinary regeneration
Cell type Origin Biological characteristics Cell isolation Applications in the genitourinary system
USCs Transitional cells
at the parietal cell–
podocyte interface
in the glomerulus55
MSC markers: CD73, CD90, CD105
(refs. 61,67,68,71)
Adhesion molecules: CD29, CD44
(refs. 54,65,66,89)
ES cell markers: low levels of SSEA4
(refs. 55,60,70,71), SOX2 (ref. 55) and
OCT4 (refs. 55,61,70)
Pericyte marker: CD146 (refs. 55,60)
Centrifugation and plastic-adherent
culture38 AKI92,93,193,204, CKD66, kidney transplantation221,
diabetic nephropathy82,94,220, nephropathic
cystinosis301, ED87,91,187,191,207,253,260, SUI259,273,
diabetic bladder dysfunction254, bladder
outlet obstruction282,283, IC196,291,
bladder tissue engineering212, urethral
tissue engineering53,80,210,294, ureter tissue
engineering210
AFSCs Fetal urinary,
respiratory or
gastrointestinal
tracts, skin, amniotic
membranes,
connective tissues99
ES cell markers: OCT4 (ref. 105),
variable expression of Nanog, SOX2,
KLF4, SSEA3, SSEA4, MYC, TRA-1-60,
TRA-1-81 (refs. 98,100)
MSC markers: CD73, CD90, CD105
(refs. 106,107)
Multipotent marker: CD133 (ref. 102)
Adhesion molecules: CD29, CD44
(ref. 106)
Centrifugation and plastic-adherent
culture102; immunoselection for
CD117+ cells directly from amniotic
luid or from adherent cells44;
expansion of unattached cells from
primary amniocytes104
AKI195,197–199,205,222, CKD223–225, diabetic
nephropathy226, ED264, SUI274–276, neurogenic
bladder208,246,250,252, overactive bladder281,
diabetic bladder dysfunction285, IC291
UCB-derived
stem cells Bone marrow HSC markers: CD34, CD45, CD133
(ref. 133)
MSC markers: CD29, CD44, CD73,
CD90, CD105 (refs. 135–137)
EPC markers: CD34, CD31, FLK1 or
KDR, vWF144
ES cell markers: OCT4, SOX2,
Nanog, SSEA3, SSEA4, TRA-1-60,
TRA-1-80 (ref. 146)
MNC, step 1: MNC isolation through
density-gradient centrifugation148
MNC, step 2: further separation
of HSCs from MNCs through
immunoselection for CD34+ or
CD133+ cells149
MSC: plastic-adherent culture150
EPC: growth selection in collagen-
coated plates with EGM-2 (ref. 153)
VSEL: multiparameter cell sorting147
AKI147,194,230,232,234–236, CKD188,201, diabetic
nephropathy231, multidrug-resistant nephrotic
syndrome237,a, ED200,247,248,261,a,262,263,267, SUI277,a,278,
diabetic bladder dysfunction251, neurogenic
bladder255, IC289,290, testicular injury302
PBSCs Bone marrow Same characteristics as
UCB-derived stem cells Same protocols as UCB-derived
stem cells AKI192,238,a,241,244, CKD239,a,240,a,245; renal artery
stenosis202, ED249, SUI279,a
MenSCs Endometrium168,169 MSC markers: CD73, CD90, CD105
(refs. 171,303)
Endometrial MSC markers: SUSD2,
CD140b, CD146 (refs. 171,172)
Variable expression of KIT167
ES cell marker: OCT4 (ref. 171)
Centrifugation and plastic-adherent
culture170; immunoselection for
CD117+ cells from adherent cells167
AKI304
AFSC, amniotic luid-derived stem cell; AKI, acute kidney injury; BFSC, body luid-derived stem cell; CKD, chronic kidney disease; ED, erectile dysfunction; EGM-2, endothelial growth
medium 2; EPC, endothelial progenitor cell; ES, embryonic stem; HSC, haematopoietic stem cell; IC, interstitial cystitis; MenSC, menstrual blood-derived stem cell; MNC, mononuclear
cell; MSC, mesenchymal stem cell; PBSC, peripheral blood-derived stem cell; SUI, stress urinary incontinence; UCB, umbilical cord blood; USC, urine-derived stem cell; VSEL, very small
embryonic-like stem cell. aStudy including human participants.
Nature Reviews Urology
Perspective
Paracrine effects of USCs
USCs have excellent immunomodulatory properties55,65,81. Cul-
tured USCs do not express human leukocyte antigen-DR isotype
(HLA-DR)
65,70,82
, indicating the low immunogenicity of these cells. In
mixed lymphocyte reaction (MLR) assays, peripheral blood mono-
nuclear cell (PB-MNC) proliferation is substantially suppressed by
immunomodulatory cytokines secreted from USCs, including IL-6,
IL-8, MCP1 and granulocyte–macrophage colony-stimulating factor
(GM-CSM)65,67,83. USCs co-cultured with CD4+ T cells effectively inhib-
ited CD4+ T cell proliferation and subsequent T helper 1-mediated
and T helper 17-mediated inflammatory responses
81,84
. Interestingly,
USC-derived extracellular vesicles (USC-EVs) exerted an inhibitory
effect on T cell activation but had a stimulatory effect on B cell function,
namely the activation, proliferation and production of IgM84,85. The
inhibitory effect of USC-EVs on T cell activation is probably ascribable
to the presence of immunomodulatory molecules on the surface of
the EVs or within the EV cargo, as USC-EVs have been shown to contain
molecules, such as transforming growth factor-β (TGFβ), that can sup
-
press T cell proliferation and activation. This evidence suggests that
USC-EVs might have potential therapeutic applications in conditions
such as autoimmune diseases or transplant rejection, in which T cell
activation needs to be controlled. Interestingly, the stimulatory effect
of USC-EVs on B cell function suggests that USC-EVs might also have
a role in enhancing the humoral immune response. The activation,
proliferation and production of IgM by B cells are crucial steps in early
stages of the immune response to an infection or a vaccine. USC-EVs
might contain factors that promote B cell activation and differentia-
tion, although the specific mechanisms are not fully understood. The
immunomodulatory effects of USCs are comparable to those of bone
marrow-derived MSCs (BM-MSCs)65,81,84 but are better than those of
ADSCs83.
USCs have crucial roles in promoting angiogenesis. In vitro,
USCs produce a series of angiogenic growth factors, including
endothelin 1, vascular endothelial growth factor (VEGF), angiogenin,
endostatin and TGFβ1 (ref. 74). Furthermore, USC-EVs contain high
levels of angiogenic growth factors82,86, pro-angiogenic microRNAs87
and angiogenesis-related proteins (DBMT1 (refs. 88,89) and ANGPTL3
(ref. 90)). These USC secretomes have shown therapeutic effects by
promoting angiogenesis and tissue regeneration in animal models of
skin wound healing89, osteonecrosis88 and critical limb ischaemia86.
USCs and USC secretomes exert a protective effect on apopto-
sis. In an in vitro model of diabetic ED, the level of cleaved caspase 3
was markedly elevated in rat corpus cavernosal vascular endothelial
cells treated with advanced glycation end products (AGEs), indicating
that AGE treatment induced apoptosis in these cells91. However, when
corpus cavernosal vascular endothelial cells were co-cultured with
USCs, the increase in cleaved caspase 3 levels observed under AGE
treatment was reversed
91
. Similarly, in in vitro ischaemia–reperfusion
injury (IRI) models, administration of USC-EVs substantially reduced
apoptosis induced by hypoxia and reoxygenation treatment in a
human normal kidney proximal tubular cell line (HK-2 cells)92,93 as
well as podocyte apoptosis induced by treatment with high glucose in
a streptozotocin-induced diabetic nephropathy rat model
82,94
. Results
from these studies provided compelling evidence that USCs inhibit
apoptosis through paracrine effects.
Oxidative stress is a detrimental process that might result in
chronic inflammation and renal fibrosis. Results from in vitro studies
in HK-2 cells showed that USC-EVs substantially reduced oxidative stress
by increasing the activity of superoxide dismutase (an anti-oxidant)
and by reducing the content of malondialdehyde (an oxidative stress
marker) after exposure to IRI93. Besides EVs, USCs secreted a high level
of Klotho, an anti-oxidant and anti-ageing protein, which could suppress
fibrosis in an in vitro model of TGFβ-induced fibrosis in HK-2 cells95.
Autophagy is a beneficial process that helps tomaintain the health
and function of cells in the body by ensuring that damaged or unneces-
sary components are efficiently removed and recycled to regenerate
newer and healthier cells
96
. AGEs activate autophagy pathways, but
excessive accumulation of AGEs was shown to contribute to autophagy
interruption and resulted in the upregulation of lysosomal biogenesis
and function in kidney proximal tubules in a streptozotocin-induced
mouse model of diabetic nephropathy
97
. In vitro, AGE treatment sig-
nificantly (P < 0.05) reduced the expression of autophagy markers in
corpus cavernosum vascular endothelial cells (for example, reduced
ratio of LC3-II to LC3-I and reduced expression of beclin 1); however, in
cells co-cultured with USCs, the levels of these autophagy markers were
elevated, indicating that USCs might protect corpus cavernosum vas-
cular endothelial cells against AGE-induced autophagic dysfunction91.
In streptozotocin-induced diabetic ED rats, the autophagic activity
was decreased in cavernosal endothelium, as shown by a lower ratio of
LC3-II to LC3-I anda lower beclin 1 expression than those observed in
untreated rats; however, intracavernous injection with USCs restored
autophagic activity and improved erectile function in these mice91.
a
b
c
Parietal epithelial cells
Podocyte
USC
Fig. 2 | USCs originating from detached parietal epithelial cells. Parietal
epithelial cells, such as renal progenitor cells, on the outer region of the Bowman
capsule tend to replace aged or injured podocytes56,57 that periodically peel
off from the parietal cell–podocyte interface in the glomerulus or the visceral
layer of the Bowman capsule owing to ageing or physiological conditions.
The detached parietal epithelial cells float in the Bowman space (part a) and are
ready to replace aged or injured podocytes that start to peel off (part b); if no
podocytes need to be replaced, the detached parietal epithelial cells (part c)
shortly persist in the original urine and then leave the kidney through urine as
urine-derived stem cells (USCs).
Nature Reviews Urology
Perspective
In summary, USCs have attracted increasing attention and interest
for use in regenerative therapy owing to the regenerative properties of
these cells as well as the simple, cost-effective and minimally invasive
cell isolation protocols.
Amniotic luid-derived stem cells
AFSCs have intermediate characteristics between ES cells and ASCs.
Indeed, these cells express pluripotency markers and exhibit high
proliferation and multilineage differentiation potential but not tumo-
rigenic potential44,98. The exact origins of amniocytes remain unclear.
These cells are mainly derived from the embryo, including from skin,
amniotic membranes and urinary, respiratory, and gastrointestinal
tracts
99
. Two main stem cell populations have been described in amni-
otic fluid: amniotic fluid-derived MSCs (AF-MSCs) and AFSCs. AF-MSCs
are isolated by plastic-adherent culture of amniocytes in serum-rich
conditions; these cells are abundant and can differentiate towards
mesenchymal lineages
46
. In addition to AF-MSCs, ~1% of amniocytes
are CD117+ (or KIT)+ cells, which were first described in 2007 (ref. 44),
and are referred to as CD117+ AFSCs in this Perspective. CD117+ AFSCs
exhibit extensive multipotency and can give rise to all three germ layer
lineages99.
Technically, the isolation of AFSCs might be achieved from fresh
amniotic fluid specimens of all gestational ages. AFSCs isolated from
amniotic fluid during the first trimester have a higher primitive phe-
notype than those from other trimesters44,98,100, but collection during
the first trimester is difficult owing to limited amniotic fluid volume
during early pregnancy stages and potential risks related to the col-
lection procedure. Thus, collecting AFSCs during the first trimester of
pregnancy is not recommended, hampering the broad clinical applica-
tion of these cells. AFSCs collected during second-trimester routine
amniocentesis98, third-trimester amnioreduction101 or caesarean sec-
tion (end of gestation)
102
maintain relevant therapeutic characteristics.
Currently, three major protocols are available for AFSC isolation. The
first is a one-step protocol based on the plastic-adherent characteristic
of MSCs
102
. The second method is an immunoselection protocol in
which amniocytes are initially selected by plastic-adherent culture and
then further selected for CD117 expression using magnetic-activated
cell sorting (MACS) or fluorescent-activated cell sorting(FACS)44.
CD117+ AFSCs can be directly obtained from amniotic fluid samples, but
this procedure requires large amounts of sample
103
. The third method
is a two-stage protocol in which unattached cells in primary amniocyte
cultures are collected and subsequently cultured104.
AFSCs have an ‘intermediate’ phenotype between pluripotent
ES cells and multipotent MSCs. Regardless of the isolation technique,
OCT4, a specific marker of pluripotency and self-renewal in PS cells,
is the most frequently reported marker in AFSCs
105
. The expression
of other pluripotency markers varies among studies
98,100
. Moreover,
AFSCs express adhesion molecules106, the multipotent marker CD133
(ref. 102) and mesenchymal markers
106,107
but do not express haema-
topoietic markers
106
and the endothelial marker CD31 (ref. 108). The
phenotype and pluripotent potential of AFSCs are influenced by several
factors, including gestational age, selection method, amniotic sample
volume and culture conditions. AFSCs isolated during the first trimes-
ter have a higher primitive phenotype than cells from the second and
third trimesters — human AFSCs from donors in the first trimester share
82% of the transcriptome with human ES cells
100
. Additionally, these
cells express pluripotency markers, including OCT4, MYC, SOX2, Nanog
and SSEA4 (ref. 100), and can generate embryo-like bodies in vitro but
do not form teratomas in vivo100. Conversely, AFSCs collected during
the second trimester do not express KLF4 and Nanog
98
and do not form
embryoid bodies
44
. Moreover, AFSCs from the early gestational period
express high levels of mesodermal and endodermal markers, which
tend to decrease in AFSCs obtained from the late gestational period
109
.
Culture conditions might partly determine AFSC fate; for example,
culturing AFSCs under ES cell conditions enhances the pluripotency
levelof these cells98. Furthermore, AFSCs isolated through CD117+
selection express higher levels of pluripotent markers than AFSCsiso-
latedby plastic-adherent culture. More than 90% of CD117+ AFSCs
express OCT4 (ref. 44) and are capable of forming all three embryonic
germ layers. Conversely, AFSCs isolated by plastic-adherent culture
alone have largely mesodermal potential
110
. Additionally, using a larger
amniotic fluid sample volume was reported to increase the probability
of successfully established AFSC cultures111.
The in vitro mesodermal differentiation potential of human
AFSCs was determined by assessing the differentiation of these cells
into myocytes
112
, chondrocytes
113
, osteoblasts
108
and adipocytes
104
.
The endodermal differentiation potential was shown by measuring
human AFSC differentiation into alveolar epithelial cells
114
, renal tubu-
lar epithelial-like cells
107
and podocytes
115
in vitro. Differentiation of
human AFSCs towards ectodermal lineages was achieved using dif-
ferent culture conditions to facilitate the growth of keratinocytes116,
neurons117 and neural stem cells118.
Immunomodulatory properties of AFSCs
AFSCs have shown immunomodulatory activity and secrete various
chemokines and cytokines that block MLR and inhibit inflammatory
responses and other immune reactions
119–121
. AFSCs are negative for
HLA-DR
113
and the costimulatory molecules CD40, CD80 and CD86
(ref. 122), indicating a low immunogenicity profile. Owing to these
characteristics, AFSCs theoretically have the potential of allogeneic
transplantation in clinical therapy, although, to date, the reported
therapeutic use of these cells in patients is rare. Thus, understand-
ing the immunogenicity profile of AFSCs is essential for developing
effective therapeutic strategies that minimize immune rejection of
the implanted cells, reduce inflammation, and promote tissue repair
and regeneration.
AFSCs have an immunosuppressive phenotype and regulate the
proliferation, activation and effector function of immune cells through
cell-to-cell contact or by secreting soluble factors. In response to
immune system activation, AFSCs secrete immunosuppressive fac-
tors, such as IL-6, IL-10, IL-1Ra, TGFβ, prostaglandin E2, indoleamine
2,3-dioxygenase, nitric oxide, HGF and growth-related oncogenes,
to inhibit effector T lymphocyte proliferation and attenuate subse-
quent inflammatory responses123,124. In addition to T cells, AFSCs exert
inhibitory effects on B cells and natural killer (NK) cells after pretreat-
ment with inflammatory cytokines122. Interestingly, AFSCs modulate
lymphocyte proliferation in a gestational age-dependent manner.
First-trimester AFSCs co-cultured with T cells and NK cells in vitro
could effectively inhibit T cell and NK cell proliferation; however,
second-trimester and third-trimester AFSCs showed reduced immuno-
suppressive effects on these cells
125
. Additionally, the proliferation
of B cells is suppressed only by pro-inflammatory cytokines produced
by second-trimester AFSCs
125
. Consistently with these in vitro findings,
AFSCs have been shown to have anti-inflammatory potential also in
animal models. Pretreatment with human AFSCs ameliorated inflam-
mation in a rat model of lipopolysaccharide-induced sepsis126, in a
mouse model of thioglycollate-induced peritonitis
127
and in a mouse
model of lipopolysaccharide-induced preterm birth128. In these models,
Nature Reviews Urology
Perspective
AFSCs inhibited inflammation-related diseases by forming aggregates
with host immune cells (such as macrophages, T cells and B cells) in the
peritoneal cavity to stimulate innate immunity
126–128
and by increasing
regulatory T cells to aid the acquired immune system127.
Collectively, AFSCs have broad potential and immunomodulatory
properties and lack the ethical and legal concerns of ES cells123; thus,
these cells are promising candidates for application in regenerative
medicine.
UCB-derived stem cells
UCB was considered a waste material before being identified as a
rich stem cell source. Human UCB is obtained through a fast, easy,
and non-invasive procedure, and the collected UCB samples can be
stored for further clinical use without losing viability or function.
UCB-derived stem cells have been used in the clinic as off-the-shelf
products because of the unique featuresof these cells, including high
self-renewal, multipotency, hypo-immunogenicity, non-tumorigenicity
and immunomodulation
129
. For example, allogeneic transplantation
with UCB was performed in paediatric patients with global develop-
mental delay or intellectual disability130, and intracerebroventricular
injection with human UCB-derived MSCs (UCB-MSCs) was conducted
in patients with mild-to-moderate Alzheimer disease dementia131.
Stem cell types present in human UCB
The UCB-MNC fraction contains highly heterogeneous stem and pro-
genitor cells, including EPCs (UCB-EPCs), CD34
+
HSCs (UCB-HSCs),
CD90
+
MSCs (UCB-MSCs) and VSELs, each with unique features and
differentiation potential.
Haematopoietic stem cells. HSCs are a population of highly
self-renewing cells at different haematopoietic commitment stages and
show haematopoietic reconstitution by producing all types of mature
blood cells
30
. Human UCB-HSCs have higher proliferative potential
in vitro than HSCs from bone marrow132. UCB-HSCs are characterized by
a diverse haematopoietic marker expression pattern in different stud-
ies, including CD34, CD45 and CD133 (ref. 133). The stemness properties
of HSCs are associated with the expression of CD34 or CD133, which
are often correlated with a primitive state, quick self-renewal and low
apoptosis rate
134
. Moreover, HSCs expressing CD133 are thought to be
more primitive than HSCs expressing CD34 (ref. 134).
Mesenchymal stem cells. UCB is an important alternative MSC source
to bone marrow. The origin of UCB-MSCs is unknown, but these cells
might be released from the bone marrow or fetal liver into the circu-
lation. Human UCB-MSCs have characteristics similar to BM-MSCs,
including plastic adherence, fibroblastic morphology, MSC immuno-
phenotypes, multipotent capacities and low immunogenicity129. Both
UCB-MSCs and BM-MSCs express mesenchymal markers135–137 and
adhesion molecules136. However, UCB-MSCs have higher proliferation
and clonality capacity and lower expression of senescence markers
than BM-MSCs135,137. UCB-MSCs are inherently capable of differentiat-
ing into mesodermal cells, including chondrogenic
138
, osteogenic
138
,
myogenic
139
and adipogenic
138
cells, as well as into non-mesodermal
cells such as neuronal140, hepatic141 and insulin-producing cell142
lineages.
Endothelial progenitor cells. EPCs are presumed to originate from
bone marrow but are also present in UCB and peripheral blood
143
. EPCs
and HSCs are postulated to arise from a common haemangioblast
precursor and share some cell surface antigens
144
. Currently, no spe-
cific marker is available for defining pure EPCs. Depending on the
isolation methods, at least two types of EPCs have been isolated from
UCB and peripheral blood: early-outgrowth EPCs (or colony-forming
unit-endothelial cells (CFU-ECs)) and late-outgrowth EPCs (or endothe-
lial colony-forming cells (ECFCs))
145
. These cells show distinct mor-
phological, phenotypical and functional characteristics. CFU-ECs are
spindle-shaped and release more angiogenic cytokines than ECFCs,
including VEGF and HGF, and show a tubular structure formation
capacity inferior to that of ECFCs145. Thus, CFU-ECs are thought not
to be actual EPCs but rather pro-angiogenic haematopoietic progeni-
tor cells. Cultured ECFCs show more endothelial cell characteristics,
such as the typical cobblestone morphology and greater nitric oxide
release, proliferation, and incorporation into endothelial networks,
than CFU-ECs145.
Very small embryonic-like stem cells. VSELs are unique pluripotent
non-HSCs that were initially identified from murine bone marrow
and subsequently confirmed to be present in human UCB
26
. VSELs
are unique pluripotent non-HSC stem cells that share embryonic-like
characteristics with ES cells. VSELs are very small (3–5 μm), are highly
enriched in a population of CXCR4
+
CD133
+
CD34
+
LIN
–
CD45
–
cells in
UCB-MNCs, contain large nuclei with unorganized euchromatin, and
express embryonic transcription factors (OCT4, SOX2 and Nanog)
as well as embryonic surface markers146. Additionally, VSELs can dif-
ferentiate into ecto-meso-endoderm lineages147. VSELs might be rep-
resentative of the most primitive stem and progenitor cell population
present in UCB.
Isolation, purification and expansion of UCB-derived stem
cells
UCB can be collected from the umbilical cord during vaginal and
caesarean deliveries. Generally, the collected UCB is processed
using density-gradient centrifugation regents, such as Ficoll-Paque,
to obtain MNCs148. After centrifugation, dead cells and high-density
cells (such as granulocytes and erythrocytes) pass through the Ficoll
reagent, whereas low-density MNCs (such as lymphocytes and mono-
cytes) remain at the blood–Ficoll interphase layer
148
. Subsequently,
the MNC layer is collected to isolate the stem cell population with
specific methods such as immunoselection of CD34
+
cells through
fluorescent-activated cell sorting for HSCs149 or plastic-adherent culture
for MSCs150.
HSCs are typically purified from unselected MNCs based on the
expression of cell surface markers, including CD34 or CD133 (ref. 149).
HSC purification procedures through MACS are relatively rapid and
simple, provide high purity, viability and recovery rates, and can be
applied in a good manufacturing practice-compliant manner149. How-
ever, immunoselection of HSCs based on a single surface marker might
lead to a loss of CD34
–
CD133
+
cells, which have been identified as a very
primitive stage of HSCs in UCB
151
. Additionally, MSCs are present in
extremely low numbers in UCB and have a low isolation efficiency
152
.
The isolation of UCB-MSCs is generally based on the plastic-adherent
propertiesof these cells
150
. Isolation of EPCs and VSELs from UCB is
difficult owing to the low number of these cell populations and to the
absence of specific markers or functional assays. A commonly used
protocol for EPC selection involves culturing unselected or CD34
+
MNCs in collagen-coated plates under endothelial-selective condi-
tions consisting of endothelial growth medium 2 with or without a
combination of endothelial growth factors
153
. The unattached cells
Nature Reviews Urology
Perspective
are removed through medium changes, and the generated adher-
ent colonies are considered EPCs. Several combinations of antigens
have been used to isolate VSELs from MNCs through multiparam-
eter cell sorting, resulting in the isolation of populations such as
CD45−CXCR4+CD133+CD34− cells147.
Immunomodulatory properties of UCB-MSCs and UCB-EPCs
Human UCB-MSCs have very low immunogenicity, as these cells are neg-
ative for HLA-DR and costimulatory molecules
154
. However, UCB-MSCs
exert immunosuppressive effects on lymphocyte proliferation137,
inhibit pro-inflammatory cytokine release135,137,155 and induce periph-
eral tolerance
156
. The mechanism underlying the immunomodulatory
properties of UCB-MSCs in multiple disease states remains elusive.
However, several mechanisms have been proposed to be potentially
responsible for the immunomodulatory activities of UCB-MSCs,
including direct cell-to-cell contact with immunomodulatory cells,
paracrine actions through secretion of immunomodulatory factors,
and generation of regulatory T cells135,137,155. In vitro, UCB-ECFCs are
hypo-immunogenic and showed immunosuppressive properties in
MLR assays through the secretion of immunoregulatory cytokines
such as TGFβ1, IL-10 and HLA-G
157
. These characteristics suggest that
these cells might have curative potential for the management of
vascular-associated diseases.
Other sources of BFSCs
In addition to USCs, AFSCs and UCB-derived stem cells, BFSCs have
been identified in other body fluids, including peripheral blood
39
, men-
strual blood40, breastmilk45, synovial fluid42 and pericardial fluid43.
BFSCs from peripheral blood (PBSCs) and menstrual blood (MenSCs)
are discussed in this Perspective.
Peripheral blood-derived stem cells
Similarly to UCB, peripheral blood also harbours a heterogeneous stem
and progenitor cell population including HSCs
158
, EPCs
144
, MSCs
39
and
VSELs159. PBSCs mainly reside in bone marrow but also migrate in low
numbers into the peripheral blood; in the setting of tissue injury, PBSCs
are mobilized from the bone marrow niche through the peripheral cir-
culation to injury sites160, indicating that these cells participate in the
regeneration of damaged tissues. This physiological phenomenon can
be leveraged by pharmaceutical mobilization, which hyper-stimulates
this process, resulting in a substantial increase in PBSC numbers in
peripheral blood, and has been widely applied in the clinic to collect
sufficient PBSCs for clinical therapy
161
such as for multiple myeloma
162
,
graft-versus-host disease
163
and cartilage repair
164
. PBSCs share biologi-
cal features with UCB, including the immunophenotype, multipotent
differentiation potential and isolation methods. PBSCs have several
advantages over BM-MSCs, ranging from basic research to clinical
applications: PBSCs can be obtained using a mildly invasive procedure;
PBSCscan be extracted in large amounts from autologous peripheral
blood; and peripheral blood is less likely than bone marrow to contain
cancer cells
39
. Moreover, PBSCs are able to rebuild the haematopoi-
etic system. Thus, peripheral blood has gradually replaced bone mar-
row as the standard graft source in allogeneic haematopoietic cell
transplantation165,166.
Menstrual blood-derived MSCs
Menstrual blood is considered a convenient source of blood from
healthy women during reproductive age. Stem cells from menstrual
blood were first identified
40
in 2007 and were subsequently termed
MenSCs
167
. MenSCsinclude mainly unpurified endometrial stromal
fibroblasts, with a minor proportion of endometrial MSCs derived from
shed endometrial tissues168,169. MenSCs are morphologically and func-
tionally similar to endometrial MSCs. Thus, these cells are considered
a surrogate for endometrial MSCs
40
. MenSCs are generally isolated
using density-gradient centrifugation and through plastic-adherent
properties170. Additionally, MenSCs have also been isolated through
MACS-mediated immunoselection of CD117+ cells by several groups167.
MenSCs fulfil the minimum MSC criteria established by the Inter-
national Society of Cell Therapy
171
; these cells also express endometrial
MSC markers, such as SUSD2, CD140b and CD146 (refs. 171,172), and
the pluripotency marker OCT4 (ref. 171) but the expression of SSEA4
and Nanog is under debate. MenSCs show a high proliferative capac-
ityand give rise to all three germ lineages40. However, the differentia-
tion capacities of MenSCs into mesodermal lineages vary depending
on the isolation methods. MenSCs isolated by plastic-adherent cul-
ture have lower osteogenic and adipogenic173 capacity but a higher
cardiomyogenic
174
capacity than BM-MSCs, whereas CD117
+
MenSCs
possess a trilineage differentiation capacity similar to or slightly better
than that of BM-MSCs
167
. Different research groups have also confirmed
the differentiation of MenSCs into non-mesodermal lineages, including
clonogenic neurosphere-like cells175, keratinocyte-like cells176, germ-like
cells177 and insulin-secreting cells178.
MenSCs exert notable immunomodulatory activities and
anti-inflammatory effects primarily through paracrine crosstalk with
innate and adaptive immune cells. Additionally, MenSCs possess a low
immunogenicity profile, as these cells express low levels of HLA-ABC
and do not express HLA-DR
179
. In vitro, MenSCs inhibit differentia-
tionand maturation of dendritic cells from human monocytes through
secretion of IL-6 and IL-10 (ref. 180). In MLR assays, MenSCs inhibit
allogeneic PB-MNC proliferation in a dose-dependent manner
179
. How-
ever, pre-activation of MenSCs with pro-inflammatory cytokines, such
as IL-1β or IFNγ, was shown to exert immunosuppressive effects on the
proliferation of PB-MNCs179 and NK cells181 in vitro, and on the recruit-
ment of macrophages in two mouse models of acute inflammation182
through the upregulation of immunomodulatory factors (including
IL-6 and TGFβ) and the release of EVs179–181,183. However, the immu-
nosuppressive activity of MenSCs seemed to be weaker than that of
BM-MSCs179. Moreover, MenSCs secrete higher amounts of angio-
genic factors, including fibroblast growth factor 2 (FGF2), VEGFA and
platelet-derived growth factor B (PDGFB), than BM-MSCs, and, there-
fore, have greater angiogenic potential both in vitro and in vivo
184
,
providing more effective therapy in the treatment of ischaemic dis
-
eases. Additionally, MenSCs suppress bleomycin-induced apoptosis
in MLE-12 cells (which are immortalized mouse lung type II epithelial
cells) in vitro
185
and ameliorate tissue fibrosis in several animal models
of fibrotic diseases185,186.
Mechanisms of BFSC action
The use of BFSCs as a regenerative therapy in the genitourinary sys-
tem typically relies on three distinct approaches: cell-based therapy,
consisting of local or systemic administration of autologous or allo-
geneic BFSCs to participate in tissue regeneration; secretome-based
therapy, based on the administration of secretomes derived from BFSCs
in various forms, such as EVs187,188, conditioned medium189,190 and cell
lysates
191
, to promote tissue regeneration; and tissue-based therapy,
in which BFSCs are used to engineer functional tissue grafts in vitro
before implantation to replace damaged tissues or organs. In the past
two decades, these BFSC-based approaches have been used in a wide
Nature Reviews Urology
Perspective
variety of preclinical studies to provide therapeutic solutions for geni-
tourinary abnormalities, some of which have also been preliminarily
assessed in preclinical studies (Fig.3).
The mechanisms of BFSC-mediated regeneration in the genitou-
rinary system have not yet been clearly elucidated, but some putative
mechanisms have been proposed. First, BFSCs exert paracrine effects
through the secretion of soluble factors, EVs and hormones to pro-
mote endogenous tissue repair. After local or systemic administra-
tion, BFSCs restore tissue functions and participate in endogenous
tissue repair by modulating the local microenvironment through
the release of bioactive factors
89,192,193
, induction of autophagy
194–196
,
cell-to-cell contact
135,137,155
or a combination of these proposed mecha-
nisms. Results from a growing number of studies in animal models
of genitourinary and other conditions have shown that BFSCs exert
therapeutic regenerative effects mainly by releasing paracrine bioac-
tive factors with immunomodulatory
93,197–199
, pro-proliferative
82,200
,
pro-angiogenic82,86,87, antifibrotic185,186,201,202, anti-oxidative93,199,203 and
anti-apoptotic82,92–94,195,197,204,205 properties. Moreover, BFSCs also pro-
duce bioactive molecules to recruit host stem cells and immune cells
to promote endogenous tissue regeneration182,206,207. Additionally,
injection with BFSC-derived secretome, EVs and conditioned medium
achieved similar outcomes to those observed with BFSC injection in
terms of tissue repair in animal models, which also suggests a paracrine
effect of BFSCs in tissue regeneration187–190. Thus, BFSC-based cell
therapy, through both systemic and local administration approaches,
exerts tissue regeneration and repair effects in the genitourinary
system mainly through paracrine effects.
Direct cell differentiation or engraftment can also mediate injured
tissue repair. BFSCs migrate or home to the site of injury and differenti-
ate into specific cell types or directly engraft into the host tissues to
repair injured tissues. However, although cell engraftment has been
observed around the damaged tissue after implantation of BFSCs in
several animal models (such as an IRI-induced CKD rat model66 and a
spinal cord injury-induced bladder dysfunction model
208
), the number
of grafted cells was shown to decrease substantially with time66,204,208,209.
Moreover, direct evidence that supports tissue repair through differen-
tiation is currently scarce. For instance, intracavernous injection with
PKH-26-labelled UCB-MSCs could effectively restore erectile function
and increase smooth muscle amount in a rat model of ED induced by
cavernous nerve injury; however, grafted PKH-26-labelled UCB-MSCs
could be detected in corpus cavernosum but did not colocalize with the
endothelial cell marker CD31 and the smooth muscle cell marker αSMA,
indicating minimal cell differentiation and engraftment in vivo200.
Another potential use of BFSCs is tissue engineering of the geni-
tourinary tract, in which BFSCs are pre-differentiated into functional
urothelial cells, smooth muscle cells and endothelial cells in vitro
Cell
proliferation
Cell
dierentiation
Engraftment Angiogenesis induction Fibrosis inhibition lmmunosuppression Autophagy
activation
Anti-oxidation Apoptosis
inhibition
Mechanisms
Approaches
BFSCs
VEGF PGE2
IL-6
HGF
NO
IDO
TGFβ1
Cell-based therapy Secretome-based and
exosome-based therapy Tissue-based therapy
Fig. 3 | Regenerative mechanisms of BFSCs. Body fluid-derived stem cells
(BFSCs) can be used as a regenerative therapy through three distinct approaches:
cell-based therapy, secretome-based therapy and tissue-based therapy. The
injected BFSCs can migrate or home to the injury sites and repair tissue damage
through direct engraftment or differentiation into functional cells (cell-based
therapy). Increasing evidence supports the hypothesis that BFSCs might exert
therapeutic effects through the local or systemic release of extracellular vesicles
or soluble factors to promote tissue regeneration (secretome-based therapy).
Moreover, BFSCs can be used to engineer functional tissue grafts in vitro before
implantation to replace damaged tissues or organs (tissue-based therapy).
The reported mechanisms of action of BFSCs in the three approaches include
promotion of cell proliferation72,211, increase of angiogenesis82,86,87, autophagy
activation91,195,225, immunosuppression93,197–199, fibrosis inhibition185,186,201,202,
apoptosis inhibition82,92–94,195,197,204,205 and anti-oxidant effects93,199,203. Additionally,
BFSCs engrafted within scaffolds and differentiated can be used in the
reconstruction of dysfunctions in genitourinary organs such as bladder and
urethra. IDO, indoleamine 2,3-dioxygenase; PGE2, prostaglandin E2; TGFβ1,
transforming growth factor-β1; VEGFA, vascular endothelial growth factor A.
Nature Reviews Urology
Perspective
before implantation53,210 or undergo in vivo differentiation and matu-
ration after implantation with or without controlled release of growth
factors after implantation72,211,212.
Moreover, mitochondrial transfer from stem cells to injured
cells has been proposed as a novel therapeutic strategy to promote
tissue repair and regeneration213 but has not yet been well investi-
gated in BFSCs. Mitochondrial impairment and dysfunction have
been observed in a variety of genitourinary disease models in rats
such as cisplatin-induced renal tubulointerstitial fibrosis201, diabetic
nephropathy
214
and lipopolysaccharide-induced acute kidney injury
(AKI)
194
. In a study in which MenSC transplantation was performed
in a rat model of myocardial infarction, mitochondrial transfer was
observed between injected MenSCs and host cardiomyocytes and
endothelial cells, which promoted host cell survival following myo-
cardial infarction and subsequently improved cardiac function215,
suggesting that BFSC mitochondrial transfer can happen and could
be explored as a potential repair method for genitourinary conditions.
Application of BFSCs to treat genitourinary
conditions
BFSCs, especially USCs, AFSCs and UCB-derived stem cells, are eas-
ily accessible and, therefore, have been investigated as potential
therapeutic agents for genitourinary diseases (Fig.4 and Table2).
Kidney diseases
The global burden of kidney diseases is substantial and growing:
~10–15% of hospital admissions are caused by AKI
216
, whereas ~10% of
adults are affected by chronic kidney disease (CKD), which results in
1.2million deaths per year worldwide
217
. Overall, more than one in seven
adults in the USA (15%, corresponding to 37 million people) are esti-
mated to have CKD218. To date, no breakthroughs have been achieved
in treating kidney diseases except for renal transplantation and haemo-
dialysis. With the emergence of stem cell therapy, transplantation of
BFSCs has been hypothesized to be a promising alternative to improve
renal function in the treatment of various kidney diseases.
AKI is characterized by tubular injury and an abrupt decrease in
kidney function. Severe, recurrent and uncontrolled AKI progresses
towards CKD or end-stage kidney disease219. The administration of USCs
was shown to be effective in reducing renal damage, improving renal
function and inhibiting renal tissue fibrosis in various animal models
of AKI
92,93,204
, CKD
66
and diabetic nephropathy
82,94,220
. Additionally,
USCs exert renoprotective effects through anti-inflammatory and
anti-apoptotic activities. For example, in a rat model of AKI induced by
cisplatin, an intravenous infusion with USCs significantly decreased the
levels of blood urea nitrogen (BUN) (P < 0.001) and serum creatinine
(sCr) (P < 0.01). Moreover, after USCs administration, the expression
of pro-inflammatory cytokines (IL-6 (P < 0.05) and TNF (P < 0.05))
and apoptosis-related proteins (cleaved caspase 3 (P < 0.01) and Bax
(P < 0.05)) was significantly reduced in renal tissues from these animals
compared with phosphate-buffered saline-treated controls
204
. Fur-
thermore, results from studies carried out in different rodent models
of kidney injury, such as diabetic nephropathy
82,94
and IRI
93,193
, showed
that intravenous infusion with USC-EVs or exosomes could effectively
protect renal function in these animals by suppressing tubular epi-
thelial cell and podocyte apoptosis
82,92,94
, suppressing ferroptosis
193
,
promoting glomerular endothelial cell proliferation82, and inhibiting
inflammatory cell infiltration93. Interestingly, the injected USCs were
detected in the tubular epithelial lining204, indicating that USC injection
might have improved renal histological damage through a paracrine
effect as well as through direct cell engraftment or differentiation
into renal cells.
AFSCs were shown to exert a renoprotective effect superior to that
of BM-MSCs and ADSCs in a cisplatin-induced rat model of AKI
203
. Intra-
venous administration of human AFSCs in rodent models of AKI led to
rapid normalization of kidney function as indicated by decreased levels
of sCr and BUN as well as by a reduced number of damaged tubules195,199;
similar results were observed in animal models of IRI197,198,205,221. AFSC
infusion was shown to delay renal interstitial fibrosis and prolong
animal survival in several animal models of IRI222, CKD223–225 and dia-
betic nephropathy
226
. AFSCs accelerate kidney function and structure
recovery in the acute injury phase by inhibiting apoptosis and pro-
moting the proliferation of tubular cells195,197, modulating the inflam-
matory response
197–199
, reducing oxidative stress
199,203
and activating
autophagy195 in rat and mouse models of AKI and CKD induced by IRI
or cisplatin treatments. In a mouse model of Alport syndrome in which
chronic fibrosis and inflammation eventually lead to CKD, injected
AFSCs were detected in the injured kidney but did not differentiate
into tubular epithelial cells or podocytes225, suggesting a local parac-
rine action that is probably based on the release of cytokines, growth
factors and EVs. For example, results from in vitro studies showed that
cultured human AFSCs secrete high levels of pro-regenerative fac-
tors, such as VEGF, IL-6, IGF1 and SDF1 (refs. 102,227), and EVs197,228,229,
which might potentially protect renal tissue from injury and induce
endogenous regeneration under various pathological conditions such
as glomerular endothelial cell damage229 and cisplatin-induced renal
tubular damage227.
UCB-MNCs are the most frequently investigated UCB stem cells
in experimental and clinical trials owing to the abundance and simple
retrieval of these cells. Injection with human UCB-MNCs was shown to
significantly decrease levels of sCr and BUN in rat models of AKI (both
P < 0.01)230 and diabetic nephropathy (both P < 0.05)231 and to prevent
AKI progression to renal tubular interstitial fibrosis in rats
201
. The under-
lying mechanism might be related to anti-inflammatory, anti-oxidant
and anti-apoptotic effects as well as to increased autophagy in the
kidney, all of which have been shown in lipopolysaccharide-induced
or cisplatin-induced AKI rat models receiving infusion with
UCB-MNCs194,201. Moreover, UBC-MNC treatment in a diabetic nephropa-
thy rat model was shown to decrease blood glucose levels and improve
renal function
231
. The renoprotective effects of MNCs are mediated
by the heterogeneous stem cell populations in UCB. In a study in which
the therapeutic effects of CD34
+
cells and MNCs from human UCB were
assessed in a rat model of glycerol-induced AKI, both cell treatments
improved renal function, but the therapeutic effects of UCB-MNCs
were superior to those of UCB-CD34+ cells with regards to the levels of
BUN, serum urea and sCr232; these results indicated that different cell
types in UCB-MNCs might exert a synergistic effect on improving renal
function. However, the effect of UCB-derived CD133
+
EPCs on renalfunc-
tion in pathological conditions remains contradictory. In a mouse
model of IRI-induced AKI, infusion with UCB-derived CD133+ cells led
to exacerbated tubular injury and augmented kidney dysfunction
compared with CD133− cell treatment233. These deleterious effects
were probably ascribable to the release of TNF observed after CD133+
cell injection, which increased the inflammatory response and tissue
injury233. Conversely, results from a study in a rat CKD model induced
by adenine-enriched diet showed that intravenous infusion with EVs
derived from UCB CD133+ cells substantially improved renal function
in terms of increased serum albumin and cystatin C
188
. Additionally,
injection with human UCB-MSCs also showed a protective effect towards
Nature Reviews Urology
Perspective
gentamicin or IRI-mediated AKI induction in rats through the release of
paracrine factors234 and EVs235. However, the injected UCB-MSCs were
also detected around tubules of the injured kidney in a cisplatin and gen-
tamycin injection-induced AKI canine model236, indicating that direct
engraftment or differentiation into renal tissue might also contribute
to the renoprotective effects of these cells. In a cisplatin-induced AKI
mouse model, VSEL‐derived renal progenitors induced endogenous
tubular cell proliferation, showing potential for renal regeneration147.
• Cell engraftment
↑ Tubular cell proliferation
↓ Epithelial cell and podocyte apoptosis
↓ Ferroptosis
↓ Oxidative stress
↓ Fibrosis
↑ Angiogenesis
↓ Inlammation
↑ Autophagy
a AKI and CKD
• USCs
• UCB-MNCs
• UCB-VSELs
• CD133+ EPCs
• PB-CD34+ cells
• MenSCs
• AFSCs
• UCB-MSCs
• UCB-CD34+ cells
• PB-MNCs
• PB-EPCs
↓ Apoptosis
↓ Oxidative stress
↓ Fibrosis
↑ Angiogenesis
↑ Autophagy
↑ Neurogenesis
↑ Myogenesis
c ED
• USCs
• USCPEDF
• AFSCs
• UCB-MSCs
• UCB-EPCs
• USCFGF2
• USCBDNF
• UCB-MNCs
• UCBSCs
• PB-EPCs
• Myogenic dierentiation
• Urothelial dierentiation
d Urinary tract defect
• USCs
• Cell engraftment
↓ Oxidative stress
↓ Fibrosis
↓ Inlammation
↓ Muscle cell apoptosis
e Overactive bladder
• USCs • AFSCs
↓ Fibrosis
↓ Oxidative stress
↓ Inlammation
↑ Neurogenesis
f Neurogenic bladder
• AFSCs • UCB-CD34+ cells
↓ Oxidative stress
↓ Fibrosis
↓ Apoptosis
↑ Neurogenesis
g Diabetic bladder
• USCs • AFSCs • USB-MNCs
• Cell engraftment
↓ Fibrosis
↓ Inlammation
↓ Apoptosis
↑ Angiogenesis
h Interstitial cystitis
• USCs • AFSCs • USB-MSCs
• Cell engraftment
↑ Host satellite cell proliferation and
↑ dierentiation
↑ Myogenesis
↑ Neurogenesis
↑ Angiogenesis
b SUI
• USCs
• UCB-MNCs
• Pre-dierentiated AFSCs (myogenic,
neurogenic and endothelial cells)
• AFSCs
• PB-TNCs
Fig. 4 | Cell-based therapy with BFSCs in genitourinary regeneration. Body
fluid-derived stem cells (BFSCs) from different sources used in the treatment of
genitourinary pathological conditions and possible regenerative mechanisms
are shown (results were obtained mainly in preclinical models). a, Urine-derived
stem cells (USCs), amniotic fluid-derived stem cells (AFSCs), umbilical cord
blood (UCB)-derived stem cells (UCBSCs), peripheral blood-derived (PB) stem
cells (PBSCs) and menstrual blood-derived stem cells (MenSCs) prevent acute
kidney injury (AKI) and ameliorate chronic kidney disease (CKD) by promoting
tubular cell proliferation82,147,192,195,197,205, enhancing angiogenesis241,247–249,
activating autophagy194,195,201,225, inhibiting epithelial cell and podocyte
apoptosis82,92,94,204, suppressing ferroptosis193, suppressing oxidative stress199,203
and inflammation93,192,197–199,233,246, and reducing tissue fibrosis in animal
models192,207,208,250–255. Moreover, in several studies, direct cell engraftment in
the injured kidney was also observed in mouse, rat and canine models of AKI
and Alport syndrome204,225,236. b, USCs, AFSCs, UCBSCs and PBSCs promote
stress urinary incontinence (SUI) recovery by inducing endogenous satellite
cell proliferation and differentiation273, and by enhancing myogenesis274–276 ,
neurogenesis275,276 and angiogenesis275 in mouse and rat models of SUI. Cell
engraftment was also observed in the host sphincter muscle after AFSC274,276
and UCBSC277 treatment. c, USCs (including genetically modified USCs),
AFSCs, UCBSCs and PBSCs) restore erectile function in animal models of
erectile dysfunction (ED) through anti-apoptotic187,207,253, anti-oxidation200,262,
antifibrosis207,253,260, pro-angiogenesis87,187,249,253, pro-neurogenesis200,247,263,267, pro-
myogenesis187,200,207,247,253 and pro-autophagy effects91. d, Undifferentiated or pre-
differentiated USCs can be seeded onto porous matrix or scaffolds to engineer
biological grafts for bladder212, urethra53 and ureter210 reconstruction. e, In mouse
and rat models of overactive bladder, USCs and AFSCs improve voiding parameters
by inhibiting oxidative stress281, inflammation281, and fibrosis282,283 and suppress-
ing muscle cell apoptosis282. Cell engraftment is observed in the injured bladder
wall after AFSC therapy281. f, Direct transplantation of AFSCs and UCB-derived
CD34+ cells into the bladder wall ameliorates bladder dysfunction in rat models
of neurogenic bladder by inhibiting inflammation246, suppressing oxidative
stress246, reducing fibrosis208,250,252,255 and promoting neurogenesis208,246,250,252,255.
g, USCs, AFSCs and UCB-derived mononuclear cells (UCB-MNCs) restore
cystometric parameters by inhibiting oxidative stress285, cellular apoptosis254
and tissue fibrosis251,254 and promoting neurogenesis285 in rat models of diabetic
bladder. h, USCs, AFSCs and UCB-derived mesenchymal stem cells (UCB-MSCs)
reduce tissue inflammation289–291 and fibrosis289,290, inhibit cellular apoptosis289,
and increase angiogenesis290 in the bladder wall of rat models of interstitial
cystitis; grafted UCB-MSCs were observed in the stromal and epithelial tissue
of the injured bladder in these models289,290. EPC, endothelial progenitor cell;
TNC, total nuclear cell; VSEL, very small embryonic stem cell; USCBDNF, USC
transfected with BDNF gene; USCFGF2, USC transfected with FGF2 gene; USCPEDF,
USC transfected with PEDF gene.
Nature Reviews Urology
Perspective
Table 2 | Preclinical application of BFSCs in the treatment of genitourinary abnormalities
Disease Cell type Experimental design Mechanism of action Highlighted results Ref.
AKI Human USCs or
USC-EVs Intravenous USC or USC-EV
injection after IRI-induced AKI
in rats
Action: exosome-mediated
secretion of factors
Mechanism: anti-oxidant,
anti-inlammatory,
anti-apoptotic effects
Both USC and USC-EV treatments led to
reduction in sCr, BUN, renal tubular cell apoptosis
and inlammatory cell iniltration; injected USCs
trapped in lung tissue, not found in the kidney;
paracrine effect instead of renal differentiation
93
Human UCB-MNCs Intravenous UCB-MNC or
NRF2 inhibitor injection after
LPS-induced AKI in rats
Mechanism: autophagy
induction, anti-oxidant,
anti-inlammatory,
anti-apoptotic effects
Reduction of inlammation, ROS, apoptosis and
mitochondrial damage; inhibition of NRF2 activity
led to weakened protection of human UCB-MNCs;
renoprotective effect through the NRF2 pathway
194
Human PB-EPCs Intravenous PB-EPC injection
after IRI-induced AKI in mice Mechanism: anti-apoptotic,
anti-inlammatory, anti-
oxidant, antiibrotic effects
Reduction in sCr, BUN, tubular injury, tubular
epithelial cell apoptosis, oxidative stress,
inlammasome activation, inlammatory cell
iniltration and renal ibrosis
244
CKD Human USCs Intrarenal USC injection after
gentamicin and IRI-induced
CKD in rats
Action: cell engraftment
Mechanism: anti-oxidant,
anti-inlammatory,
antiibrotic effects
Reduction of sCr, glomerular sclerosis, atrophic
renal tubules, ibrosis, monocyte iniltration;
increased GRF; amount of injected USCs
substantially decreased post-injection but
persisted within renal tubules for the entire study
period
66
Rat PB-EPCs Transfusion of autologous
PB-EPCs in the renal artery
and penile vein in a rat CKD
model induced by subtotal
nephrectomy plus renal arterial
ligation
Action: cell engraftment
Mechanism:
pro-angiogenic,
anti-oxidant,
anti-inlammatory,
antiibrotic and
anti-apoptotic effects
Reduction in sCr, BUN, kidney injury score,
apoptosis and ibrosis; injected EPCs engraft to
interstitial, peritubular and glomerular areas of
the kidney on day 46
245
Diabetic
nephropathy Human USC-EVs Intravenous USC-EV injection
after streptozotocin-induced
diabetic nephropathy in rats
Mechanism: proliferative,
pro-angiogenic,
anti-inlammatory,
antiibrotic and
anti-apoptotic effects
Reduction of urine volume, urinary microalbumin
excretion, podocyte and tubular epithelial cell
apoptosis; increased glomerular endothelial
cell proliferation; blood glucose levels not
improved; USC-EVs contained potential factors
promoting angiogenesis and cell survival
82
ED Human UCB-MSCs
or ADSCs Intracavernous UCB-MSC or
ADSC injection after bilateral
CNI-induced ED in rats
Action: secretome-mediated
release of factors
Mechanism: anti-oxidant,
anti-apoptotic and
proliferative effects
Both UCB-MSC and ADSC treatments led to
increased ICP-to-MAP ratio, smooth muscle-to-
collagen ratio, nNOS and desmin; both
UCB-MSCs and ADSCs did not colocalize with
endothelial cells and smooth muscle cells in the
corpus cavernosum; UCB-MSCs showed better
therapeutic effects than ADSCs
200
Human USCs Co-culture of USCs with
AGE-treated CCECs in vitro;
intracavernous USC injection
after streptozotocin-induced
diabetic ED in rats
Mechanism: autophagy
activation, anti-apoptotic
effect
In vitro: increased ratio of LC3-II to LC3-I, beclin1,
autophagosomes, cleaved caspase 3; reduced
p62 and PCNA In vivo: increased ICP, ICP-to-
MAP ratio, CD31, eNOS, phospho-eNOS, VEGFA,
VEGFR2
91
SUI Endothelial,
myogenic and
neurogenic
differentiated
human AFSCs
Single (myogenic), double
(myogenic and neurogenic) or
triple (myogenic, neurogenic
and endothelial) cell injection
into the urethral sphincter
region after pudendal nerve
transection-induced SUI
in mice
Action: cell engraftment
Mechanism:
pro-angiogenesis,
pro-neurogenesis,
pro-myogenesis
Triple-cell combination treatment showed the
highest amount of new striated muscle ibres and
neuromuscular junction formation compared with
single-cell and double-cell treatments; no sign of
inlammation and teratoma formation; injected
AFSCs could be tracked in vivo for up to 14 days
after injection
276
Bladder outlet
obstruction Human USC-EVs Intrabladder USC-EV injection
after urethra ligation-induced
bladder outlet obstruction
model in mice
Action: exosome-mediated
release of factors
Mechanism: antiibrotic
effect
Reduction in bladder weight-to-body weight ratio,
collagen volume fraction, αSMA, collagenIII;
NRF1 carried by USC-EVs ameliorated bladder
ibrosis
283
Overactive
bladder Human AFSCs Intravenous AFSC
administration in an
atherosclerosis-induced
overactive bladder model
in rats
Mechanism: anti-oxidant
and anti-inlammatory
effects
Increased voided volumes, intercontraction
and intervals; reduced residual volumes, wall
thickness of iliac artery, bladder wall arterioles,
8OHdG, MDA, TNF
281
Nature Reviews Urology
Perspective
However, clinical studies to evaluate the therapeutic effects of UCB
stem cells in patients with kidney diseases are scarce. To the best of
our knowledge, only one trial in which UCB-MSCs were used as an
immunosuppressive therapy for the treatment of multidrug-resistant
nephrotic syndrome in paediatric patients is available237. In this prospec-
tive, open-label, single-arm phase I/II pilot study, 11 patients under-
went three intravenous infusions with allogeneic UCB-MSCs. Results
after a 12-month follow-up time showed that UCB-MSC treatment was
safe and no patients experienced any infusion-related adverse or toxic
effects; however, only three of nine assessable patients had partial or
complete remission of the urine protein-to-creatinine ratio
237
. In the
future, additional high-level evidence-based studies will be required to
bridge the gap between the promising preclinical results obtained with
UCB-derived stem and progenitor cells and the modest performance
observed in clinical studies.
PBSCs have shown safety and preliminary efficacy in the clinical
treatment of AKI238 and CKD239,240. In 2021, autologous transplanta-
tion of mobilized peripheral blood CD34+ cells was shown to success-
fully improve kidney function for the first time in humans in a patient
with severe AKI, and no substantial adverse events were observed238.
Additionally, various clinical trials were conducted to investigate the
safety and efficacy of autologous peripheral blood CD34+ cell therapy
for patients with stage III and IV CKD
239,240
. In a phase I clinical trial,
the safety of intrarenal artery infusion with autologous peripheral
blood CD34
+
cells was assessed in 10 patients with CKD
240
. In a phaseII
randomized controlled trial including 52 consecutive patients with
stage III or IV CKD, peripheral blood CD34+ cell treatment did not
improve kidney function in terms of urine protein-to-creatinine ratio
(P = 0.178) and creatinine clearance (P = 0.446) 1 year after treatment,
probably owing to the small sample size, but significantly reduced
Disease Cell type Experimental design Mechanism of action Highlighted results Ref.
Neurogenic
bladder
dysfunction
Human AFSCs and
HEK293 cells Local injection with AFSCs or
HEK293 cells into the injured
spinal cord after SCI-induced
neurogenic bladder
dysfunction in rats
Action: cell engraftment,
secretome-mediated
release of factors
Mechanism:
pro-neurogenesis,
anti-autoimmunity,
anti-inlammatory,
antiibrotic effects
Reduction of peak voiding pressure, voiding
volume, bladder capacity, residual volume,
non-voiding contraction, collagen concentration
and bladder weight; increased total elastin and
collagen amount; expression of β3-adrenoceptor,
muscarinic receptors and BDNF increased
after AFSC transplantation; injected AFSCs
were present in the spinal cord and colocalized
with neural cell markers but AFSCs number
decreased over time
208
Interstitial
cystitis Human USCs,
human BM-MSCs,
human ADSCs and
human AFSCs
Interstitial cystitis was induced
by uroplakin II injection in
rats; four stem cell types were
used: USCs, BM-MSCs, ADSCs
or AFSCs; three injection
routes were used: submucosa,
intravenous or transurethral
instillation
Mechanism:
anti-inlammatory,
antiibrotic effects
All stem cell treatments decreased inlammatory
reactions, improved uroplakin II-induced ibrotic
changes in the bladder wall and promoted
bladder function recovery; USC treatment
showed the greatest anti-inlammatory effect;
submucosa injection showed the greatest
improvement in bladder function and tissue
regeneration compared with other injection
routes
291
Urinary
tract tissue
engineering
Human USCs USCs were differentiated
into urothelial cells and
smooth muscle cells, and
seeded onto vessel ECM;
subsequently, constructs
were cultured in vitro for
2 weeks, pre-vascularized by
omentum for 3 weeks and
transplanted for ureter defect
reconstruction in a rabbit
model of unilateral ureter
excision
Action: differentiation After 2 weeks of in vitro culture, cells inilled
into the matrix pores and formed 3–6 layers of
smooth muscle cells and 3–4 layers of urothelial
cells under dynamic culture conditions; after
3 weeks of omental maturation, the constructs
were revascularized and transplanted for ureteral
defect reconstruction; 2 months after ureter
reconstruction, the implanted graft formed a
clearly layered ureter structure with a multilayer
epithelium over the organized smooth muscle
tissue
210
Rabbit USCs Urethral grafts were
engineered by culturing
autologous USCs on small
intestinal submucosa scaffolds
in vitro and transplanted to
repair a urethral defect in
rabbits
Action: differentiation Autologous USCs differentiated into smooth
muscle cells and urothelial cells in vivo
3 months after transplantation;one urethra
graft-treated rabbit showed urethral stricture,
whereas all small intestinal submucosa-treated
controls underwent urethral stricture;urethra
graft-treated rabbits showed lower degrees of
inlammation and ibrosis than small intestinal
submucosa-treated controls
53
8OHdG, 8-hydroxy-2-deoxyguanosine; ADSC, adipose-derived stem cell; AFSC, amniotic luid-derived stem cell; AGE, advanced glycation end product; AKI, acute kidney injury; BDNF,
brain-derived neurotrophic factor; BFSC, body luid-derived stem cell; BM-MSC, bone marrow-derived mesenchymal stem cell; BUN, blood urea nitrogen; CCEC, corpus cavernosal vascular
endothelial cell; CKD, chronic kidney disease; CNI, cavernous nerve injury; ECM, extracellular matrix; ED, erectile dysfunction; eNOS, endothelial nitric oxide synthase; EV, extracellular vesicle;
HEK293cell, human embryonic kidney 293cell; ICP, intracavernosal pressure; IRI, ischaemia–reperfusion injury; LPS, lipopolysaccharide; MAP, mean arterial pressure; MDA, malondialdehyde;
MNC, mononuclear cell; MSC, mesenchymal stem cell; nNOS, neuronal nitric oxide synthase; PB-EPC, peripheral blood-derived endothelial progenitor cell; PCNA, proliferating cell
nuclear antigen; ROS, reactive oxygen species; SCI, spinal cord injury; sCr, serum creatinine; SUI, stress urinary incontinence; TNF, tumour necrosis factor; UCB, umbilical cord blood; USC,
urine-derived stem cell; VEGFA, vascular endothelial growth factor A; VEGFR2, vascular endothelial growth factor receptor 2.
Table 2 (continued) | Preclinical application of BFSCs in the treatment of genitourinary abnormalities
Nature Reviews Urology
Perspective
the 1-year incidence of dialysis or death (P = 0.038)239. Results from
these trials highlighted the potential extrarenal benefits of intrarenal
artery infusion of autologous CD34
+
cells in the setting of advanced
kidney disease. Interestingly, the renoprotective effects of human
PB-MNCs against IRI-induced AKI in a mouse model were shown to be
substantially enhanced by increasing the number and colony-forming
potential of EPCs
241
, indicating that the curative effect of PBSCs in
CKD mice is probably mediated by EPCs; indeed, EPCs were shown to
have crucial roles in maintaining vascular homeostasis and restoring
endothelial injury in animal models of vascular-related diseases (such
as a mouse model of liver sinusoidal endothelial cell injury242 or a mouse
model of ischaemic hindlimb)243. In addition to improving angiogen-
esis, PBSC therapy effectively preserves renal function and amelio-
rates renal tissue injury by exerting pro-proliferative, anti-oxidant,
anti-inflammatory, antifibrosis and anti-apoptotic effects
192,244,245
. For
instance, in a mouse model of AKI, pretreatment with human PB-EPCs
before reperfusion ameliorated IRI-induced renal dysfunction and tis-
sue damage, measured as decreased levels of BUN and sCr as well as by
attenuated tubular atrophy, cast formation and brush border loss244.
Similarly, autologous transfusion with rat PB-EPCs through the renal
artery or penile vein effectively ameliorated renal dysfunction, reversed
renal tissue injury score and inhibited the progression of CKD induced
by 5/6 nephrectomy in rats
245
. Moreover, the pro-proliferative and
anti-inflammatory effects of PB-MNCs were shown to be enhanced by
polarization of the monocytes into an M2 phenotype through pretreat-
ment with repetitive anoxia–reoxygenation cycles, which increased
the release of anti-inflammatory cytokines and other bioactive factors
supporting cell proliferation during renal tissue implantation under
both in vitro and in vivo conditions
192
. Indeed, polarized PB-MNCs
(given intravenously) were more effective than untreated PB-MNCs in
protecting renal tissue against ischaemia-mediated injury and fibrosis
in a mouse model of IRI-induced AKI192.
Overall, BFSCs have shown promising renoprotective effects
in terms of improving renal dysfunction in AKI and inhibiting pro-
gression to CKD in several preclinical studies. BFSCs exert these
renoprotective effects through multiple mechanisms, mainly con-
sisting of immunomodulatory93,192,197–199,233,246, anti-apoptotic82,92,94,204,
pro-angiogenic
241,247–249
and antifibrotic
192,207,208,250–255
paracrine effects.
BFSC engraftment was observed in several studies, butsolid evidence
indicating differentiation at the site of injury is rare. Moreover, most
available studies in this field have been conducted in animal mod-
els; thus, conclusions drawn from these studies still require further
confirmation in high-level evidence trials before clinical translation.
Erectile dysfunction
ED is defined as the consistent or recurrent inability to attain and/or
maintain an erection sufficient for satisfactory sexual intercourse
256
and affects up to 30% of men. ED incidence increases with age257. ED is
caused by insufficient blood flowing into the penis. Thus, common
causes of ED are conditions that lead to alterations in this blood flow,
including diabetes and nerve damage
258
. Current pathways for ED man-
agement aim to increase penile blood flow through self-administered
oral medications (phosphodiesterase type 5 inhibitors), intra-urethral
or intracavernous injections (with alprostadil, papaverine, phentola-
mine and vasoactive intestinal polypeptide), vacuum erection devices,
and surgical implants258. However, current treatments for ED mainly
palliate the symptoms rather than restore natural erectile physiology.
The overall aim of ED treatment is to reverse the underlying cor-
pus cavernosum damage caused by various metabolic conditions or
physical effectors, which eventually result in changes to the penis
blood flow. In a rat model of diabetic ED, intracavernous injection
with human USCs significantly (P < 0.01) enhanced erectile function,
as indicated by an increased intracavernous pressure-to-mean arte-
rial pressure (ICP-to-MAP) ratio compared with phosphate-buffered
saline-treated controls207. In this study, few human USCs were detected
within the injection sites; however, histological results showed an
increased number of endothelial and smooth muscle cells within
the cavernous tissue following USC injection207. Additionally, in dia-
betic ED rats, intracavernous injection with USCs from healthy rats
improved copulatory functions
191
by ameliorating penile fibrosis and
increasing smooth muscle content in the corpora cavernosum. Similar
results were observed in a rat model of cavernous nerve injury-induced
ED receiving treatment with human USCs; in this study, a significant
increase in the ICP-to-MAP ratio (P < 0.05) was observed 28 days after
transplantation
253
. Results from in vitro studies have shown that USCs
were able to differentiate into endothelial cells, but no USCs were
tracked in penile tissue following USC transplantation
207,253
, indicat-
ing a paracrine mechanism of action. However, the exact mechanisms
underlying these paracrine effects have not been completely eluci-
dated. A plausible mechanism could be the USC-mediated secretion
of pro-angiogenic trophic factors (such as PDGF, FGF2 and VEGF)
and immunomodulatory factors (such as IL-8 and IL-10), which has
been observed in vitro
207,259
but might also happen in vivo; indeed,
in a study in a rat ED model253, USCs transplanted into the corpora
cavernosum were shown to recruit resident endothelial cells, sup-
press cell apoptosis, increase smooth muscle content and decrease
fibrosis in cavernous tissues. Administration of EVs and cell lysate
derived from USCs also resulted in erectile function recovery in rat
models of diabetes-induced ED
87,187,191
and Peyronie disease
260
, further
supporting the hypothesis of a paracrine mechanism of action of
these cells. In another study91, autophagic dysfunction was reported
to be associated with cavernosal endothelial dysfunction in rats with
diabetic ED; an intracavernous injection with USCs in these mice could
increase autophagy in cavernous endothelial cells, which ameliorated
endothelial dysfunction and eventually improved ED91.
UCB-derived stem cells showed some curative effects on ED,
mostly in animal models. In a study performed in 2010 (ref. 261), seven
patients with diabetic ED received allogeneic UCB-derived stem cell
treatment. After a single intracavernous infusion, three and six partici-
pants regained morning erections within 1 and 2 months, respectively,
and these results were maintained for >6 months; six participants
experienced an increase in penile rigidity with time, although this
was still insufficient for effective penetration; blood glucose levels
decreased after 2 weeks in six of seven participants and were main-
tained for 4–7 months; and no adverse events after stem cell therapy
were observed in this study, even without immunosuppression261.
In ED rat models, infusion with human UCB-MNCs and UCB-MSCs
improved ED by reducing tissue fibrosis, ameliorating hypoxia and
promoting neuronal regeneration
193,262,263
. However, in one of these
studies, although UCB-MSCs were directly injected into the corpus cav-
ernosum immediately after cavernous nerve injury in a rat ED model,
the injected UCB-MSCs did not colocalize with endothelial cells and
smooth muscle cells in the corpus cavernosum, indicating that these
cells might function through a paracrine mechanism rather than by
direct differentiation
193
. In another study in which an ED rat model was
established through cavernous nerve injury, infusion with AFSCs pro-
duced the most significant (P < 0.05) improvement in the ICP-to-MAP
ratio compared with umbilical vein endothelial cells and ADSCs264,
Nature Reviews Urology
Perspective
which might be related to a higher neurogenic, myogenic and vascular
tissue regenerative capacity of AFSCs than thatof other stem cells.
Interestingly, a decreased number of circulating EPCs has been
observed in the peripheral circulation of patients with ED, which
is related to weak endothelial function265 and low-grade chronic
inflammation266, indicating that increasing the number of circulating
EPCs might ameliorate ED. Results from a 2012 study in diabetic rats
249
showed that EPCs mobilized from the marrow into the circulation
through chronic melatonin administration effectively increased the
number of circulating EPCs and prevented ED in these rats, as indicated
by greater ICP-to-MAP ratios observed in melatonin-treated rats than
in diabetic control rats. In another study, an intracavernous injection
with UCB-EPCs restored erectile function in a rat model of diabetic
ED by restoring corpus cavernosal endothelial and smooth muscle
cell numbers and improving cavernous nerve functions247. Moreover,
in a study in rats, the combined transplantation with UCB-EPCs and
BM-MSCs exerted a synergistic effect on erectile function recovery
compared with the single cell types transplanted alone248.
The therapeutic effects of BFSCs on ED might be enhanced by gene
modification. For example, human USCs transfected with a lentivirus
carrying FGF2 could spontaneously differentiate into endothelial cells
without extra induction in vitro
207
. Infusion of these cells in rats with
diabetic ED led to a greater improvement in the ICP-to-MAP ratio than
that caused by unmodified USCs
207
. Similar results were observed in
studies in which rat models of cavernous nerve injury-induced ED
were treated with rat USCs hypersecreting the neurotrophic factor
pigment epithelium-derived factor (PEDF)253 or withhuman UCB-MSCs
hypersecreting brain-derived neurotrophic factor (BDNF)267.
The performance of BFSCs in animal models of ED is promising,
but additional clinical trials are required in the future to translate this
potential into the clinical treatment of ED.
Stress urinary incontinence
SUI, the involuntary loss of urine on effort or physical exertion268, is a
prevalent urological disorder affecting ~46% of adult women, and
increases with age
269
. Overall, two main, often overlapping, mecha-
nisms of SUI exist: urethral hypermobility caused by a weakening of
the pelvic floor muscles or vaginal connective tissue; and intrinsic
sphincter deficiency caused by a loss of urethral mucosal and muscular
tone
270
. Current SUI treatments include pelvic floor muscle exercises,
bladder retraining, pharmacological therapy, injection of periurethral
bulking agents and vaginal sling surgery
271,272
. Sling surgery is success-
ful but only serves as symptomatic treatment, without treating the
underlying disorder
271
. Thus, stem cell-based therapies, which aim
to recover deficient urethral sphincter function and target the patho-
physiological mechanisms underlying SUI, are a promising alternative
for correcting SUI272.
Improving local blood circulation, enhancing muscle regenera-
tion and improving peripheral nerve function are essential to restor-
ing urethral sphincter muscle defects
259
. USCs have been shown to
differentiate into skeletal muscle, smooth muscle, endothelial and
neurogenic lineages in vitro38, suggesting the potential of these cells
in the treatment of SUI. Thus, a series of USC-containing hydrogels
were established containing different combinations of USCs206, USCs
hypersecreting VEGF259, endothelial cells259, growth factors206, collagenI
gel
259
or heparin–hyaluronic acid gel
206
. When these USC-containing
hydrogels were subcutaneously implanted into nude mice, the grafts
maintained a larger volume of tissue mass and produced a greater num-
ber of blood vessels and neurons than those generated by the controls
(hydrogels without any cells, growth factors or genetic alterations).
These hydrogels can be a novel cell-delivery system for SUI and other
injured muscle tissue models, but further studies on SUI animal models
are needed. In another study, USC-EVs have been shown to accelerate
pubococcygeus muscle injury recovery in a rat model of SUI induced by
vaginal hyperextension by prompting the activation and differentiation
of endogenous muscle satellite cells273.
AFSCs have shown preclinical efficiency and safety in correcting
SUI. In a mouse model of SUI generated through bilateral pudendal
nerve transection, periurethral administration of undifferentiated
human AFSCs effectively restored the histological structure and func-
tion of the injured urethral sphincter to normal levels274. The injected
AFSCs were detected in periurethral tissue up to 10 days after injec-
tion, and human myogenic gene expression gradually decreased over
time, whereas mouse myogenic gene expression steadily increased
274
,
indicating that the grafted human AFSCs might have undergone myo-
genic differentiation in situ and induced host muscle regeneration. In
two studies in mice, an injection with early myogenic differentiated
human AFSCs275 or a triple combination of myogenic, neurogenic and
endothelial differentiated human AFSCs
276
showed greater reparative
effects (in terms of improved leak point pressure and formation of new
striated muscle fibres and neuromuscular junctions at the injection
sites) than infusion with undifferentiated AFSCs. These results sug-
gest that AFSCs might integrate into the host sphincter muscle and
accelerate urethral sphincter regeneration through in situ myogenic
differentiation or stimulation of host cell differentiation.
MNCs have been used to correct SUI. For example, in a rat SUI
model established by electrocauterization of periurethral soft tissue
277
,
periurethral injection with human UCB-MNCs improved the function
and histological structure of the damaged urethral sphincter. Based on
these results, the same authors conducted a study in which 39 women
with all SUI types, including urethral hypermobility, intrinsic sphinc-
ter deficiency and mixed urinary incontinence, received allogeneic
UCB-MNC injection into the submucosal layer of the proximal ure-
thra. Results after a 12-month follow-up time showed that 13 women
were fully satisfied, the condition improved in another 13 women, and
10 women still had incontinence278. Additionally, results from a 3-month
postoperative urodynamic study including 5 women with SUI and
5women with mixed urinary incontinence showed that the maximal
urethral closing pressure values were increased by >30 cm H
2
O in all
10 women after UCB-MSC injection compared with the preoperative
values
278
. In another clinical pilot study
279
, a periurethral injection with
PB-derived total nucleated cells containing EPCs was evaluated for the
treatment of severe SUI in women. After a single injection, 8 of 9 women
were clinically completely cured, and one woman experienced a marked
improvement in SUI measured through the International Consultation
on Incontinence Questionnaire-Urinary Incontinence and International
Consultation on Incontinence Modular Questionnaire-Quality of Life
(P < 0.05)279.
Results from preclinical studies on animal models have shown
that BFSCs are a promising treatment for SUI; however, currently avail-
able clinical studies are limited and show moderate efficacy. Thus,
additional preclinical studies to optimize the use of BFSCs should be
conducted before further clinical trials including patients with SUI
are carried out.
Bladder dysfunction
Bladder dysfunctions, including detrusor under-activity, overac-
tivebladder and interstitial cystitis, are often chronic and have a high
Nature Reviews Urology
Perspective
prevalence, resulting in a substantial negative effect on quality of life.
An overactive bladder is characterized by complex storage symptoms,
but the exact aetiology remains unclear280. Human AFSCs have shown
protective effects against bladder overactivity. In a rat model of chronic
bladder ischaemia induced by atherosclerosis, intravenous injection
with human AFSCs ameliorated histological damage and altered void-
ing parameters, probably by downregulating the expression profiles of
oxidative stress inducers and of TNF within the injured bladder wall
281
.
Bladder outlet obstruction often results in bladder tissue inflammation
and fibrosis. In a rat model of partial bladder outlet obstruction, intra-
venous injection with human USCs could effectively elevate bladder
maximal voiding pressure, decrease end-filling pressure and improve
detrusor muscle contractility
282
. Histological analysis showed that USC
treatment alleviated tissue fibrosis and inhibited muscle cell apoptosis
in the bladder wall of these rats
282
. Additionally, in a mouse model of
bladder outlet obstruction, direct intrabladder injection with human
USC-EVs induced bladder function improvement and histological
changes similar to those observed with USC infusion
283
, indicating
that USCs might function through EVs.
Diabetic bladder dysfunction affects approximately half of all
patients with diabetes284. The pathogenesis has not been completely
clarified but is probably related to urothelial dysfunction, detru-
sor decompensation and bladder nerve damage
284
. In a rat model of
streptozotocin-induced diabetic bladder dysfunction, human AFSCs
were directly injected into the bladder wall aiming to correct this
dysfunction285. AFSC-treated rats showed decreased body weight but
increased bladder weight compared with untreated controls; most
importantly, significantly improved cystometric parameters, such
as non-voiding contraction (P < 0.05), residual volume (P < 0.05),
voided volume (P < 0.05) and intercontraction interval (P < 0.05), were
observed 12 weeks after diabetes mellitus induction in AFSC-treated
rats. Moreover, the expression of NGF and muscarinic receptors, which
are involved in nerve regeneration, was restored in these rats285, indi-
cating a potential mechanism underlying bladder function recovery
following AFSC treatment. Similarly, in other studies in diabetic rats,
treatment with human UCB-MNCs251 and human USCs254 significantly
improved cystometric parameters by inhibiting apoptosis and reduc-
ing fibrosis in the bladder wall of these animals. Interestingly, in dia-
betic rats, intravenously injected USCs were observed in the kidney and
pancreas but not in the bladder
254
. Thus, USCs might exert therapeutic
actions not by homing into damaged target organs and differentiat-
ing in situ into functional cells, but by releasing various trophic and
immunomodulatory factors (such as VEGF, PDGF, FGF, IL-8 and IL-10)
to systemic circulation to regulate tissue repair and regeneration in
a distant place, suggesting a systemic rather than local mechanism
of regeneration and/or a paracrine effect of USCs in the treatment of
chronic diseases such as diabetes.
Neurogenic bladder is a dysfunctional bladder condition caused
by nervous system damage, including stroke, Parkinson disease, spinal
cord injury and multiple sclerosis
286
. Results from a study in which a rat
Parkinson model was injected with human AFSC246 showed that cysto-
metric parameters, such as bladder capacity (P < 0.001), spontaneous
activity (P = 0.003), micturition frequency (P < 0.001) and threshold
pressure (P = 0.001), were significantly improved after 14 days, and the
functional improvement subsided at 28 days
246
. The injected AFSCs
repaired injured neural tissue by modulating local inflammation and
oxidative stress246. Similarly, in rat models of bladder dysfunction
caused by cerebral ischaemia
252,255
, pelvic nerve transection
250
and
spinal cord injury
208
, direct transplantation of human AFSCs
208,250,252
or
human UCB-derived CD34
+
cells
255
into the bladder wall ameliorated
dysfunctional bladder parameters such as bladder capacity, residual
volume, number of non-voiding contractions, peak voiding pressure
and leak point pressure. The therapeutic effects of BFSCs in these
studies were probably related to the paracrine effects of these cells in
the injured bladder, including increased expression of neurotrophins
and muscarinic receptors, which are involved in nerve regeneration, and
decreased release of HIF1α, IGF1 and TGFβ1, which mediate bladder
wall fibrosis208,250,252,255.
Interstitial cystitis is a chronic condition characterized by the
symptoms of urinary urgency, frequency, nocturia and pelvic pain in
the absence of any identifiable pathology such as bacterial infection
287
.
The possible pathophysiology of this condition includes glycosamino-
glycan layer defects, permeability disruption, autoimmune disorders,
infection and inflammation
288
. In preclinical studies, rat models of
interstitial cystitis induced through ketamine289 or HCl290 instillation
received a single injection with human UCB-MSCs into the bladder
submucosal layer. The injected UCB-MSCs successfully engrafted into
the stromal and epithelial tissue of the injured bladder and relieved
interstitial cystitis by alleviating mast cell infiltration and inducing
bladder epithelial regeneration and angiogenesis
289,290
. Similarly, intra-
venous injection with human USCs also restored bladder histology and
micturition function by reducing oxidative stress, inflammation
andapoptosis in a rat model of interstitial cystitis induced by prota-
mineand lipopolysaccharide
196
. In another study
291
, the curative effects
of different stem cells and cell-delivery routes on interstitial cystitis were
assessed in an interstitial cystitis rat model. In this study, USCs exerted
a greater anti-inflammatory effect than ADSCs, AFSCs and BM-MSCs,
and submucosal injection had a higher therapeutic efficiency than
transurethral instillation and intravenous injection291.
Taken together, results from studies in various preclinical models
have shown the efficacy and safety of BFSC therapy for different blad-
der dysfunction conditions. However, to our knowledge, no clinical trial
has been conducted in this area. Future research should be focused on
translating these experimental research advancements from bench
to bedside.
Bioengineering of the genitourinary tract
Several pathological conditions of the genitourinary tract require
repair or replacement of the affected organs, such as ureters, bladder
and urethra, to reconstruct biological functions. The bladder is a hol-
low muscular organ with three principal tissue layers covered with a
multi layered, highly specialized urothelium. These complex structures
are challenging for reconstructive surgery. Surgical management of
bladder defects, damage or loss relies on cystoplasty with transplan-
tation of viscoelastic autogenous gastrointestinal segments to the
bladder to achieve adequate capacity and low-pressure storage292.
However, transplantation with gastrointestinal tissue might result in
long-term complications such as urolithiasis, vesicoureteral reflux,
metabolic disorders and carcinoma292. Tissue-engineered bladder
substitutes are an attractive option for bladder reconstruction, par-
ticularly for paediatric patients. An ideal bladder substitute should
reconstruct urinary barrier function and smooth muscle compliance.
To engineer a functional bladder, urothelial cells, smooth muscle
cells and endothelial cells are required to build the bladder mucosa,
bladder wall and blood vessels, respectively. USCs originate in the
urinary region and, therefore, are capable of giving rise to urothelial
cells, smooth muscle cells and endothelial cells; thus, these cells are a
potentially excellent cell source for urinary tract bioengineering
38,52,60
.
Nature Reviews Urology
Perspective
A multilayered urothelium with permeability barrier properties was
successfully engineered in vitro by seeding human USCs onto small
intestinal submucosa scaffolds and culturing these cells in a urothelial
induction medium under dynamic conditions80, suggesting that USCs
could be potentially used in urological tissue repair or urethra and blad-
der modelling. In another study
212
, a bladder substitute was engineered
by directly seeding undifferentiated USCs onto composite scaffolds.
These USC-seeded grafts were successfully used to reconstruct the
bladder wall in a partial bladder cystectomy rat model, as indicated by
the formation of well-differentiated smooth muscle and a multilayered
urothelium in the reconstructive areas212.
The primary function of the ureters and urethra is to provide a
strictly waterproof barrier that prevents toxic urine diffusion beneath
the epithelium. This function must be preserved when reconstructing
these structures; otherwise, fibrosis will occur. Similarly to the bladder,
ureters and urethra mainly consist of two functional layers: the urothelial
layer and the smooth muscle coat293. In a mouse study, the feasibility of
using USCs as a single-cell source for multilayered urinary graft engi-
neering was assessed. Human USCs were first induced to differentiate
into urothelial cells and smooth muscle cells. Subsequently, these cells
were seeded onto porous small intestinal submucosa scaffolds
294
. After
2 weeks of culture under 3D dynamic conditions, multilayered urothe-
lium with colonized USCs was obtained in the matrix
294
. In another study,
the feasibility of using USC-seeded scaffolds for urethral defect repair
after autologous transplantation was assessed
53
. The urethral grafts
were engineered by seeding rabbit USCs onto porous scaffolds and
transplanted to repair a 2 cm-urethral defect in a rabbit model. After
3 months, the seeded USCs differentiated into smooth muscle cells and
urothelial cells in the defect sites; urethral stricture was observed in all
control rabbits treated with non-seeded scaffolds but only in one rabbit
treated with USC-seeded scaffolds
53
, suggesting that USCs could be used
as autologous BFSCs for urinary tract reconstruction. This strategy was
also adopted for ureter tissue engineering. An engineered ureter graft
was constructed by seeding human USC-induced urothelial cells and
smooth muscle cells onto vessel extracellular matrix210. After 2 weeks
of dynamic culture in vitro, the graft formed multilayered epithelium
over the organized smooth muscle tissue. The tabularized graft was
then transferred between the two layers of the omentum of the rabbit
for further in vivo maturation. After 3 weeks of omental maturation, the
graft was vascularized and could achieve ureter reconstruction in
the same rabbit. After two months following the ureter reconstruction, the
implanted graft formed a clearly layered structure of the ureter with
multilayered urothelium over the organized smooth muscle tissue
210
.
However, a mechanical test on the reconstructed ureter and a long-term
follow-up analysis of ureter function should be performed in large
animals in future studies.
Future perspectives and challenges
BFSCs have shown therapeutic potential in preclinical models of geni-
tourinary conditions; however, translation of the promising results
obtained in animal models into clinical practice remains challenging
for several reasons. The first issue is low cell density; indeed, harvesting
body fluid samples and extracting stem cells from these samples is quite
simple, but the number of stem cells in body fluids is relatively low com-
pared with that in solid tissues. Thus, expansion of BFSCs under exvivo
conditions is usually required before clinical application. Another
problem is cell diversity; body fluids are present in a dynamic and
circulating system, and the interpopulation and intrapopulation cel-
lular composition of these fluids are highly heterogeneous and usually
influenced by the age, sex, health status and genetic background of the
donor. This heterogeneity might hinder the characterization of body
fluid cell composition and hamper a consistent therapeutic response.
Microorganism contamination might also affect BFSC use. Urine sam-
ples collected from female donors might be contaminated by microor-
ganisms present in the pudendum and the labia; menstrual blood and
breastmilk samples might also be contaminated by microorganisms
presented in the pudendum and the nipple, owing to the direct contact
of these samples with the skin in these areas during collection and stor-
age. An additional point to consider for the clinical translation of BFSCs
is that BFSC production must be standardized and performed under
good manufacturing practice guidelines to respect safety and regula-
tory issues. Thus, banking, shipping, in vitro expansion, administra-
tion, delivery, and homing of BFSCs must be optimized. Another issue
connected with the use ofBFSC in clinics is the lack of a uniform and
clarified nomenclature. Discovery and research on BFSCs are rapidly
advancing in this decade, but the nomenclature of BFSCs identified
in different species is inconsistent owing to different experimental
purposes and isolation protocols, which makes the interpretation and
comparison of experimental results challenging. BFSC use is also associ-
ated with safety and efficacy concerns. Lastly, the underlying mecha-
nisms of action of BFSCs in vivo remain to be further investigated.
Differentiation and engraftment of BFSCs in damaged tissue have been
observed in preclinical models, but convincing evidence supporting
differentiation into functional cells in vivo is currently scarce. Results
from current studies suggest that the therapeutic effects of BFSCs
might mainly rely on paracrine action rather than on differentiation
and engraftment. However, identifying all the components of BFSCs
is complicated, and understanding the effects of these secretomes on
tissue regeneration in vivo requires further investigation. Thus, BFSCs
have shown promising results in repairing damaged or diseased geni-
tourinary tissues in animal models but, owing to all these issues, a gap
exists between these preclinical results and the application of BFSCs
in the treatment of clinical conditions. Well-designed clinical trials
are required to prove the safety and efficacy, as well as to optimize the
dosage and timing of BFSC treatment before therapeutic translation.
Understanding the characteristics and therapeutic mechanisms of
each BFSC type can help toopen doors to new applications. Emerging
new technologies might help toelaborate the therapeutic regenerative
effects of these cells. Most BFSCs are heterologous cell populations
with undefined tissue origin; thus, single-cell RNA sequencing, which
has been widely adopted to deconvolute the heterogeneity of stem
cells
295,296
, can also be used to investigate the cellular composition and
possible tissue origin of different subpopulations in BFSCs. Similarly,
proteomic analysis of BFSC secretomes might enable the identification
of putative effectors of the therapeutic regenerative effects of BFSCs,
whereas single-cell lineage tracing might help understanding the fate of
grafted BFSCs during tissue regeneration in the genitourinary system.
Sorting particular BFSC subpopulations through well-characterized
surface markers and biological prosperities might help togenerate
reliable results in preclinical studies and facilitate clinical translation.
Additionally, factors such as donor health status and age, in vivo
resident conditions, and ex vivo culture conditions have a negative
influence on the regenerative potential of BFSCs; thus, preconditioning
strategies can be used to boost the differentiation and immunomodu-
latory potentials of BFSCs before in vivo administration. For exam-
ple, increasing evidence has shown that preconditioning of human
USC-laden porous scaffolds under hypoxic culture conditions could
enhance the regenerative potential of these cells, in turn accelerating
Nature Reviews Urology
Perspective
angiogenesis in several animal models such as skin wound healing
297
and right ventricular outflow tract reconstruction
298
. Preconditioning
with IL‐1β and IFNγ substantially enhanced the in vitro immunosup-
pressive ability of human UCB-MSCs
299
, which might be beneficial in
the treatment of immune-mediated kidney diseases. However, these
preconditioning protocols should be well defined and standardized
in the future.
Importantly, optimization of the different steps of stem cell-based
therapy is needed to improve efficacy and safety
300
before translating
BFSCs into clinical practice. Several crucial factors for cell preparation
need to be optimized, including the selection of optimal cell sources,
number of cell passages, number of injected cells, appropriate combi-
nation of exogenous growth factors, appropriate route of administra-
tion (local versus systematic), timing of cell injection (early versus late
stage of injury), single or multiple injections, and single or multiple
sites of injection. Different diseases might require different cell therapy
strategies. Pending the resolution of these issues,in the future, cell
therapy with BFSCs could become more efficient than gene editing and
drug-based therapies in the treatment of genitourinary tract diseases.
Conclusions
The discovery of BFSCs provided a series of versatile stem cell sources
to treat genitourinary abnormalities. BFSCs have shown unique features
in genitourinary disease modelling and treatment in preclinical studies.
BFSCs are relatively abundant and easier to harvest— in a non-invasive
or minimally invasive manner, and without enzymatic digestion—than
stem cells derived from solid tissues. Moreover, BFSCs have stemness
properties similar to those of other ASCs, and have cell surface markers,
multiple differentiation plasticity, and immunomodulatory effects.
In preclinical studies, BFSC-based therapies have shown promising
therapeutic potential for the treatment of various genitourinary
abnormalities in animal models. Some BFSCs, such as UCB-derived
stem cells
261,278,279
and PBSCs
238–240
, have also shown promising pre-
liminary results in patients with AKI238, CKD239,240, ED261 and SUI278,279 in
early-phase clinical trials and case reports. BFSC-based therapies were
shown to be safe and effective in tissue repair in rodent models, but
additional in vivo experiments in large animal models as well as clinical
trials in patients are needed to confirm both clinical value and safety
of these therapies. Overall, BFSCs show promise in the treatment and
modelling of genitourinary diseases; in the future, extensive combined
efforts will be needed to facilitate bench-to-bedside translation.
Published online: xx xx xxxx
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Author contributions
R.L.H. researched data for the article. All authors contributed substantially to discussion of the
content. Y.Z. and R.L.H. wrote the article. All authors reviewed and/or edited the manuscript
before submission.
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Reviews Urology thanks Tetsuro Tamaki, Margot Damaser and
the other, anonymous, reviewers for their contribution to the peer review of this work.
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