ArticlePDF AvailableLiterature Review

Body fluid-derived stem cells - an untapped stem cell source in genitourinary regeneration



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 identified in various body fluids, including urine, peripheral blood, umbilical cord blood, amniotic fluid, synovial fluid, breastmilk and menstrual blood. These body fluid-derived stem cells (BFSCs) have stemness properties comparable to those of other adult stem cells and, similarly to tissue-derived stem cells, show cell surface markers, multi-differentiation potential and immunomodulatory effects. 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 differentiation or paracrine mechanisms such as pro-angiogenic, anti-apoptotic, antifibrotic, anti-oxidant and anti-inflammatory effects. However, optimization of protocols is needed to improve the efficacy and safety of BFSC therapy before therapeutic translation.
Nature Reviews Urology
nature reviews urology
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
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 identied 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-
dierentiation potential and immunomodulatory eects. 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 dierentiation or paracrine
mechanisms such as pro-angiogenic, anti-apoptotic, antibrotic,
anti-oxidant and anti-inammatory eects. However, optimization of
protocols is needed to improve the ecacy and safety of BFSC therapy
before therapeutic translation.
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
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:
Nature Reviews Urology
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-uptime
. 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
. 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
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
. 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
. 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
Direct use of the stromal vascular fraction has been reported in clini-
cal studies, forexample for knee osteoarthritis
, wound healing
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
, synovial fluid
, pericardial fluid
, amniotic fluid
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 Table1). 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
. First, harvesting body fluid samples
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
. 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
and uterus transplantation
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
. 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
, 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
. 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
. 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
. 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
. 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
. 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
. 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
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 tissues4951.
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. Consistentlywith 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
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
. USCs collected from young individu-
als presented higher cell viability, clonogenicity and proliferation
but lower senescence than cells collected from older donors
. Fur-
thermore, the efficiency of USC collection in male donors was higher
than in female donors
. The culture contamination rate is higher in
a Tissue-derived stem cells b Body fluid-derived stem cells
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
Amniotic luid
Umbilical cord blood
Peripheral blood
Menstrual blood
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
urine samples collected from females than those from males, but the
risk of contamination can be decreased by collecting urine in sterile
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
. 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
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)
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
, SOX2 (ref. 55), MYC
, 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
. Ectodermal neural lineages have been
obtained by culturing USCs in neural induction medium
. Endodermal
lineages, such as hepatocytes
, insulin-producing cells
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
AFSCs Fetal urinary,
respiratory or
tracts, skin, amniotic
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
AKI195,197199,205,222, CKD223225, diabetic
nephropathy226, ED264, SUI274276, neurogenic
bladder208,246,250,252, overactive bladder281,
diabetic bladder dysfunction285, IC291
stem cells Bone marrow HSC markers: CD34, CD45, CD133
(ref. 133)
MSC markers: CD29, CD44, CD73,
CD90, CD105 (refs. 135137)
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
AKI147,194,230,232,234236, 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
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
Paracrine effects of USCs
USCs have excellent immunomodulatory properties55,65,81. Cul-
tured USCs do not express human leukocyte antigen-DR isotype
, 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
. 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
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
. 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
. 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 tomaintain 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
. 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
. 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 anda 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.
Parietal epithelial cells
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
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
. 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
. 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
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)
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
. 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
. 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
. The expression
of other pluripotency markers varies among studies
. Moreover,
AFSCs express adhesion molecules106, the multipotent marker CD133
(ref. 102) and mesenchymal markers
but do not express haema-
topoietic markers
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
. 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
and do not form
embryoid bodies
. 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
Culture conditions might partly determine AFSC fate; for example,
culturing AFSCs under ES cell conditions enhances the pluripotency
levelof these cells98. Furthermore, AFSCs isolated through CD117+
selection express higher levels of pluripotent markers than AFSCsiso-
latedby 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
. 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
, chondrocytes
, osteoblasts
and adipocytes
The endodermal differentiation potential was shown by measuring
human AFSC differentiation into alveolar epithelial cells
, renal tubu-
lar epithelial-like cells
and podocytes
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
. AFSCs are negative for
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
. Additionally, the proliferation
of B cells is suppressed only by pro-inflammatory cytokines produced
by second-trimester AFSCs
. 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
and in a mouse
model of lipopolysaccharide-induced preterm birth128. In these models,
Nature Reviews Urology
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
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
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 featuresof these cells, including high
self-renewal, multipotency, hypo-immunogenicity, non-tumorigenicity
and immunomodulation
. 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
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
. 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
. 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 markers135137 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
, osteogenic
and adipogenic
cells, as well as into non-mesodermal
cells such as neuronal140, hepatic141 and insulin-producing cell142
Endothelial progenitor cells. EPCs are presumed to originate from
bone marrow but are also present in UCB and peripheral blood
. EPCs
and HSCs are postulated to arise from a common haemangioblast
precursor and share some cell surface antigens
. 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))
. 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
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
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
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
. 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
cells, which have been identified as a very
primitive stage of HSCs in UCB
. Additionally, MSCs are present in
extremely low numbers in UCB and have a low isolation efficiency
The isolation of UCB-MSCs is generally based on the plastic-adherent
propertiesof these cells
. 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
. The unattached cells
Nature Reviews Urology
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
CD45CXCR4+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
. However, UCB-MSCs
exert immunosuppressive effects on lymphocyte proliferation137,
inhibit pro-inflammatory cytokine release135,137,155 and induce periph-
eral tolerance
. 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
. 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
, 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
, EPCs
, MSCs
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
such as for multiple myeloma
graft-versus-host disease
and cartilage repair
. 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;
PBSCscan be extracted in large amounts from autologous peripheral
blood; and peripheral blood is less likely than bone marrow to contain
cancer cells
. 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
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
in 2007 and were subsequently termed
. MenSCsinclude 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
. 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
; 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-
ityand 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
capacity than BM-MSCs, whereas CD117
possess a trilineage differentiation capacity similar to or slightly better
than that of BM-MSCs
. 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
. In vitro, MenSCs inhibit differentia-
tionand 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
. 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 EVs179181,183. 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
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
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
, 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
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
, induction of autophagy
cell-to-cell contact
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
, pro-proliferative
pro-angiogenic82,86,87, antifibrotic185,186,201,202, anti-oxidative93,199,203 and
anti-apoptotic82,9294,195,197,204,205 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 regeneration187190. 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
), 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
Engraftment Angiogenesis induction Fibrosis inhibition lmmunosuppression Autophagy
Anti-oxidation Apoptosis
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, immunosuppression93,197199, fibrosis inhibition185,186,201,202,
apoptosis inhibition82,9294,195,197,204,205 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
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
and lipopolysaccharide-induced acute kidney injury
. 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
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 Table2).
Kidney diseases
The global burden of kidney diseases is substantial and growing:
~10–15% of hospital admissions are caused by AKI
, whereas ~10% of
adults are affected by chronic kidney disease (CKD), which results in
1.2million deaths per year worldwide
. 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
and diabetic nephropathy
. 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
. Fur-
thermore, results from studies carried out in different rodent models
of kidney injury, such as diabetic nephropathy
and IRI
, 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
, suppressing ferroptosis
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
. 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, CKD223225 and dia-
betic nephropathy
. 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
, reducing oxidative stress
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
. 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
. 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 renalfunc-
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
. Additionally,
injection with human UCB-MSCs also showed a protective effect towards
Nature Reviews Urology
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, VSELderived renal progenitors induced endogenous
tubular cell proliferation, showing potential for renal regeneration147.
Cell engraftment
Tubular cell proliferation
Epithelial cell and podocyte apoptosis
Oxidative stress
a AKI and CKD
CD133+ EPCs
PB-CD34+ cells
UCB-CD34+ cells
Oxidative stress
c ED
Myogenic dierentiation
Urothelial dierentiation
d Urinary tract defect
Cell engraftment
Oxidative stress
Muscle cell apoptosis
e Overactive bladder
Oxidative stress
f Neurogenic bladder
AFSCs UCB-CD34+ cells
Oxidative stress
g Diabetic bladder
Cell engraftment
h Interstitial cystitis
Cell engraftment
Host satellite cell proliferation and
Pre-dierentiated AFSCs (myogenic,
neurogenic and endothelial cells)
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 angiogenesis241,247249,
activating autophagy194,195,201,225, inhibiting epithelial cell and podocyte
apoptosis82,92,94,204, suppressing ferroptosis193, suppressing oxidative stress199,203
and inflammation93,192,197199,233,246, and reducing tissue fibrosis in animal
models192,207,208,250255. 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 myogenesis274276 ,
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 inflammation289291 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.