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

July 2023
Nature Reviews Urology 20(12)

DOI:10.1038/s41585-023-00787-2

Authors:

Ru-Lin Huang

Shanghai Jiao Tong University

Qingfeng Li

Shanghai Jiao Tong University

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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.

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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 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.

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-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

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

, UCB

, 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

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

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

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

, 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

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

. 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

. 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. 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

donor

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

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

. 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

, 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

75,76

. 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

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

. 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 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

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

. 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

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

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

109

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

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 featuresof 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

. 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

. 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

propertiesof 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

, 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

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;

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

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

in 2007 and were subsequently termed

MenSCs

167

. 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

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-

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

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-

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

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

dierentiation

Engraftment Angiogenesis induction Fibrosis inhibition lmmunosuppression Autophagy

activation

Anti-oxidation Apoptosis

inhibition

Mechanisms

Approaches

BFSCs

VEGF PGE2

IL-6

HGF

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 Table2).

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

1.2million 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

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 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

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

↓ Inlammation

↑ 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 dierentiation

• Urothelial dierentiation

d Urinary tract defect

• USCs

• Cell engraftment

↓ Oxidative stress

↓ Fibrosis

↓ Inlammation

↓ Muscle cell apoptosis

e Overactive bladder

• USCs • AFSCs

↓ Fibrosis

↓ Oxidative stress

↓ Inlammation

↑ Neurogenesis

f Neurogenic bladder

• AFSCs • UCB-CD34+ cells

↓ Oxidative stress

↓ Fibrosis

↓ Apoptosis

↑ Neurogenesis

g Diabetic bladder

• USCs • AFSCs • USB-MNCs

• Cell engraftment

↓ Fibrosis

↓ Inlammation

↓ Apoptosis

↑ Angiogenesis

h Interstitial cystitis

• USCs • AFSCs • USB-MSCs

• Cell engraftment

↑ Host satellite cell proliferation and

↑ dierentiation

↑ Myogenesis

↑ Neurogenesis

↑ Angiogenesis

b SUI

• USCs

• UCB-MNCs

• Pre-dierentiated 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.