Anti-fibrotic effect of a selective estrogen receptor modulator in systemic sclerosis

Abstract

Background
The rarity of systemic sclerosis (SSc) has hampered the development of therapies for this intractable autoimmune disease. Induced pluripotent stem cell (iPSC) can be differentiated into the key disease-affected cells in vitro. The generation of patient-derived iPSCs has opened up possibilities for rare disease modeling. Since these cells can recapitulate the disease phenotypes of the cell in question, they are useful high-throughput platforms for screening for drugs that can reverse these abnormal phenotypes.

Methods
SSc iPSC was generated from PBMC by Sendai virus. Human iPSC lines from SSc patients were differentiated into dermal fibroblasts and keratinocytes. The iPSC-derived differentiated cells from the SSc patients were used on high-throughput platforms to screen for FDA-approved drugs that could be effective treatments for SSc.

Results
Skin organoids were generated from these cells exhibited fibrosis that resembled SSc skin. Screening of the 770-FDA-approved drug library showed that the anti-osteoporotic drug raloxifene reduced SSc iPSC-derived fibroblast proliferation and extracellular matrix production and skin fibrosis in organoids and bleomycin-induced SSc-model mice.

Conclusions
This study reveals that a disease model of systemic sclerosis generated using iPSCs-derived skin organoid is a novel tool for in vitro and in vivo dermatologic research. Since raloxifene and bazedoxifene are well-tolerated anti-osteoporotic drugs, our findings suggest that selective estrogen receptor modulator (SERM)-class drugs could treat SSc fibrosis.

Full text

Kimetal. Stem Cell Research & Therapy (2022) 13:303
https://doi.org/10.1186/s13287-022-02987-w
RESEARCH
Anti-brotic eect ofaselective estrogen
receptor modulator insystemic sclerosis
Yena Kim1,2, Yoojun Nam2, Yeri Alice Rim1 and Ji Hyeon Ju1,2,3*
Abstract
Background: The rarity of systemic sclerosis (SSc) has hampered the development of therapies for this intractable
autoimmune disease. Induced pluripotent stem cell (iPSC) can be differentiated into the key disease-affected cells
in vitro. The generation of patient-derived iPSCs has opened up possibilities for rare disease modeling. Since these
cells can recapitulate the disease phenotypes of the cell in question, they are useful high-throughput platforms for
screening for drugs that can reverse these abnormal phenotypes.
Methods: SSc iPSC was generated from PBMC by Sendai virus. Human iPSC lines from SSc patients were differenti-
ated into dermal fibroblasts and keratinocytes. The iPSC-derived differentiated cells from the SSc patients were used
on high-throughput platforms to screen for FDA-approved drugs that could be effective treatments for SSc.
Results: Skin organoids were generated from these cells exhibited fibrosis that resembled SSc skin. Screening of
the 770-FDA-approved drug library showed that the anti-osteoporotic drug raloxifene reduced SSc iPSC-derived
fibroblast proliferation and extracellular matrix production and skin fibrosis in organoids and bleomycin-induced SSc-
model mice.
Conclusions: This study reveals that a disease model of systemic sclerosis generated using iPSCs-derived skin orga-
noid is a novel tool for in vitro and in vivo dermatologic research. Since raloxifene and bazedoxifene are well-tolerated
anti-osteoporotic drugs, our findings suggest that selective estrogen receptor modulator (SERM)-class drugs could
treat SSc fibrosis.
Keywords: Systemic sclerosis, Induced pluripotent stem cells, 3D skin organoid, Disease modeling, High-throughput
screening, Drug repositioning, Raloxifene
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Background
Scleroderma (SSc) is heterogeneous disease, and the
pathogenesis is characterized by three hallmarks of small
vessel vasculopathy, autoantibodies, and fibroblast dys-
function resulting in increased deposition of extracellular
matrix [1, 2]. SSc patients exhibit progressive fibrosis and
vascular abnormalities in the skin and multiple internal
organs that can lead to fatal systemic complications [3,
4]. ere are two major classifications of scleroderma:
localized sclerosis and systemic sclerosis. Localized scle-
rosis is usually founded in only a few places on the skin or
muscles, and rarely spread elsewhere. Localized sclerosis
is rarely developed to systemic scleroderma. Systemic
sclerosis is affected the connective tissue in many parts
of the body. Systemic scleroderma is involved the skin,
esophagus, gastrointestinal tract, lung, kidney, heart, and
other internal organs and affected blood vessels, muscle,
and joint. Systemic sclerosis is recognized of two major
types as diffuse scleroderma and limited scleroderma.
In diffuse scleroderma, skin thickness and fibrosis are
occurred more rapidly and spread to other skin area than
limited scleroderma [5]. e exact pathophysiological
Open Access
*Correspondence: juji@catholic.ac.kr
1 Catholic iPSC Research Center, College of Medicine, The Catholic University
of Korea, Seoul, South Korea
Full list of author information is available at the end of the article
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
mechanism of systemic sclerosis is not known, and the
treatment of SSc continues to present a major challenge
[6, 7], partly because of its heterogeneity and partly
because it is an orphan disease and patient-derived bio-
materials are scarce. e latter has greatly hampered
research on SSc pathophysiology and the development of
effective treatments.
A promising strategy for overcoming the biomaterial
scarcity in orphan diseases is to generate induced pluri-
potent stem cells (iPSCs) by transducing the somatic
cells of patients with reprogramming factor genes (e.g.,
OCT4, SOX2, KLF4, and c-MYC) [8–10]. e resulting
iPSCs can be differentiated into the key disease-affected
cells invitro. Since these cells can recapitulate the disease
phenotypes of the cell in question, they are useful high-
throughput platforms for screening for drugs that can
reverse these abnormal phenotypes.
Organoid is a 3D multicellular invitro tissue construct
that mimics its corresponding in vivo organ, such that
can be derived from adult cells or pluripotent stem cells
[11]. Organoid can be used to study aspects of that organ
in the culture dish [12, 13]. us, we generated iPSC lines
from peripheral blood mononuclear cells (PBMCs) from
SSc patients and healthy controls and differentiated them
into skin cells, namely keratinocytes and fibroblasts. We
generated the 3D skin using iPSC-derived keratinocytes
and fibroblasts and that skin mimics its correspond-
ing in vivo skin. When these cells were co-cultured in
a three-dimensional layered system, they formed skin
organoids, whether the skin organoids recapitulated the
SSc-skin phenotype, both invitro and when xenografted
onto immunodeficient mice, was assessed.
We also used the iPSC-derived fibroblasts from the SSc
patients as high-throughput platforms to screen for FDA-
approved drugs that could be effective treatments for
SSc. e iPSC-derived fibroblasts were used because the
main therapeutic target in SSc is its rampant fibrosis, and
therapies that can halt fibrosis in SSc are not yet available
[14]. Our experiments showed that raloxifene, a selective
estrogen receptor antagonist, may be a candidate treat-
ment for SSc. us, the SSc iPSC-based platform was
useful for disease modeling, drug screening, and drug
repositioning.
Materials andmethods
PBMC isolation
Blood was collected into heparin tubes, diluted with
phosphate-buffered saline (PBS), and centrifuged
through a Ficoll gradient (GE Healthcare) for 30min at
850g. e PBMCs were washed, resuspended in Stem-
Span medium (STEMCELL Technologies) supplemented
with CC110 cytokine cocktail (STEMCELL Technolo-
gies), and cultured for 5days at 37℃ in 5% CO2 before
being reprogrammed. PBMCs were collected from five
SSc patients subjects, and the clinical information of the
patients is shown in Table1.
Generation ofiPSCs fromPBMC bySendai virus‑based
reprogramming
Human iPSCs were generated from the PBMCs by using
the CytoTune-iPS Sendai Reprogramming kit (Life Tech-
nologies) as described previously [15, 16]. us, Sendai
viruses that expressed the four Yamanaka factors were
added to the PBMCs at a multiplicity of infection of 7.5
per 3 × 105 cells. After transduction, the PBMCs were
centrifuged at 1160g for 30min and then incubated at
37℃ in 5% CO2. e next day, the cells were transferred
to a vitronectin-coated plate (ermo Fisher Scien-
tific) and settled by centrifugation for 10min at 1160g.
ese reprogrammed iPSCs were maintained in vitron-
ectin-coated culture dishes containing TeSR-E8 medium
(STEMCELL Technologies) that was changed daily. Cell
morphology was examined by Leica microscopy.
Alkaline‑phosphatase andimmunocytochemical staining
ofiPSCs
e iPSCs were expanded for 5–7 days with daily
medium changes. e resulting large iPSC colonies were
subjected to alkaline-phosphatase staining and immuno-
cytochemical analysis. For alkaline-phosphatase staining,
the cells were fixed in 4% paraformaldehyde for 1–2min,
after which the staining solution (Fast Red Violet, Naph-
thol AS-BI phosphate solution, and water in a 2:1:1 ratio)
was added. After incubation in the dark for 15min, the
stained colonies were examined by bright-field micros-
copy. For immunocytochemical staining, the iPSCs were
washed with PBS and then fixed with 4% paraformalde-
hyde for 30min. e cells were permeabilized with 0.1%
Triton X-100 for 10min, blocked for 30 min at room
temperature in PBS containing 2% bovine serum albumin
(Sigma-Aldrich) (PBA), and stained at room temperature
for 2h with primary antibodies diluted in PBA as fol-
lows: Oct4 (Santa Cruz Biotechnology, 1:100 dilution),
Klf4 (Abcam, 1:250), Sox2 (BioLegend, 1:100), TRA-1–60
(EMD Millipore, 1:100), TRA-1–81 (EMD Millipore,
1:100), and SSEA4 (EMD Millipore, 1:200). e cells were
then treated with the secondary Alexa Fluor 594- and
488-conjugated secondary antibodies (Life Technolo-
gies) diluted 1:400 in PBA. After 1h incubation at room
temperature, the cells were washed, and counterstaining
was conducted with DAPI (blue). e cells were mounted
by using ProLong Antifade mounting reagent (ermo
Fisher Scientific) and analyzed by Leica immunofluores-
cence microscopy.
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
Table 1 Clinical information of systemic sclerosis patients
1, Female; 2, Male; ILD, interstitial lung disease; PAH, pulmonary artery hypertension; ANA, antinuclear antibody; NSIP, nonspecic interstitial pneumonia; UIP, usual interstitial pneumonia; GERD, gastroesophageal reux
disease
No Sex Age range Disease
duration,
years
Skin involvement ILD PAH GI involvement ANA Anti‑centromere Anti‑scl70
1 2 40–49 20 Limited Yes, NSIP No GERD 1:80, nucleolar Negative Negative
2 1 40–49 13 Diffuse Yes, NSIP + UIP Yes Recurrent paralytic ileus 1:1280, speckled Negative Negative
3 1 50–59 2 Diffuse Yes, UIP No GERD 1:1280, cytoplasmic Negative Positive
4 2 60–69 5 Diffuse Yes, NSIP No GERD 1:80, speckled Negative Negative
5 1 50–59 16 Diffuse No No GERD, scleroderma esophagus 1:640, speckled Negative Negative
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
Teratoma formation
All procedures involving animals were conducted in
accordance with the Laboratory Animals Welfare Act,
the Guide for the Care and Use of Laboratory Animals,
and the Guidelines and Policies for Rodent Experimenta-
tion of the Institutional Animal Care and Use Committee
of the College of Medicine of the Catholic University of
Korea. e study protocol was approved by the Institu-
tional Review Board of the Catholic University of Korea
(CUMC-2016-0291-02). To further assess iPSC pluri-
potency, their ability to form teratomas was examined
[17–19]. Pluripotency of the cell lines is confirmed by
whether the teratoma contains tissues derived from each
of the embryonic germ layers: endoderm, mesoderm,
and ectoderm. us, the iPSC colonies were dissociated
and 1 × 106 cells were resuspended in a 1:1 solution of
Matrigel (BD Biosciences) and Dulbecco’s Modified Eagle
Medium (DMEM, Gibco) mixed 1:1 with F12 medium
(Gibco). e iPSCs were then injected into the testis cap-
sule of immunodeficient mice (NOD/SCID, Jackson).
After 6–12weeks, the tumor tissue was excised and sub-
jected to H&E staining and histology to assess whether
all three germ layers were present. Histological analysis
was examined by Leica microscopy.
Dierentiation ofiPSCs intokeratinocytes andbroblasts
e iPSCs were induced to differentiate into keratino-
cytes and fibroblasts as described previously [20–24]. In
our previous study, we established the protocol for dif-
ferentiating iPSCs into fibroblast and keratinocyte [25,
26]. Briefly, the iPSCs were first induced to form EBs by
using the hanging drop method. is method ensures
uniform and well-controlled differentiation. e iPSCs
were differentiated into keratinocytes by attaching the
EBs to a plate coated with type IV collagen (Santa Cruz
Biotechnology) on day 0. Over the next 21days, the EBs
were cultured sequentially with three keratinocyte differ-
entiation media on days 1–7, 8–11, and 12–30, respec-
tively. Keratinocyte differentiation medium 1 was a 3:1
mixture of DMEM and F12 medium supplemented with
2% FBS, 0.3 mmol/l L-ascorbic acid, 5 μg/ml insulin,
24μg/ml adenine, 1μg/ml retinoic acid (Sigma-Aldrich),
25 ng/ml BMP4, and 20 ng/ml EGF. Keratinocyte dif-
ferentiation medium 2 consisted of defined keratinocyte
serum-free medium (Gibco) without FBS but supple-
mented with all the other supplements in keratinocyte
differentiation medium 1 (the concentrations were all the
same except adenine was present at 10μg/ml). Keratino-
cyte differentiation medium 3 consisted of a 1:1 mixture
of keratinocyte serum-free medium (Gibco) and defined
keratinocyte serum-free medium supplemented with
25ng/ml BMP4 and 20ng/ml EGF.
To induce iPSCs to differentiate into fibroblasts, the
EBs were attached to a Matrigel-coated plate on day
0. e cells were then incubated sequentially in three
fibroblast differentiation media on days 1–3, 4–6, and
7–14, respectively. Fibroblast differentiation medium 1
consisted of a 3:1 mixture of DMEM and F12 medium
supplemented with 5% fetal bovine serum (FBS), 5 μg/
ml insulin, 24 μg/ml adenine, and 10 ng/ml epidermal
growth factor (EGF; R&D). On days, 4–6 fibroblast dif-
ferentiation medium 1 was supplemented with 6.5ng/ml
bone morphogenetic protein-4 (BMP4; R&D). Fibroblast
differentiation medium 2 consisted of a 1:1 mixture of
DMEM and F12 medium supplemented with 5% FBS and
1% non-essential amino acids on day 7–14. On days 14
and 21, the cells were passaged onto non-coated and type
I collagen-coated dishes (BD Biosciences), respectively, in
fibroblast differentiation medium 1. For each experiment,
the eight iPSC cell lines were subjected to the differentia-
tion process five times.
RNA isolation andqRT‑PCR ofcells dierentiated
fromiPSCs
Total mRNA was extracted from the iPSC-derived fibro-
blasts and keratinocytes by using Trizol (Life Technolo-
gies) and cDNA was synthesized by using a RevertAid™
First Strand cDNA Synthesis kit (ermo Fisher Scien-
tific). RT-PCRs were performed with the LightCycler®
480 instrument (Roshe), the SYBR Green Real-time PCR
Master Mix (Roshe), and the OCT4, CD44, COL1A1,
COL1A2, COL3A1, vimentin, PAX6, SOX1, Np63, KRT5,
and KRT14 primer sequences shown in Table2. e gene
expression levels were normalized to GAPDH expression
levels.
Immunocytochemical staining ofcells dierentiated
fromiPSCs
e differentiated cells were fixed with 4% paraformalde-
hyde, permeabilized by using 0.1% Triton X-100, stained
with primary antibodies against Keratin14 (Abcam,
1:200), Np63 (Abcam, 1:100), vimentin (Abcam, 1:200)
or fibronectin (Abcam, 1:200) for 1 h, and then incu-
bated with Alexa488/594-conjugated goat anti-mouse or
goat anti-rabbit secondary antibodies. Counterstaining
was conducted with DAPI (blue). e stained cells were
observed by immunofluorescence microscopy.
Flow cytometric analysis ofcells dierentiated fromiPSCs
e differentiated cells were fixed by using a Foxp3 Tran-
scription Factor Staining Buffer Kit (Affymetrix) and then
stained with antibodies against CD73, CD105, CD45, and
CD34. e stained cells were then analyzed by using an
LSR Fortessa Cell Analyzer (BE Biosciences). e data
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
were analyzed by using Flowjo V10 single-cell analysis
software (TreeStar Inc).
Proliferation assay ofiPSC‑derived broblasts
e iPSCs before and after their differentiation into
fibroblasts were seeded onto 96-well plates, incubated
for 24h, and then incubated for 1–4 h with 10μl/well
of Cell Counting Kit-8 solution (Dojindo). Absorbance
at 450nm was measured by using a microplate reader
(VersaMax).
Western blot analysis ofiPSC‑derived broblasts
Cellular protein was harvested by using RIPA buffer
(Sigma) and quantified by using a BCA protein assay
(GenDEPOT). Equal protein amounts were resolved by
10% SDS-PAGE and transferred to polyvinylidene dif-
luoride membranes (Amersham Pharmacia Biotech).
e membranes were incubated overnight with specific
antibodies against α-SMA (Abcam), pSMAD2 (er-
mofisher), SMAD2 (ermofisher) and anti-GAPDH
(Abcam). e next day, the membranes were washed,
incubated with a peroxidase-linked IgG (Abcam), and
visualized by using an ECL kit (WESTSAVE Gold).
Hydroxyproline assay ofiPSC‑derived broblasts
Measurement of collagen concentrations was performed
using hydroxyproline assay kit (Sigma-Aldrich). Homog-
enize cells or supernatant were mixed with hydrochlo-
ric acid and incubated at 120 ℃ for 3h. Add 5mg of
activated charcoal and centrifuge at 13,000g for 2min.
Transfer supernatant to a 96 well plate and dry all wells
under vacuum. Add the Chloramine T/Oxidation Buffer
mixture to each well and incubate at RT for 5 minutes.
After 5 minutes, add the Diluted DMAB Reagent to each
sample and incubated for 90min at 60 ℃. Absorbance
at 560nm was measured by using a microplate reader
(VersaMax).
Three‑dimensional skin organoid culture
Differentiated fibroblasts were resuspended in neu-
tralized type 1 collagen solution (BD Biosciences), and
2 × 105 cells were added to each insert of a Transwell
plate (Corning) with fibroblast differentiation medium 1.
After 5–7days of incubation, 1 × 106 keratinocytes were
seeded onto the fibroblast layer in low-calcium epithelial
medium for 2days. e bilayer was then submerged for
2days in normal calcium medium. After another 4days,
the normal calcium medium was only added to the bot-
tom of the insert so that an air/liquid interface was
created.
Histological analysis oftheskin organoids
e organoids were fixed in 4% paraformaldehyde for 1h
at room temperature and then dehydrated and cleared
with graded ethanol and xylene. After paraffin infiltration
and embedding in paraffin blocks, the organoids were
sectioned into 8μm slices by using a microtome. e skin
area and thickness of the organoids were determined by
image j program. Quantify was performed according to
the manufacturer’s instruction.
Phospho‑Kinase Array
Proteome Profiler human Phospho-Kinase Array Kit
(R&D Systems) was performed according to the manu-
facturer’s instructions for analyzing proteomics analysis.
Briefly, phospho-kinase array detects relative phospho-
rylation levels of individual analytes. Parts A and B of
each array were incubated with 200μg of cell lysate. Each
Table 2 Sequence of primers used for quantitative RT-PCR
Target name Direction Primer sequence Size
hOCT4 Forward ACC CCT GGT GCC GTGAA 190
Reverse GGC TGA ATA CCT TCC CAA ATA
hCD44 Forward AAG GTG GAG CAA ACA CAA CC 151
Reverse AGC TTT TTC TTC TGC CCA CA
hCOL1A1 Forward CCC CTG GAA AGA ATG GAG ATG 148
Reverse TCC AAA CCA CTG AAA CCT CTG
hCOL1A2 Forward GGA TGA GGA GAC TGG CAA CC 77
Reverse TGC CCT CAG CAA CAA GTT CA
hCOL3A1 Forward CGC CCT CCT AAT GGT CAA GG 161
Reverse TTC TGA GGA CCA GTA GGG CA
hVimentin Forward GAG AAC TTT GCC GTT GAA GC 170
Reverse TCC AGC AGC TTC CTG TAG GT
hPAX6 Forward GTC CAT CTT TGC TTG GGA AA 110
Reverse TAG CCA GGT TGC GAA GAA CT
hSOX1 Forward CAC AAC TCG GAG ATC AGC AA 133
Reverse GGT ACT TGT AAT CCG GGT GC
hNp63 Forward GGA AAA CAA TGC CCA GAC TC 294
Reverse GTG GAA TAC GTC CAG GTG GC
hKRT5 Forward ACC GTT CCT GGG TAA CAG AGC CAC 198
Reverse GCG GGA GAC AGA CGG GGT GATG
hKRT14 Forward GCA GTC ATC CAG AGA TGT GACC 181
Reverse GGG ATC TTC CAG TGG GAT CT
hGAPDH Forward ACC CAC TCC TCC ACC TTT GA 110
Reverse CTG TTG CTG TAG CCA AAT TCGT
mCOL1A1 Forward GCA ACA GTC GCT TCA CCT ACA 138
Reverse CAA TGT CCA AGG GAG CCA CAT
mCOL3A1 Forward TGA GCG TGG CTA TTC CTT CGT 76
Reverse GCC GTG GCC ATC TCA TTT TCAA
mACTA2 Forward GTT CTA GAG GAT GGC TGT ACTA 108
Reverse TTG CCT TGC GTG TTT GAT ATTC
mGAPDH Forward ACC CCA GCA AGG ACA CTG AGC AAG 92
Reverse TGG GGG TCT GGG ATG GAA ATT GTG
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
spotted in duplicate with antibodies against 37 different
kinases.
Xenografting ofiPSC‑derived skin organoids inmice
All procedures involving animals were conducted in
accordance with the Laboratory Animals Welfare Act,
the Guide for the Care and Use of Laboratory Animals,
and the Guidelines and Policies for Rodent Experimenta-
tion of the Institutional Animal Care and Use Committee
of the College of Medicine of the Catholic University of
Korea. e study protocol was approved by the Institu-
tional Review Board of the Catholic University of Korea
(CUMC-2017-0150-01). About 1 × 2 cm of the dorsal
skin of male NOD/SCID mice (n = 5 per group, 6weeks
old, Jackson Laboratories) was removed and the orga-
noids from SSc patients and healthy controls were placed
in the defects and sutured with silk sutures by using the
tie-over dressing method. After 2weeks, the mice were
killed and their organoids were subjected to histology
using Leica microscopy.
Primary screening oftheFDA‑approved drug library
For the primary screen, iPSC-derived fibroblasts from
SSc patients were seeded into 96-well plates at 1 × 104
cells/well, incubated for 24h in fibroblast differentiation
medium 1, and then treated with each FDA-approved
drug (ENZO life Science) or the vehicle control (DMSO)
for 1 h. Cell proliferation was detected by using Cell
Counting Kit-8 (Dojindo). e drugs selected in the pri-
mary screen were then assessed for their ability to reduce
the total collagen content by using the hydroxyproline
assay.
Treatment ofmice withbleomycin‑induced SSc
withraloxifene
All procedures involving animals were conducted in
accordance with the Laboratory Animals Welfare Act,
the Guide for the Care and Use of Laboratory Animals,
and the Guidelines and Policies for Rodent Experimenta-
tion of the Institutional Animal Care and Use Committee
of the College of Medicine of the Catholic University of
Korea. e study protocol was approved by the Institu-
tional Review Board of the Catholic University of Korea
(CUMC-2017-0128-05). To generate mice with bleomy-
cin-induced SSc, C57BL/6 mice were injected subcuta-
neously with 1μg bleomycin (NIPPON KAYAKU) every
day for 3weeks. e model was generated by daily sub-
cutaneous bleomycin injections. Starting 3days after the
bleomycin injections began, the mice were also injected
subcutaneously with 10mg/kg raloxifene (Takeda). Start-
ing three days later, the mice were also treated with daily
subcutaneous injections of raloxifene. e mice were
killed. e double injections were continued for the
remaining 21days of the experiment. e skin was sub-
jected to histology and western blot analysis of fibrosis
markers expression.
Histological analysis
Skin was fixed in 4% paraformaldehyde at room tem-
perature, and then dehydrated and cleared using graded
ethanol and xylene. After paraffin infiltration and embed-
ding, paraffin blocks of the skin were sectioned at a thick-
ness of 8μm using a microtome. Slides were dried for
60min at 60 °C and deparaffinized by two incubations
with xylene. Sections were rehydrated using a decreasing
sequential ethanol series and rinsed under tap water for
5min. For hematoxylin and eosin (H&E) staining, sec-
tions were incubated in Harris’ hematoxylin solution for
10min, washed with 1% HCl-ethanol, neutralized in 0.2%
ammonia water, and counterstained with eosin for 1min.
For Picrosirius Red (PSR) staining, slides were incubated
in PSR solution for 1 h and washed with acetic acid.
For Masson’s trichrome staining, slides were re-fixed
in Bouin’s solution overnight at room temperature and
incubated in Weigert’s hematoxylin for 10min, Biebrich
scarlet-acid fuchsin for 5min, and a mixture of phospho-
tungstic acid, phosphomolybdic acid, and distilled water
(1:1:2) for 10min. ereafter, slides were directly trans-
ferred to 2% aniline blue, incubated for 5min, washed
with 1% acetic acid, and then incubated in an increas-
ing sequential ethanol series. Ethanol was cleared using
xylene and slides were mounted using VectaMount per-
manent mounting medium (Vector laboratories Burl-
ingame, CA, USA). Staining was examined underneath a
bright-field microscope.
Statistical analyses
e results are expressed as mean and standard error
of the mean. Error bars indicate the standard error of
the mean. Groups were compared by using Student’s
t test and calculating the one-tailed p value (*p < 0.05,
**p < 0.01, ***p < 0.001 indicated statistical significance).
All statistical analyses were performed by using Graph-
Pad Prism 9 (GraphPad Software).
Results
Generation ofiPSCs fromSSc patients andcontrol subjects
Stable iPSC lines were generated from five SSc patients
and three healthy-control subjects by reprogramming
their PBMCs with Sendai viruses containing the four
Yamanaka factors (Klf4, Oct3/4, Sox2, and c-Myc) and
then passaging the virus-transduced cells. e clini-
cal information of the SSc patients is shown in Table1.
Regardless of whether they were from patients or healthy
controls, the iPSC lines resembled human embryonic-
stem cells in terms of morphology (Fig.1a and Additional
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
file1: Fig.S1a). ey also expressed alkaline phosphatase
(Fig. 1b and Additional file 1: Fig. S1b) and expressed
multiple pluripotency-marker proteins, namely Oct4,
SSEA4, Sox2, TRA-1-60, Klf4, and TRA-1-81 (Fig. 1c
and Additional file1: Fig.S1c). To confirm the pluripo-
tency of the iPSCs, they were injected into the testicular
capsule of immunodeficient mice to determine whether
they could induce teratomas [17–19]. Indeed, SSc iPSCs
proliferated and differentiated into all three germ lay-
ers (Fig.1d). Moreover, they expressed several pluripo-
tent-marker genes (OCT4, SOX2, NANOG, and LIN28)
(Fig. 1e and Additional file 1: Fig. S1d). us, iPSCs
were successfully generated from the PBMCs of the SSc
patients and the healthy controls.
Generation ofiPSC‑derived keratinocytes andbroblasts
e iPSC lines were induced to form embryoid bodies
(EBs), after which the EBs were transferred to type IV
collagen- or Matrigel-coated dishes and subjected to cul-
ture conditions that caused their outgrowing cells to dif-
ferentiate into keratinocytes or fibroblasts [20, 22, 24, 27].
On day 14, the iPSC-derived keratinocytes had a pri-
mary keratinocyte-like cobblestone morphology when
cultured on type IV collagen-coated dishes (Fig. 2a).
ese cells expressed the keratinocyte-marker Np63
and KRT14 protein (Fig.2b). eir expression of OCT4,
the pluripotency-marker gene, and the neuroectoderm-
marker genes PAX6 and SOX1 was decreased compared
to iPSCs (Fig.2c). us, the iPSC-derived keratinocytes
lacked the characteristics of iPSCs and did not differen-
tiate along the neuroectoderm lineage. ese cells also
expressed the keratinocyte-marker genes Np63, KRT5,
and KRT14 (Fig.2c).
On day 21, the iPSC-derived fibroblasts had a simi-
lar morphology as 3T3 cells, which is an established
fibroblast-cell line [25]. SSc iPSC-derived fibroblasts
had a similar morphology as flat, elongated and spindle-
shaped (Fig.2d). ey expressed fibronectin and vimen-
tin protein, which are well-known markers of fibroblasts
(Fig.2e). Furthermore, flow cytometric analyses showed
that their expression of the hematopoietic stem cell
markers CD34 and CD45 was lowed and their expres-
sion of the fibroblast-surface markers CD73 and CD105
was high (Fig. 2f). ey also expressed OCT4, a pluri-
potency-marker gene, at lower levels than undifferenti-
ated iPSCs and expressed numerous fibroblast-marker
genes (i.e., COL1A1, COL1A2, COL3A1, ACTA2 and
vimentin). Especially, the gene expression was increased
in SSc iPSC-derived fibroblast than health-control
(Fig.2g). us, these results confirmed that iPSC-derived
keratinocytes and fibroblasts resembled primary cell
lines in terms of gene expression, protein expression and
morphology.
In vitro andinvivo modeling ofSSc disease byusing
dierentiated iPSC‑derived cells
e iPSC lines from the SSc patients and the healthy
controls did not differ in terms of proliferation (Fig.3a).
After differentiation, however, the fibroblasts derived
from the SSc iPSC lines proliferated more rapidly
than the equivalent cells from the fibroblast derived
from healthy-control iPSC lines (Fig. 3b). e SSc
Fig. 1 Characterization of induced pluripotent stem cells (iPSCs) from patients with systemic sclerosis (SSc) and healthy controls. a Morphology of
iPSCs, as determined by Leica microscopy. Scale bars, 200 μm. b Alkaline phosphatase staining of iPSCs. Scale bars, 200 μm. c Immunocytochemical
analysis of iPSC expression of the pluripotency-marker proteins Oct4, SSEA4, Sox2, TRA-1-60, Klf4, and TRA-1-81. Scale bars, 200 μm. d H&E staining
of the teratomas generated from the iPSCs. e RT-PCR analysis of iPSC expression of the pluripotency-marker genes OCT4, SOX2, NANOG, and LIN28.
All graphs show the mean and standard error of the mean of five replicates of each of the iPSC lines (*p < 0.05, **p < 0.01, ***p < 0.001, as determined
by Student’s t test)
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
iPSC-derived fibroblasts also expressed more alpha
smooth muscle actin(α-SMA) protein (Fig.3c–e) and
had higher total collagen concentrations (Fig. 3f).
us, the iPSC-derived fibroblasts from SSc patients
recapitulated the fibrotic phenotype of SSc fibroblasts.
Analyzing the phosphorylation profiles of kinase and
their protein substrates is essential for understanding
how cells recognize and respond to changes in their
environment. Severe fibrotic tissues from patients had
increased levels of phosphorylation [28–34]. e SSc
iPSC-derived fibroblasts increased phosphorylated
protein than HC iPSC-derived fibroblasts. In particu-
lar, the phosphorylation was increased in ERK1/2,
GSK-3a/b, CREB, c-Jun, PRAS40, and HSP60 (Fig.3g,
h). Furthermore, the iPSC-derived fibroblasts were
cultured to produce three-dimensional (3D) fibroblast
layer as dermal equivalent. e SSc-derived fibroblast
layers were thicker, had a greater surface area, and had
more cells than the healthy-control organoids (Fig.3i).
us, the SSc patient-derived 3D fibroblast layer reca-
pitulated the fibrotic phenotype of the skin in SSc.
Fig. 2 Differentiation of induced pluripotent stem cells (iPSCs) into fibroblasts and keratinocytes. a–c Characteristics of the iPSC-derived
keratinocytes on day 21. a Morphology, as determined by Leica microscopy. Scale bars, 100 μm. b Immunocytochemical analysis of Np63 (red) and
Keratin14 (green), together with DAPI staining (blue). Scale bars, 100 μm. c Gene expression of an iPSC marker (OCT4), neuroectoderm markers
(PAX6 and SOX1), and keratinocyte markers (Np63, KRT5, and KRT14). d–f Characteristics of the iPSC-derived fibroblasts on day 21. d Morphology,
as determined by Leica microscopy. Scale bars, 100 μm. e Immunocytochemical analysis of the fibroblast-marker proteins fibronectin (red) and
vimentin (red), together with DAPI staining (blue). Scale bars, 100 μm. f Flow cytometric analysis of CD34, CD45, CD73, and CD105 expression.
All graphs show the mean and standard error of the mean. g Expression of the pluripotency-marker gene OCT4 and the fibroblast-marker genes
COL1A1, COL1A2, COL3A1, ACTA2, and vimentin (*p < 0.05, **p < 0.01, ***p < 0.001, as determined by Student’s t-test)
Fig. 3 In vitro and in vivo modeling of systemic sclerosis using the induced pluripotent stem cells (iPSCs). a, b Proliferation assay of iPSCs (a)
and their differentiation into fibroblasts (b). c Immunocytochemical analysis of α-SMA expression (green), together with DAPI staining was also
performed (blue). Scale bars, 200 μm. d Expression of the fibrosis marker α-SMA, as determined by western blot. e Quantification of the relative
band intensity by using imageJ. f Quantification of the total collagen contents by using the hydroxyproline assay. g Proteome profiler human
phospho-kinase array of HC iPSC-derived fibroblast. h Proteome profiler human phospho-kinase array of SSc iPSC-derived fibroblast. i Histological
analysis of the organoids and quantification of their culture area and thickness. Scale bars, 200 μm. j Histological analysis of the skin thickness and
area of the transplanted organoids after 2 weeks. All graphs show the mean and standard error of the mean
(See figure on next page.)
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Fig. 3 (See legend on previous page.)
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
e iPSC-derived fibroblasts and keratinocytes were
cultured together to produce three-dimensional skin
organoids. We asked whether the SSc patient-derived
organoids continued to exhibit accelerated fibrosis when
they were xenografted onto immunodeficient mice. us,
the organoids from the SSc patients and healthy controls
were transplanted in the dorsal skin of NOD/SCID mice.
After 2weeks, the mice were killed, and the organoids
were subjected to histology. Indeed, the SSc patient-
derived organoids transplanted mice skin were thicker
and had greater surface areas than the healthy control-
derived organoids (Fig.3j). e expression of collagen3
and α-SMA, fibrosis marker, was increased in SSc iPSC-
derived skin organoid transplanted mice than health-
control (Fig.3j). erefore, the SSc patient-derived skin
organoids reflect the skin pathology of SSc invitro and
invivo.
FDA‑approved drug library screening using iPSC‑derived
disease model
e most prominent feature of SSc is elevated fibroblast
proliferation [23, 35]. e ability of 770 FDA-approved
drugs to reduce the proliferation of iPSC-derived fibro-
blasts from the SSc patients was examined by using the
Cell Counting Kit-8 (CCK-8) assay. In total, 48 drugs
reduce the proliferation of SSc iPSC-derived fibroblasts
(Fig.4a).
Since fibroblasts from SSc patients also express higher
collagen levels [36–38], we subjected the 48 selected
drugs to a second screen for their ability to reduce
the total collagen content of the iPSC-derived fibro-
blasts from the SSc patients. e hydroxyproline assay
showed that 13 of the 48 drugs had this effect (Fig.4b).
Two particularly effective drugs were dactinomycin and
raloxifene.
Transforming-growth factor (TGF)-β1 plays a major
augmenting role in fibrosis [39–41]. Indeed, when we
treated the iPSC-derived fibroblasts from SSc patients
and healthy controls with TGF-β1, their total collagen
content rose significantly. However, co-treatment with
dactinomycin or raloxifene abrogated the ability of TGF-
β1 to augment the total collagen levels in the fibroblasts
(Fig.4c).
Finally, the fibrosis in SSc associates with upregu-
lated α-SMA expression in fibroblasts [42, 43]. When
the iPSC-derived fibroblasts from the SSc patients were
treated with raloxifene, their elevated α-SMA expres-
sion dropped. Dactinomycin was not more effective than
raloxifene in reducing α-SMA expression (Fig. 4d, e).
erefore, we selected raloxifene as the hit drug for fur-
ther analysis.
Anti‑brotic eect ofraloxifene invitro
To confirm that raloxifene has anti-fibrotic effects,
the iPSC-derived fibroblasts were expanded to conflu-
ency, treated with TGF-β1 with or without raloxifene,
and scratched. e wound length was measured 12 and
24 h later as an estimate of proliferation. Indeed, the
TGF-β1-augmented proliferation of the fibroblasts was
significantly reduced when raloxifene was also present
(Fig. 5a–c). When the three-dimensional iPSC-derived
fibroblast layers were treated with TGF-β1, the skin
thickness increased. Treatment with raloxifene decreased
this effect of TGF-β1 (Fig. 5d–f). Analyzing the phos-
phorylation profiles of kinase with treatment of ralox-
ifene, the phosphorylation was decreased in GSK-3a/b
(Fig.5g, h). Similarly, when iPSC-derived fibroblasts from
SSc patients were treated with various concentrations
of raloxifene. is treatment also decreased the total
collagen concentration in the cells in a raloxifene dose-
dependent manner (Fig.5i). Expression level of α-SMA
dropped in a concentration-dependent manner (Fig.5j,
k). Raloxifene belongs to a class of selective estrogen
receptor modulators (SERMs) that, depending on the tar-
get tissue, can act on the estrogen receptor as either an
agonist or an antagonist [44]. SERMs include tamoxifene,
raloxifene, lasofoxifene, bazedoxifene, and clomiphene
citrate. Bazedoxifene has been shown to be relatively
safe and well tolerated. To confirm SERMs has anti-
fibrotic effects, raloxifene or bazedoxifene was treated
iPSC-derived fibroblasts. Also, TGF-β1/SMAD signaling
plays an important role in the pathogenesis of SSc [45].
e relative expression ratio of pSMAD2/SMAD2 was
increased by treatment TGF-β1. By treating raloxifene or
bazedoxifene, the expression of phosphorylated SMAD2
was downregulated (Fig. 5l, m). us, raloxifene could
reduce the proliferation and fibrotic-factor expression
(See figure on next page.)
Fig. 4 Screening of the FDA-approved drug library for an agent that reduces the accelerated fibrosis of induced pluripotent stem cells
(iPSCs)-derived fibroblasts from systemic sclerosis patients. a Primary screen searching for drugs that reduced iPSC-derived fibroblast proliferation,
as measured by using the Cell Counting Kit-8 assay. b Secondary screen of the drugs selected in primary screening. The ability of the drugs to
reduce the total collagen content of the iPSC-derived fibroblasts was examined by using the hydroxyproline assay. c The ability of TGF-β1 to
increase the total collagen levels of iPSC-derived fibroblasts, and the ability of dactinomycin and raloxifene to reduce this augmenting ability
of TGF-β1, was examined by using the hydroxyproline assay. d, e The effect of TGF-β1 treatment with dactinomycin or raloxifene on the α-SMA
expression of iPSC-derived fibroblasts was examined by western blot analysis. All graphs show the mean and standard error of the mean (*p < 0.05,
**p < 0.01, ***p < 0.001, as determined by Student’s t test)
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Fig. 4 (See legend on previous page.)
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
of iPSC-derived fibroblasts in a dose-dependent man-
ner. is suggests that raloxifene could have anti-fibrotic
effects in iPSC-derived SSc model.
Furthermore, to confirm that raloxifene had anti-
fibrotic effect in primary cells, health control and patient-
derived fibroblasts were collected and expended. When
the patient-derived fibroblasts were treated with baze-
doxifene or raloxifene, their morphology was not changed
(Additional file2: Fig.S2a). Bazedoxifene and raloxifene
reduce the proliferation of primary fibroblast (Additional
file 2: Fig. S2b). e gene expression of fibrosis mark-
ers was increased in SSc fibroblast than health-control
(Additional file2: Fig.S2c, d). By treating bazedoxifene
or raloxifene, wound length was reduced (Additional
file2: Fig.S2e–g) and total collagen concentration in the
cells was also decreased than vehicle (Additional file 2:
Fig.S2h). Similarly, when the three-dimensional primary
fibroblast layers were treated with bazedoxifene or ralox-
ifene. Raloxifene was more effective than bazedoxifene
in reducing thickness of 3D fibroblast layers (Additional
file2: Fig.S2i–k). Total collagen dropped in a concen-
tration-dependent manner with various concentrations
of raloxifene (Additional file2: Fig. S2l). is suggests
that raloxifene could have anti-fibrotic effects in patient-
derived primary fibroblasts.
Anti‑brotic eect ofraloxifene inamice model ofSSc
e bleomycin-induced mice model is a commonly used
chemical model of SSc [46, 47]. erefore, to confirm
that raloxifene has anti-fibrotic effects in mice model of
SSc, we generated the bleomycin-induced model of SSc
by daily subcutaneous bleomycin injections. Starting
3days later, the mice were also treated with daily sub-
cutaneous injections of raloxifene. Raloxifene is anti-
osteoporotic drug and used for the treatment prevention
of osteoporosis in postmenopausal women. Raloxifene
is a selective estrogen receptor modulator (SERM). We
treated bazedoxifene, which is a kind of SERM-class drug
in bleomycin mice model. After 3weeks, the mice were
killed, and their skin thickness was examined by histol-
ogy (Fig. 6a, b). Also, the gene expression of fibrosis
markers COL1A1, COL3A1 and ACTA2 was increased
by bleomycin induction and this effect was attenuated by
raloxifene treatment (Fig.6c–e). eir skin expression of
pSMAD was also assessed by western blot analysis. e
expression level of pSMAD2/SMAD2 was increased by
treatment bleomycin. By treating raloxifene, the expres-
sion was downregulated (Fig.6f, g).
Many variables of fibrosis marker were increased by the
bleomycin injections but the raloxifene injections signifi-
cantly reduced these effects. erefore, these results sug-
gested that raloxifene therapy had anti-fibrotic effects in
the bleomycin-induced model of SSc.
Discussion
Fibrosis is a pathological symptom of chronic inflamma-
tory disease. Fibrosis, or scarring, is determined by the
accumulation of excess extracellular matrix component
(ECM) such as collagen and fibronectin [48, 49]. Fibrosis
becomes dysregulated following tissue injury. When tis-
sues are injured, fibroblast becomes activated, increasing
their contractility, secretion of inflammatory cytokines,
and synthesis of ECM components. Fibroblasts are
the common cell type of the connective tissues and the
major source of the ECM. Also, fibroblasts are the effec-
tive mediator of the pathological fibrotic accumulation of
ECM, proliferation and differentiation that results in tis-
sue injury and chronic inflammation [50]. Skin fibrosis
occurs locally in response to dermal injury according to
burn, surgery, trauma, infection, or radiation, or in asso-
ciation with systemic diseases such as scleroderma and
graft-versus-host disease [51–53]. Scleroderma (SSc) is
an autoimmune disease associated with high morbidity
and mortality [51, 54]. SSc is heterogeneous and rare dis-
ease, and the pathogenesis is characterized by three hall-
marks of small vessel vasculopathy, autoantibodies, and
fibroblast dysfunction resulting in increased deposition
of extracellular matrix [1, 2]. In SSc, fibrosis occurs in the
skin and progress to many organs, including esophagus,
gastrointestinal track, lung, kidney, heart [3, 4]. Fibrosis
is the result of activation of fibroblast and deposition of
excessive ECM, which are critical features of SSc [55].
Development of effective treatment of SSc has been ham-
pered by a lack of sufficient understanding of its patho-
physiology, partly because of its heterogeneity and partly
because it is an orphan disease and patient-derived bio-
materials are scarce [6, 7].
Fig. 5 Anti-fibrotic effects of raloxifene in vitro. a–c The iPSC-derived fibroblasts from SSc patients were subjected to the scratch assay and the
wound length was measured 12 (b) and 24 (c) hours later. Scale bars, 100 μm. d–f The three-dimensional dermal equivalent derived from SSc
patients were treated with TGF-β1 in the presence or absence of raloxifene and the skin thickness and area were determined by histology. Scale
bars, 100 μm. g Proteome profiler human phospho-kinase array of GSK-3α/β with treatment raloxifene. h Quantification of the relative intensity by
using imageJ. i Total collagen concentration with various concentrations of raloxifene was assessed by the hydroxyproline assay. j α-SMA expression
with various concentrations of raloxifene was assessed by western blot analysis. k Quantification of the relative band intensity by using imageJ.
l Expression of phosphorylated SMAD2 signaling with treatment raloxifene and bazedoxifene in iPSC-Fs by western blot assay. m Quantification
of the relative band intensity by using imageJ. All graphs show the mean and standard error of the mean (*p < 0.05, **p < 0.01, ***p < 0.001, as
determined by Student’s t test)
(See figure on next page.)
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Recently, a promising strategy for overcoming the
biomaterial scarcity in orphan diseases is to gener-
ate induced pluripotent stem cells (iPSCs) [56–58]. e
present study showed that the paucity of biomaterials in
dermatology can be overcome by generating iPSCs from
PBMCs of patients and inducing them to differentiate
Fig. 5 (See legend on previous page.)
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
into skin cells, particularly the fibroblasts, which play a
key role in the pathophysiology [23, 35, 43]. We observed
that the fibroblasts derived from iPSCs of SSc patients
exhibited significantly greater proliferation and produced
more ECM than the equivalent fibroblasts from healthy-
control subjects. Severe fibrotic tissues from patients had
increased levels of phosphorylation [28–34]. Phosphoryl-
ation of fibrosis related proteins was increased SSc iPSC-
derived fibroblasts than HC iPSC-derived fibroblast.
Moreover, when they were used together with keratino-
cytes derived from the same iPSC lines to generate skin
organoids, the organoids from the SSc patients were
thicker and larger than those from the healthy controls.
In addition, when these organoids were transplanted
into the dorsal skin of immunodeficient mice, they were
thicker than the healthy-control organoids. us, fibro-
blasts derived from SSc iPSCs bear the same pathologi-
cal features as fibrosis of SSc patients. is suggests that
these cells may be useful for studying the pathological
mechanism underlying SSc and for identifying effective
therapies. is notion is supported by the fact that SSc is
not a Mendelian genetic disorder: environmental changes
and the resulting epigenetic modifications can be impor-
tant factors that drive the development of this disease
[59, 60]. Moreover, it has been shown that since epige-
netic modifications can remain after somatic cell repro-
gramming, cells that are differentiated from the iPSCs of
somatic cells can reflect the disease phenotypes that lead
to diseases such as cardiomyopathy and neuronal disease
[61, 62].
In recent years, considerable effort has gone into iden-
tifying anti-fibrotic therapies [14]. Nevertheless, only a
few therapies that can suppress fibrosis have been dis-
covered [63]. One may be mesenchymal stem cell (MSC)
therapy. However, the anti-fibrotic effects of MSC are
either small or only effective in the early stage of fibro-
sis [64]. Human cells that are derived from iPSCs are
increasingly being used as high-throughput platforms for
the screening of diverse compounds [9, 10, 65]. We used
this approach to identify drugs that can suppress the pro-
fibrotic activity of SSc fibroblasts. us, iPSC-derived
fibroblasts from SSc patients were tested with the FDA-
approved drug library, which consists of 770 drugs. e
first screen showed that 48 drugs effectively suppressed
SSc-fibroblast proliferation. e second screen then
showed that 13 drugs efficiently reduced not only SSc-
fibroblast proliferation but also ECM protein synthesis.
Interestingly, of these 13 drugs, raloxifene was the most
effective anti-fibrotic agent. Raloxifene belongs to a class
of selective estrogen receptor modulators (SERMs) that,
depending on the target tissue, can act on the estro-
gen receptor as either an agonist or an antagonist [44].
Raloxifene has strong anti-estrogenic effects and is thus
a very well-known and effective anti-osteoporotic drug
[66]. Two of the most common SERMs are tamoxifene
and raloxifene. ere are several others as well, includ-
ing lasofoxifene, bazedoxifene, and clomiphene citrate.
Bazedoxifene has been shown to be relatively safe and
well tolerated. SERMs are competitive partial agonist of
the ER. Different tissues have different degrees of sensi-
tivity to the activity of endogenous estrogens, so SERMs
produce estrogenic or anti-estrogenic effects depending
in the specific tissue in question as well as the percent-
age of intrinsic activity (IA) of the SERM [67]. Further-
more, affinities of estrogen receptor ligands for the ERα
and EBβ are used depending on the type of SERMs [68–
71]. e effects and affinities acting on the skin are dif-
ferent; it is expected that the effects will also be different
depending on the type of SERMs.
In this study, we suggest that the anti-estrogen drug
of raloxifene has anti-fibrotic properties. As men-
tioned above, it reduced the excessive proliferation
and ECM production of iPSC-derived fibroblasts from
SSc patients. It also reversed the pro-fibrotic effects of
TGF-β treatment on these cells. is cytokine partici-
pates in a pathway with SMAD that converts SSc fibro-
blasts into myofibroblasts [39–41]. Since these cells
proliferate strongly and produce high levels of α-SMA
and ECM proteins (e.g., collagen types I and III, vimen-
tin, and fibronectin), the TGF-β1/SMAD pathway plays
an important role in the pathogenesis of SSc [45]. Also,
Wnt signaling was aberrantly activated and produced of
TGF-β1 in fibrosis tissues. GSK-3 is a key factor as gly-
cogen synthase kinase in Wnt signaling. e phospho-
rylation of GSK-3 can diminish the activity of GSK-3 and
activate β-catenin. us, we observed that while TGF-β1
treatment increased the thickness of SSc-derived skin
organoids and the ECM production, α-SMA expression,
and proliferation of SSc iPSC-derived fibroblasts, treat-
ment of raloxifene decreased all these effects of TGF-β1.
TGF-β1 treatment increased pSMAD2 expression of SSc
iPSC-derived fibroblast that was decreased by raloxifene
treatment. Furthermore, raloxifene reduced the phos-
phorylation of GSK-3. To confirm that raloxifene has
(See figure on next page.)
Fig. 6 Anti-fibrotic effects of raloxifene in the bleomycin-induced model of systemic sclerosis. a Their dorsal skin thickness was examined by
histology, Scale bars, 100 μm. b Quantification of the skin thickness by histology of the bleomycin induction model. c‑e Expression of the fibrosis
marker genes of COL1A1, COL3A1 and ACTA2 in bleomycin model. f, g After 3 weeks of raloxifene treatment, bleomycin mice skin expression
of pSMAD was assessed by western blot analysis. All graphs show the mean and standard error of the mean (*p < 0.05, **p < 0.01, ***p < 0.001, as
determined by Student’s t test)
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Fig. 6 (See legend on previous page.)
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Kimetal. Stem Cell Research & Therapy (2022) 13:303
anti-fibrotic effects in SSc, we generated the bleomycin-
induced murine model of SSc and injected the mice with
raloxifene. Raloxifene effectively reduced the skin thick-
ness of the mice along with the expression by the skin
of the fibrotic-marker genes COL1A1, COL3A1, and
ACTA2. Furthermore, bleomycin increased pSMAD2
expression of mice skin that was decreased by ralox-
ifene treatment. erefore, this result suggested that the
GSK-3β and TGF-β1/Smad2/3 signaling may be impor-
tant pathways as antifibrotic activity of raloxifene.
Conclusions
In conclusion, raloxifene reduced SSc-related fibrosis
by downregulating fibroblast proliferation and ECM
production. is is a clinically important observation
because raloxifene is a very safe and well-tolerated anti-
osteoporotic drug and our data suggest that it may be
useful for intractable fibrosis in SSc. We also observed
that a closely related SERM, bazedoxifene, had a simi-
lar anti-fibrotic effect. us, SERM-class drugs might be
candidate therapeutic drugs for SSc in the near further.
Abbreviations
α-SMA: Alpha smooth muscle actin; BMP4: Bone morphogenetic protein 4; EB:
Embryonic body; ECM: Extracellular matrix; EGF: Epidermal growth factor; ESC:
Embryonic stem cell; iF: IPSC-derived fibroblast; iK: IPSC-derived keratinocyte;
iPSC: Induced pluripotent stem cell; iSO: Induced pluripotent stem cell derived
skin organoid; MTS: Masson’s trichrome staining; PBMC: Peripheral blood mon-
onuclear cell; PSR: Picrosirius red; RA: Retinoic acid; SERM: Selective estrogen
receptor modulator; SSc: Systemic sclerosis; TGF: Transforming growth factor.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13287- 022- 02987-w.
Additional le1: Figure S1. Characterization of induced pluripotent
stem cells (iPSCs) from patients with systemic sclerosis (SSc) and healthy
controls. a Morphology of iPSCs, as determined by Leica microscopy. Scale
bars, 200 μm. b Alkaline phosphatase staining of iPSCs. Scale bars, 200 μm.
c Immunocytochemical analysis of iPSC expression of the pluripotency-
marker proteins Oct4, SSEA4, Sox2, TRA-1–60, Klf4, and TRA-1–81. Scale
bars, 200 μm. d RT-PCR analysis of iPSC expression of the pluripotency-
marker genes OCT4, SOX2, NANOG, and LIN28. All graphs show the mean
and standard error of the mean (*p < 0.05, **p < 0.01, ***p < 0.001, as
determined by Student’s t test).
Additional le2: Figure S2. Anti-fibrotic effects of raloxifene in systemic
sclerosis (SSc) patient-derived primary fibroblasts. a Morphology of
primary fibroblast with bazedoxifene and raloxifene treatment, as
determined by Leica microscopy. Scale bars, 100 μm. b Proliferation assay
of primary fibroblasts with bazedoxifene and raloxifene treatment. c, d
RT-PCR analysis of fibrosis marker genes COL1A1, and ACTA2. e HC and
Patient-derived fibroblasts were subjected to the scratch assay with baze-
doxifene and raloxifene treatment and the wound length was measured
24 h later. Scale bars, 100 μm. f, g Quantification of the wound length by
using imageJ. h Total collagen concentration with bazedoxifene and ralox-
ifene was assessed by the hydroxyproline assay. i–k The three-dimensional
dermal equivalent derived from primary fibroblasts were treated with
bazedoxifene and raloxifene and the skin thickness were determined by
histology. Scale bars, 100 μm. l Total collagen concentration with various
concentrations of raloxifene was assessed by the hydroxyproline assay.
All graphs show the mean and standard error of the mean (*p < 0.05,
**p < 0.01, ***p < 0.001, as determined by Student’s t test).
Acknowledgements
Not applicable.
Author contributions
YK designed and performed the experiment and analyzed the results. YAR
and YN carried out the experiments and data analysis. YK and JHJ wrote the
manuscript. JHJ helped in analyzing the results. All authors read and approved
the final draft of the manuscript.
Funding
This work was supported by a grant from the Korean Healthcare Technology
R&D Project of the Ministry for Health, Welfare, and Family Affairs of Korea (Nos.
HI16C2177 and HI20C0495). This work was supported by a grant from the
Basic Science Research Program through the National Research Foundation
of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning
(Grant Nos. NRF-2019R1A5A2027588, NRF-2020R1A2C3004123, and NRF-
2021R1C1C2004688). This research was also supported by a grant from the
Catholic Institute of Cell Therapy in 2021 (CRC).
Availability of data and materials
All datasets of this article are included within the article.
Declarations
Ethics approval and consent to participate
This study was approved by the Institutional Review Board (IRB) of the Catholic
University of Korea. Written informed consent was obtained from all partici-
pants involved in this study.
Consent for publication
Not application.
Competing interests
The authors declare that there is no competing interest.
Author details
1 Catholic iPSC Research Center, College of Medicine, The Catholic Univer-
sity of Korea, Seoul, South Korea. 2 YiPSCELL Inc., 47-3, Banpo-dearo 39-gil,
Seocho-gu, Seoul, Republic of Korea. 3 Division of Rheumatology, Department
of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catho-
lic University of Korea, Seoul 137-040, Republic of Korea.
Received: 14 March 2022 Accepted: 17 June 2022
References
1. van den Hoogen F, Khanna D, Fransen J, Johnson SR, Baron M, Tyndall A,
et al. 2013 classification criteria for systemic sclerosis: an American Col-
lege of Rheumatology/European League against Rheumatism collabora-
tive initiative. Arthritis Rheum. 2013;65:2737–47.
2. Wollheim FA. Classification of systemic sclerosis. Visions and reality. Rheu-
matology (Oxford). 2005;44:1212–6.
3. Allanore Y, Simms R, Distler O, Trojanowska M, Pope J, Denton CP, et al.
Systemic sclerosis. Nat Rev Dis Primers. 2015;1:15002.
4. LeRoy EC, Trojanowska M, Smith EA. The pathogenesis of scleroderma
(systemic sclerosis, SSc). Clin Exp Rheumatol. 1991;9:173–7.
5. Gottschalk P, Vasquez R, Lopez PD, Then J, Tineo C, Loyo E. Scleroderma in
the Caribbean: characteristics in a Dominican case series. Reumatol Clin.
2014;10:373–9.
6. Nihtyanova SI, Ong VH, Denton CP. Current management strategies for
systemic sclerosis. Clin Exp Rheumatol. 2014;32:156–64.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 17 of 18
Kimetal. Stem Cell Research & Therapy (2022) 13:303
7. Affandi AJ, Radstake TR, Marut W. Update on biomarkers in systemic
sclerosis: tools for diagnosis and treatment. Semin Immunopathol.
2015;37:475–87.
8. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell.
2006;126:663–76.
9. Diecke S, Jung SM, Lee J, Ju JH. Recent technological updates and clinical
applications of induced pluripotent stem cells. Korean J Intern Med.
2014;29:547–57.
10. Yamanaka S. Induced pluripotent stem cells: past, present, and future.
Cell Stem Cell. 2012;10:678–84.
11. Takebe T, Wells JM. Organoids by design. Science. 2019;364:956–9.
12. Sharma A, Sances S, Workman MJ, Svendsen CN. Multi-lineage human
iPSC-derived platforms for disease modeling and drug discovery. Cell
Stem Cell. 2020;26:309–29.
13. Kim J, Koo BK, Knoblich JA. Human organoids: model systems for human
biology and medicine. Nat Rev Mol Cell Biol. 2020;21:571–84.
14. Distler O, Cozzio A. Systemic sclerosis and localized scleroderma–cur-
rent concepts and novel targets for therapy. Semin Immunopathol.
2016;38:87–95.
15. Rim YA, Nam Y, Ju JH. Induced pluripotent stem cell generation
from blood cells using sendai virus and centrifugation. J Vis Exp.
2016;118:e54650.
16. Kim Y, Rim YA, Yi H, Park N, Park SH, Ju JH. The generation of human
induced pluripotent stem cells from blood cells: an efficient protocol
using serial plating of reprogrammed cells by centrifugation. Stem Cells
Int. 2016;2016:1329459.
17. Muller FJ, Goldmann J, Loser P, Loring JF. A call to standardize teratoma
assays used to define human pluripotent cell lines. Cell Stem Cell.
2010;6:412–4.
18. Gropp M, Shilo V, Vainer G, Gov M, Gil Y, Khaner H, et al. Standardization
of the teratoma assay for analysis of pluripotency of human ES cells and
biosafety of their differentiated progeny. PLoS ONE. 2012;7:e45532.
19. Nelakanti RV, Kooreman NG, Wu JC. Teratoma formation: a tool for
monitoring pluripotency in stem cell research. Curr Protoc Stem Cell Biol.
2015;32:4A – 8.
20. Bilousova G, Chen J, Roop DR. Differentiation of mouse induced pluripo-
tent stem cells into a multipotent keratinocyte lineage. J Invest Dermatol.
2011;131:857–64.
21. Sakurai M, Hayashi R, Kageyama T, Yamato M, Nishida K. Induction of
putative stratified epithelial progenitor cells in vitro from mouse-induced
pluripotent stem cells. J Artif Organs. 2011;14:58–66.
22. Yang R, Zheng Y, Burrows M, Liu S, Wei Z, Nace A, et al. Generation of
folliculogenic human epithelial stem cells from induced pluripotent stem
cells. Nat Commun. 2014;5:3071.
23. Usategui A, del Rey MJ, Pablos JL. Fibroblast abnormalities in the patho-
genesis of systemic sclerosis. Expert Rev Clin Immunol. 2011;7:491–8.
24. Guenou H, Nissan X, Larcher F, Feteira J, Lemaitre G, Saidani M, et al.
Human embryonic stem-cell derivatives for full reconstruction of the
pluristratified epidermis: a preclinical study. Lancet. 2009;374:1745–53.
25. Kim Y, Park N, Rim YA, Nam Y, Jung H, Lee K, et al. Establishment of a
complex skin structure via layered co-culture of keratinocytes and fibro-
blasts derived from induced pluripotent stem cells. Stem Cell Res Ther.
2018;9:217.
26. Kim Y, Ju JH. Generation of 3D skin organoid from cord blood-derived
induced pluripotent stem cells. J Vis Exp. 2019;146:e59297.
27. Hewitt KJ, Shamis Y, Carlson MW, Aberdam E, Aberdam D, Garlick JA.
Three-dimensional epithelial tissues generated from human embryonic
stem cells. Tissue Eng Part A. 2009;15:3417–26.
28. Zheng H, Yang Z, Xin Z, Yang Y, Yu Y, Cui J, et al. Glycogen synthase kinase-
3beta: a promising candidate in the fight against fibrosis. Theranostics.
2020;10:11737–53.
29. Wang P, Deng L, Zhuang C, Cheng C, Xu K. p-CREB-1 promotes hepatic
fibrosis through the transactivation of transforming growth factor-beta1
expression in rats. Int J Mol Med. 2016;38:521–8.
30. Mercer BA, D’Armiento JM. Emerging role of MAP kinase pathways
as therapeutic targets in COPD. Int J Chron Obstruct Pulmon Dis.
2006;1:137–50.
31. Matsuda T, Zhai P, Maejima Y, Hong C, Gao S, Tian B, et al. Distinct roles of
GSK-3alpha and GSK-3beta phosphorylation in the heart under pressure
overload. Proc Natl Acad Sci U S A. 2008;105:20900–5.
32. Lal H, Ahmad F, Woodgett J, Force T. The GSK-3 family as therapeutic
target for myocardial diseases. Circ Res. 2015;116:138–49.
33. Chan EC, Dusting GJ, Guo N, Peshavariya HM, Taylor CJ, Dilley R, et al.
Prostacyclin receptor suppresses cardiac fibrosis: role of CREB phospho-
rylation. J Mol Cell Cardiol. 2010;49:176–85.
34. Barlow CA, Barrett TF, Shukla A, Mossman BT, Lounsbury KM. Asbestos-
mediated CREB phosphorylation is regulated by protein kinase A and
extracellular signal-regulated kinases 1/2. Am J Physiol Lung Cell Mol
Physiol. 2007;292:L1361–9.
35. Sollberg S, Mauch C, Eckes B, Krieg T. The fibroblast in systemic sclerosis.
Clin Dermatol. 1994;12:379–85.
36. Black MM, Bottoms E, Shuster S. Skin collagen content and thickness in
systemic sclerosis. Br J Dermatol. 1970;83:552–5.
37. Rodnan GP, Lipinski E, Luksick J. Skin thickness and collagen content in
progressive systemic sclerosis and localized scleroderma. Arthritis Rheum.
1979;22:130–40.
38. Harrison NK, Argent AC, McAnulty RJ, Black CM, Corrin B, Laurent GJ. Col-
lagen synthesis and degradation by systemic sclerosis lung fibroblasts.
Responses to transforming growth factor-beta. Chest. 1991;99:71S-S72.
39. Wan YN, Wang YJ, Yan JW, Li XP, Tao JH, Wang BX, et al. The effect of
TGF-beta1 polymorphism on systemic sclerosis: a systematic review and
pooled analysis of available literature. Rheumatol Int. 2013;33:2859–65.
40. Cotton SA, Herrick AL, Jayson MI, Freemont AJ. TGF beta: a role in sys-
temic sclerosis? J Pathol. 1998;184:4–6.
41. Derk CT. Transforming growth factor-beta (TGF-beta) and its role in the
pathogenesis of systemic sclerosis: a novel target for therapy? Recent Pat
Inflamm Allergy Drug Discov. 2007;1:142–5.
42. Sappino AP, Masouye I, Saurat JH, Gabbiani G. Smooth muscle differentia-
tion in scleroderma fibroblastic cells. Am J Pathol. 1990;137:585–91.
43. Gilbane AJ, Denton CP, Holmes AM. Scleroderma pathogenesis: a pivotal
role for fibroblasts as effector cells. Arthritis Res Ther. 2013;15:215.
44. Bryant HU. Mechanism of action and preclinical profile of raloxifene,
a selective estrogen receptor modulation. Rev Endocr Metab Disord.
2001;2:129–38.
45. Verrecchia F, Mauviel A, Farge D. Transforming growth factor-beta signal-
ing through the Smad proteins: role in systemic sclerosis. Autoimmun
Rev. 2006;5:563–9.
46. Moeller A, Ask K, Warburton D, Gauldie J, Kolb M. The bleomycin animal
model: a useful tool to investigate treatment options for idiopathic
pulmonary fibrosis? Int J Biochem Cell Biol. 2008;40:362–82.
47. Yamamoto T. The bleomycin-induced scleroderma model: what
have we learned for scleroderma pathogenesis? Arch Dermatol Res.
2006;297:333–44.
48. Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation
for fibrotic disease. Nat Med. 2012;18:1028–40.
49. Henderson NC, Rieder F, Wynn TA. Fibrosis: from mechanisms to medi-
cines. Nature. 2020;587:555–66.
50. Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and
mediators. Front Pharmacol. 2014;5:123.
51. Griffin MF, desJardins-Park HE, Mascharak S, Borrelli MR, Longaker MT.
Understanding the impact of fibroblast heterogeneity on skin fibrosis. Dis
Model Mech. 2020;13.
52. Pedroza M, To S, Assassi S, Wu M, Tweardy D, Agarwal SK. Role of STAT3 in
skin fibrosis and transforming growth factor beta signalling. Rheumatol-
ogy (Oxford). 2018;57:1838–50.
53. Song J, Zhang H, Wang Z, Xu W, Zhong L, Cao J, et al. The role of FABP5 in
radiation-induced human skin fibrosis. Radiat Res. 2018;189:177–86.
54. Elhai M, Meune C, Avouac J, Kahan A, Allanore Y. Trends in mortal-
ity in patients with systemic sclerosis over 40 years: a systematic
review and meta-analysis of cohort studies. Rheumatology (Oxford).
2012;51:1017–26.
55. Brown M, O’Reilly S. The immunopathogenesis of fibrosis in systemic
sclerosis. Clin Exp Immunol. 2019;195:310–21.
56. Shi Y, Inoue H, Wu JC, Yamanaka S. Induced pluripotent stem cell technol-
ogy: a decade of progress. Nat Rev Drug Discov. 2017;16:115–30.
57. Karagiannis P, Takahashi K, Saito M, Yoshida Y, Okita K, Watanabe A, et al.
Induced pluripotent stem cells and their use in human models of disease
and development. Physiol Rev. 2019;99:79–114.
58. Rowe RG, Daley GQ. Induced pluripotent stem cells in disease modelling
and drug discovery. Nat Rev Genet. 2019;20:377–88.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 18 of 18
Kimetal. Stem Cell Research & Therapy (2022) 13:303
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59. Yagi M, Yamanaka S, Yamada Y. Epigenetic foundations of pluripo-
tent stem cells that recapitulate in vivo pluripotency. Lab Investig.
2017;97:1133.
60. Ciechomska M, van Laar JM, O’Reilly S. Emerging role of epigenetics in
systemic sclerosis pathogenesis. Genes Immun. 2014;15:433–9.
61. Roessler R, Smallwood SA, Veenvliet JV, Pechlivanoglou P, Peng SP,
Chakrabarty K, et al. Detailed analysis of the genetic and epigenetic sig-
natures of iPSC-derived mesodiencephalic dopaminergic neurons. Stem
Cell Rep. 2014;2:520–33.
62. Wu H, Lee J, Vincent LG, Wang Q, Gu M, Lan F, et al. Epigenetic regulation
of phosphodiesterases 2A and 3A underlies compromised beta-adrener-
gic signaling in an iPSC model of dilated cardiomyopathy. Cell Stem Cell.
2015;17:89–100.
63. Furue M, Mitoma C, Mitoma H, Tsuji G, Chiba T, Nakahara T, et al. Patho-
genesis of systemic sclerosis-current concept and emerging treatments.
Immunol Res. 2017;65:790–7.
64. Liu D, Kong F, Yuan Y, Seth P, Xu W, Wang H, et al. Decorin-modified
umbilical cord mesenchymal stem cells (MSCs) attenuate radiation-
induced lung injuries via regulating inflammation, fibrotic factors, and
immune responses. Int J Radiat Oncol Biol Phys. 2018;101:945–56.
65. Hosoya M, Czysz K. Translational prospects and challenges in human
induced pluripotent stem cell research in drug discovery. Cells. 2016;5:46.
66. Muchmore DB. Raloxifene: a selective estrogen receptor modulator
(SERM) with multiple target system effects. Oncologist. 2000;5:388–92.
67. Miller CP. SERMs: evolutionar y chemistry, revolutionary biology. Curr
Pharm Des. 2002;8:2089–111.
68. Komm BS. A new approach to menopausal therapy: the tissue selective
estrogen complex. Reprod Sci. 2008;15:984–92.
69. Martinkovich S, Shah D, Planey SL, Arnott JA. Selective estrogen recep-
tor modulators: tissue specificity and clinical utility. Clin Interv Aging.
2014;9:1437–52.
70. Kharode Y, Bodine PV, Miller CP, Lyttle CR, Komm BS. The pairing of a
selective estrogen receptor modulator, bazedoxifene, with conjugated
estrogens as a new paradigm for the treatment of menopausal symp-
toms and osteoporosis prevention. Endocrinology. 2008;149:6084–91.
71. Berrodin TJ, Chang KC, Komm BS, Freedman LP, Nagpal S. Differential bio-
chemical and cellular actions of Premarin estrogens: distinct pharmacol-
ogy of bazedoxifene-conjugated estrogens combination. Mol Endocrinol.
2009;23:74–85.
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