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Human Menstrual Blood Stem Cell-Derived Granulosa Cells
Participate in Ovarian Follicle Formation in a Rat Model
of Premature Ovarian Failure In Vivo
Parastoo Noory,
1
Shadan Navid,
2
Bagher Minaee Zanganeh,
1
Ali Talebi,
3,4
Maryam Borhani-Haghighi,
1
Keykavos Gholami,
1
Marjan Dehghan Manshadi,
1
and Mehdi Abbasi
1
Abstract
We recently reported the application of human menstrual blood stem cells’ (HuMenSCs) transplantation as a
treatment modality in a rat model of premature ovarian failure (POF). We continued to investigate further in this
respect. Female rats were injected intraperitoneally with 36 mg/kg busulfan. HuMenSCs were obtained, grown,
and analyzed for immunophenotypic features at passage three. The cells were labeled with CM-Dil and infused
into the rats. There were four groups: normal, negative control, treatment, and Sham. One month after treat-
ment, the ovaries were collected and weighed. Histological sections were prepared from the ovary and Hu-
MenSCs were tracking. Subsequently, we examined the changes of expression of Bax and B cell lymphoma 2
(Bcl2) genes by real-time polymerase chain reaction assay. One month after HuMenSCs transplantation, these
cells were located in the ovarian interstitium and granulosa cells (GCs). The number of TUNEL-positive cells
significantly decreased in the treatment group. Also the expression level of Bax genes, unlike Bcl2 gene,
significantly decreased compared with negative and sham groups. In our study, HuMenSCs were tracked in
ovarian tissues within 2 months after transplantation, and they differentiated into GCs. Therefore, the use of
these cells can be a practical and low-cost method for the treatment of POF patients.
Keywords: POF, HuMenSCs, busulfan, Bax, Bcl2, ovary
Introduction
Menopause or the last menstrual cycle of women
occurs at an average age of 50.7 years. Menopause
before the age of 40 is called premature ovarian failure (POF)
(Goswami and Conway, 2007). One percent of women
under the age of 40 and 0.1% of women under the age of 30
experience POF (Meskhi and Seif, 2006). Patients with
POF have symptoms such as stopping follicular ovarian
activity, a follicle-stimulating hormone (FSH) concentra-
tiongreaterthan20to40mIU/mLinthepresenceofpri-
mary or secondary amenorrhea, hypergonadotropinemia,
and hypoestrogenemia (Falsetti et al., 1999; Kovanci and
Schutt, 2015).
Symptoms of POF patients are like physiological men-
opause, such as: infertility with palpitations, heat intoler-
ance, flushes, anxiety, depression, and fatigue. In addition
to infertility, hormone defects may cause neurological,
metabolic, or cardiovascular complications, and ultimately
lead to osteoporosis (Beck-Peccoz and Persani, 2006).
POF in each stage reduces the number of initial primor-
dial follicles, increases apoptosis or follicular degeneration,
and induces inability of follicles in response to gonado-
tropin stimulation (Welt, 2008). Several factors contribute
to the regulation of apoptosis and guarantee the survival of
preovulation follicles, including: gonadotropins, estrogen,
growth factors, cytokines, reorganization of the actin cy-
toskeleton, and nitric oxide and any changes in them can
lead to early ovarian failure. In contrast, tumor necrosis
factor-a,F
as
ligand, and androgens are stimulants of apo-
ptosis (Sinha and Kuruba, 2007).
POF can be due to several factors, such as infection,
metabolic disease, autoimmune disorders, or iatrogenic
cause such as radiation, chemotherapy, or physical damage
to the ovary (Chapman et al., 2015). One of the side effects
of chemotherapy or radiation therapy in treating patients
1
Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2
Department of Anatomy, Gonabad University of Medical Sciences, Gonabad, Iran.
3
School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran.
4
Clinical Research Development Unit, Bahar Hospital, Shahroud University of Medical Sciences, Shahroud, Iran.
CELLULAR REPROGRAMMING
Volume 21, Number 5, 2019
ªMary Ann Liebert, Inc.
DOI: 10.1089/cell.2019.0020
249
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with cancer is to increase the likelihood of ovarian damage
and, consequently, infertility (Schmidt et al., 2005). There are
several ways to preserve women’s fertility before treatment
with chemotherapeutic agents. The common method is hor-
monal stimulation of the ovary followed by in vitro fertiliza-
tion, and finally embryo cryopreservation. The second method
is ovarian tissue cryopreservation and its transplantation after
treatment. Nevertheless, both methods delay the treatment of
cancer, and cryopreserved tissue transplantation also has the
potential of reintroducing cancer cells (Xu et al., 2009).
One of the other methods is hormonal replacement ther-
apy (HRT). But the results of several recent studies have
indicated that HRT increases the risk of breast cancer, heart
attacks, and stroke. Therefore, it is recommended that
treatment of menopausal women with HRT be stopped
(Shelling, 2010).
In recent years, several studies have been done on the
therapeutic potential of stem cells. Mesenchymal stem cells
(MSCs) are capable of self-renewal, and have the capability
of differentiating into three germ layers and therapeutic
potential. Multipotent stem cells can now be obtained from
various sources such as adipose tissue, amniotic fluid, um-
bilical cord, bone marrow etc. (Ding et al., 2011; Lai et al.,
2015; Lv et al., 2018).
Studies with MSCs, such as bone marrow stem cells (Lee
et al., 2007), umbilical stem cells (Song et al., 2016), adi-
pose tissue stem cells (Sun et al., 2013), and amniotic fluid
stem cells (AFSCs) (Xiao et al., 2016), have shown that
these cells have the ability to restore ovarian function and
prolonged fertility in chemotherapy agents-induced labora-
tory animals. However, difficult access with invasive pro-
cedures and low proliferation capacity, has limited their
application.
Recently, endometrial-derived, highly proliferative stem
cell population has been identified in menstrual blood
(Xiang et al., 2017). Human menstrual blood stem cells
(HuMenSCs) were first extracted and described by Gargett
(2004). HuMenSCs have characteristics, such as spindle-
shaped appearance in culture, differentiation into three
germinal layers, and expression of surface markers similar
to those of the bone marrow-derived MSCs (Chen et al.,
2017). Previous studies have shown that HuMenSCs ex-
press some of the pluripotency markers, including Oct-4,
SSEA-4, nanog, c-kit, and STRO-1, as well as some of the
specific markers of MSCs, such as CD9, CD29, CD44,
CD49f, CD90, CD105, and CD117 (Lin et al., 2011; Ro-
drigues et al., 2012).
These cells are able to differentiate into chrondrogenic,
adipogenic, osteogenic, neurogenic, endothelial, pulmonary
epithelial, hepatic/pancreatic, and cardiogenic cell lineages
(Borlongan et al., 2010).
The therapeutic potential of HuMenSCs has been dem-
onstrated in several disease models, such as Duchenne
muscular dystrophy (Cui et al., 2007), stroke (Borlongan
et al., 2010), diabetes (Santamaria et al., 2011), myocardial
infarction (Zhang et al., 2013), and hepatic failure (Chen
et al., 2017). Previous studies indicated that Menstrual-
derived mesenchymal cells have a strong potential for re-
storing the function of cardiovascular disorders through
cardiomyogenesis in vitro (Hida et al., 2008).
Previous clinical studies have shown that alkylating
agents have the highest risk of infertility (Oktem and Oktay,
2007b). Cyclophosphamide, chlorambucil, melphalan, bu-
sulfan, nitrogen mustard, and procarbazine can be men-
tioned as alkylating agents (Oktem and Oktay, 2007a).
Busulfan is one of them that affects the reproductive process
of rat by its cytotoxic effect on the ovary (Sakurada et al.,
2009). In a previous study on rats treated with busulfan, it
has been reported that the number of oogonia reduces during
germ cell proliferation, resulting in a decrease in the number
of primordial follicles in the ovary (Shirota et al., 2003). In
another study, Brinster et al. (2003) reported that busulfan
targets granulosa cells (GCs) in the follicle.
According to the therapeutic properties and noninvasive
and easy access to HuMenSCs, we injected these cells
through the tail vein into chemotherapy-induced POF rat
and then measured restorative effects on ovarian function
with TUNEL assay and real-time technique. Since the death
of GCs occurs during the process of injection of busulfan as
chemotherapy agent, we decided to measure the differenti-
ation of these cells into ovarian-like cells (particularly GCs).
Materials and Methods
Experimental animals
Forty female Wistar albino rats (200–250 g, 6–8 week-old)
were purchased from Pharmacy Faculty of Tehran University
of Medical Sciences, Tehran, Iran. All procedures were ap-
proved by the Ethics Committee of Tehran University of
Medical Sciences, which corresponds to the national and
institutional guidelines for animal care and use.
Animal model establishment
and experimental grouping
To establish the POF model of chemotherapy-induced
ovarian damage, adult female rats were administered bu-
sulfan (Sigma, St. Louis, MO) at a dose of 36 mg/kg through
intraperitoneal injection. To confirm the POF model 7 days
after injection, the ovaries were collected. After Hematox-
ylin and Eosin (H&E) staining, the samples were examined
under light microscope and images were taken from the
slides for POF confirmation.
After establishing the POF model, we randomly divided
the rats into four equal groups (n=8): The control group
consisted of normal control rats that received no treatment. In
the negative control group, the rats were administered
busulfan. In the treatment group, after 7 days, POF rats were
injected intravenously with HuMenSCs (1·10
6
cells per
200 lL) in 1 mL phosphate-buffered saline (PBS; Sigma,
Steinheim, Germany). In Sham group, POF rats were injected
intravenously with 1mL PBS through the tail vein.
Isolation and culture of cells
HuMenSCs was collected from 20- to 30-year-old five
healthy women on the second day of the period with the
help of a special cup. This study was approved by the Ethics
Committee of Tehran University of Medical Sciences (ref.
no. 25110, approved 28 April 2014) and was performed
according to national and international guidelines.
HuMenSCs were isolated and centrifuged at 1500 gfor 10
minutes to obtain cellular pellets. The cellular pellets were
cultured in a T25 flask containing DMEM/F12 supple-
mented with 10% fetal bovine serum (Gibco), 100 mg/mL
250 NOORY ET AL.
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streptomycin, and 100 U/mL penicillin (both from Gibco) in
a humidified incubator at 37C with 5% carbon dioxide. The
culture medium was changed every 3 days. When the cells
reached 80%–90% confluence, they were detached using
trypsin–EDTA (Gibco) for 5 minutes and passaged to cul-
ture flasks and observed under an inverted microscope
(Olympus, Tokyo, Japan) for evaluation of morphologic
features. For transplantation, the cells were prepared after 14
days of culture in passage 3 and were labeled with DiI
(Invitrogen, Carlsbad, CA).
For transplantation of these cells to the rats, vaginal
smears were obtained from rats daily. Only rats showing at
least two consecutive normal 4- to 5-day vaginal estrus
cycles were used. The injection of these cells was done in
the first cycle of estrus.
Flow cytometry for identification of HuMenSCs’
characterization and confirmation by the Homing assay
in ovary after transplantation
HuMenSCs were characterized by flow cytometry analysis
of specific surface antigen expression. The suspension cells
were incubated with FITC-conjugated monoclonal antibodies
against CD90, CD44, CD34, CD45, CD146, CD105, CD73,
CD10, and CD29 (all from eBioscience, CA), with a 1:100
dilution for 1 hour at room temperature. We used an isotype
antibody (mouse IgG1-FITC; BD Biosciences) as a negative
control for the measurement of nonspecific binding. The
expression levels of OCT4 and C-kit genes were assessed
(Abcam) followed by a 30-minute incubation with FITC-
conjugated sheep anti-rabbit antibody (Sina Biotech, Iran).
Finally, the cells were analyzed by FC500 flow cytometry
(based on the method used in our previously published arti-
cles) (Manshadi et al., 2019; Rajabi et al., 2018).
In addition, we used flow cytometry technique for homing
assessment 14 days after transplantation to the POF rat to
confirm the survival of HuMenSCs. Briefly, ovary samples
were collected and homogenized with collagenase IV (In-
vitrogen). The suspension cells were centrifuged and exposed
to lysis buffer. Samples were suspended in PBS and evaluated
for red fluorescence CM-Dil (570nm). The percentage of the
Dil cells in ovary was analyzed by flow cytometry. Our
finding was presented by FlowJo software (Navid et al., 2017;
Szilvassy et al., 2001).
Hoechst staining
One month after the cell injection, ovaries were collected,
fixed and embedded in paraffin, and cut into 5 lm sections.
The slides were then permeabilized by 0.02% Triton X-100
(Merck, Germany) and were then stained with Hoechst dye
(33258, Sigma, 25MG) to label the nuclei. The slides were
visualized with an inverted microscope (Olympus).
Ovarian follicle counts and histological analysis
One month after transplantation, the ovaries were col-
lected from all groups and the surrounding fat tissues were
removed and weighed. Ovaries were then embedded in
paraffin, serial sections (5 lm) were taken from five con-
secutive 100 lm intervals in the middle third of each ovary
and stained with H&E (Muskhelishvili et al., 2005; Picut
et al., 2008). Slides were observed using a light microscope.
The number of oocyte-containing follicles with a distinct
oocyte nucleus at each developmental stage was classified
and counted. The follicles were classified as follows: pri-
mordial follicle, primary follicle, secondary follicle, early
antral follicles, and preovulatory follicles (Myers et al.,
2004; Pedersen et al., 1968).
Tunel assay
To detect apoptosis in the cell of ovaries, TUNEL stain-
ing kits (Fluorescein Roche, 11684795910) were used on
paraffin-embedded ovaries according to the manufacturer’s
instructions. Briefly, 2 lm sections were cut and the slides
were deparaffinized and washed thrice with PBS for 5 min-
utes, immersed twice in 10% aqueous hydrogen peroxide for
10 minutes, and incubated with proteinase K at 37Cfor30
minutes. The slides were then exposed to 3% Triton X-100
for about 10 minutes at room temperature. Twenty-five mi-
croliters of terminal deoxynucleotidyl transferase (TdT) was
added to the samples, and the whole setting was incubated for
2 hours at 37C in a humidified atmosphere in the dark.
After washing thrice with PBS, the nuclei were stained
using 5 lg/mL propidium iodide (PI; Invitrogen) for a few
seconds. Apoptotic cells in the ovary were stained green.
Images were observed with fluorescence microscope
(Olympus). The percentage of TUNEL-positive cells was
determined by counting five random fields from each sam-
ple. The results are expressed as the percentages of apo-
ptotic cells in each section (Kraupp et al., 1995).
Real-time polymerase chain reaction
One month after transplantation, expression levels of ap-
optosis regulator (Bax, proapoptotic) and B cell lymphoma 2
(Bcl2, antiapoptotic) were examined by real-time polymerase
chain reaction (PCR). Total RNA was isolated from ovaries
using TRIzol reagent (Ready Mini Kit, Qiagen), according to
the manufacturer’s instruction. To ensure cDNA, 1 lgRNA
was used to prepare a single-strand cDNA using Oligo (dT)
primer (MWG-Biotech, Germany) and reverse transcription
enzyme (Fermentas), based on the protocol.
Each PCR was performed using PCR master mix and SYBR
GreenontheABI(AppliedBiosystems)StepOnemachine
(Sequences Detection Systems, Foster City, CA), according to
the manufacturer’s protocol. The ratio of expression of the genes
examined in this study was evaluated using the comparative CT
method (DDCT) and glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) used as internal control. The nucleotide se-
quences of primers are listed in Table 1 (Navid et al., 2017).
Statistical analysis
All data were expressed as mean –standard deviation.
Statistical analysis of the results of all data were performed
using one-way analysis of variance (ANOVA) followed by
Tukey’s post hoc test. p£0.05 was considered statistically
significant.
Results
HuMenSCs morphology
The cells were cultured in cell culture flasks and rapidly
proliferated in vitro. In passage 0, the cells with a spindle
HUMENSC-DERIVED GRANULOSA CELLS PARTICIPATE IN PREMATURE OVARIAN FAILURE 251
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fibroblast-like morphology was observed (Fig. 1a) and these
cells obtained a homologous single layer of the specific
mesenchymal colonies (passage 3) (Fig. 1b).
Immunophenotypic characterization of HuMenSCs
After three passages, the flow cytometry analysis of the
cells revealed that HuMenSCs typically express CD44,
CD90 CD34, CD146, CD105, CD73, CD10, and OCT4
(specific MSC markers) but they failed to express c-kit.
Also, the lack of expression of CD45, CD34, and CD29
cultured cells showed a nonhematopoietic stem cell origin
(based on our article published in the microscopy research
and techniques journal and Reproductive Biology journal).
Specifications of POF-induced rats
Initially, the POF model was confirmed. One week after
injection of busulfan, ovaries of POF rats and normal rats
were collected and cut and subjected to H&E staining for
pathological evaluation. According to observations, ovarian
size in the POF group was smaller than the normal group.
Microscopic examination showed that in POF-induced rats,
the ovaries were atrophied, as a result of busulfan toxicity,
and they contain fibrous tissue, follicular atresia, and a small
number of follicles with damaged oocytes in all stages of
development as a result of follicular evacuation. In contrast,
ovaries of normal rats contain a large number of follicles
with healthy oocytes in each stages of development (Fig. 2).
Tracing of HuMenSCs in the ovaries with fluorescent
microscopy and flow cytometry
HuMenSCslabeledwithDil(redfluorescence)weretrans-
planted into the busulfan-induced female rats after a week.
The rat ovary examination by fluorescence microscopy con-
firmed HuMenSC’s implantation. According to our results,
HuMenSCs labeled with Dil showed fluorescent signals
1 month after injection. These cells localized particularly into
GCs of follicle (Fig. 3a–c). Moreover, cell homing was as-
sessed by flow cytometry. Dil (red fluorescence)-positive
HuMenSCs were observed along the injection tract after 2
months in the ovaries of POF rat. So, this date indicates that
Dil-positive HuMenSCs could survive transplantation within
the POF rat ovaries for at least 2 months in vivo.However,
HuMenSCs were capable of in vivo survival after implanta-
tion (Fig. 3e).
Thus, our finding showed that HuMenSC transplantation
can play a pivotal role in the improved structure and
function of the ovaries in the rat. Also, Dil-positive Hu-
MenSCs could not be detected along the injection tract in
the ovaries of POF rat.
Ovarian weight and follicle number increased
after HuMenSC transplantation
One month after injection of HuMenSCs, H&E staining
showed an increase in the number of follicles at each stage of
development in the treatment group (Fig. 4a–c). Therefore, in-
travenous treatment significantly reduced ovarian damage.
There was no significant difference between the number of
sham group and negative control group in all stages of fol-
licular evolution, and both groups showed a significant de-
crease compared with the normal group. This decrease was
significant for primordial follicle ( p£0.0001 vs. negative con-
trol and sham group), secondary follicle ( p<0.1 vs. negative
control and sham group), but this decrease was not signifi-
cant for the primary, early antral, and preovulatory follicles.
After 1 month, the number of primordial follicles in the
treatment group (293.66 –37.16) was significantly in-
creased compared with the negative group (164 –4.35) and
sham group (189 –26.6). The number of secondary folli-
cles in the treatment group (47.33 –1.52) were increased
compared with the negative group (27.33 –5.50) and the
sham group (21.33 –1.52). Also, the number of primary
Table 1. The Primers Sequence Bax, Bcl2, GAPDH of Genes
Gene name Sequence Product size (bp) Annealing temp
Bcl2 For: 5¢-GCAAACTGGTGCTCAAGG-3¢183 56.63
Rev: 5¢-CAGCCACAAAGATGGTCA-3¢
Bax For: 5¢-GAGTGGGATACTGGAGATGAAG-3¢233 57.4
Rev: 5¢-TGGTAGCGACGAGAGAAGTCC-3¢
GAPDH For: 5¢-AAGTTCAACGGCACAGTCAAGG-3¢121 61.58
Rev: 5¢-CATACTCAGCACCAGCATCACC-3¢
Bcl2, B cell lymphoma 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIG. 1. Morphology characteris-
tics of HuMenSCs. (a) Spindle-
shaped HuMenSCs sticking to the
flask floor in early stages of
growth: passage 0 (b) Colony-
forming HuMenSCs with multi-
faceted appearance after 2 weeks:
passage3 (inverted microscope,
scale bar =200 lm). HuMenSCs,
human menstrual blood stem cells.
252 NOORY ET AL.
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follicles (182.66 –29.95), early antral follicles (23 –5.29),
and preovulatory follicles (8.33–2.8) in the treatment
group increased compared with the negative groups, but
this increase was not significant (Fig. 4e).
To examine the effect of HuMenSCs on ovarian function,
ovarian weight changes were evaluated in all four groups.
There were no mortality due to the transplantation. One
month after injection of HuMenSCs, the ovaries of all four
groups were collected and weighed. The weight of the
ovaries in the negative control and sham group showed a
significant difference relative to the positive control and
treatment group. Ovarian weight increased in the treatment
group and was close to the normal (positive control) group.
The weight of ovaries in the treatment group (41.4 –6.95),
compared with the negative control group (20.88 –5.14),
indicated a significant increase ( p£0.001) and also showed
a significant increase ( p£0.01) compared with the sham
group (25.09 –4.66) (Fig. 4f ).
The morphological changes in the ovaries have shown that
Dil-positive HuMenSCs cells could increase a higher number
of follicles at each stage of development in the ovaries in the
treatment group compared with the negative group (POF). In
addition, ovarian weight changes in the treatment group in
comparison with the negative group confirmed this result.
TUNEL assay
Since the alkylating agents (busulfan) induce ovarian
degeneration through GC’s apoptosis, we evaluated the
protective effects of HuMenSCs against apoptosis in ovaries
FIG. 2. Microscopic morphology
of ovarian after induction of POF
(H&E staining, scale bar =200
lm). (a) Follicular atresia and de-
pletion, 7 days after intraperitoneal
injection of busulfan. (b) Ovaries
of normal rats contain a large
number of follicles with healthy
oocytes in each stages of develop-
ment. H&E, Hematoxylin and Eo-
sin; POF, premature ovarian
failure.
FIG. 3. In vivo homing of CM-Dil-labeled HuMenSCs in busulfan-injured ovarian. (a) Nuclei were labeled with Hoechst.
(b) CM-Dil-labeled HuMenSCs. (c) Merged. HuMenSCs were traced in ovarian, 1 month after injection using CM-Dil-
labeling. These cells were located in the ovarian interstitium and GCs (arrow) (Fluorescent microscope, scale bar =200 lm).
(d) Cell homing was assessed by flow cytometry for red fluorescence CM-Dil (570 nm) 2 months after transplantation
in vivo. GCs, granulosa cells.
HUMENSC-DERIVED GRANULOSA CELLS PARTICIPATE IN PREMATURE OVARIAN FAILURE 253
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of POF rat model. One month after the HuMenSC’s trans-
plantation, the TUNEL staining was performed and then the
number of TUNEL-positive cells was calculated in each
sections. There was no significant difference between the
number of TUNEL-positive cells in the sham group and the
negative group, and both groups showed a significant in-
crease ( p<0.0001) in comparison with the normal group
(Fig. 5). The mean number of TUNEL-positive cells in the
treatment group (23.58% –1.12%) was significantly lower
than the negative control group (46.67% –0.89%) and sham
group (45.49% –0.83%) ( p<0.0001).
Real-time PCR analysis
To further investigate the changes in apoptosis after bu-
sulfan induction, 1 month after injection of HuMenSCs, the
changes in the expression of Bax and Bcl2 genes involved
in apoptosis were investigated by quantitative real-time
PCR. As shown in Figure 6, in the negative control and
sham groups, the expression of Bax ( proapoptosis) and Bcl2
(antiapoptotic) genes was significantly ( p<0.0001) differ-
ent from that of the normal group, indicating an increase in
the level of apoptosis in the negative control group after
busulfan induction. Also, there was no significant difference
in the expression of these genes in the sham group compared
with the negative control group, which indicates that the
ovarian function is not improved in the sham group and the
negative group without cell therapy in these rats.
The graph indicates that the level of expression of these
genes in the negative group and sham group is at one level,
and not seen any difference in terms of reducing or in-
creasing of them. In the treatment groups, the level of ex-
pression of Bax gene was significantly decreased
(p<0.0001) compared with the negative group and sham
group. However, the expression of Bcl2 gene increased in
the treatment group in comparison with the negative group
and sham group, but this increase was not significant. These
changes indicate that the reduction in apoptosis after cell
therapy was mostly through a reduction in the expression of
Bax genes in the treatment group (Fig. 6).
Discussion
Previous study clearly demonstrated that HuMenSCs can
play an important role in the treatment of POF rat ( Man-
shadi et al., 2019), where HuMenSCs can improve and re-
store ovarian function and reduce apoptosis in damaged
ovarian tissue caused by busulfan toxicity. We also found
that there was the presence of effective homing of infused
cells into the ovary is crucial in cell-based therapies.
Today, the use of MSCs in the treatment of a wide range
of diseases has been studied. These cells are multipotent,
capable of self-renewal and high proliferative potential, and
differentiate into mesodermic and nonmesodermic lineages
(Emmerson and Gargett, 2016). MSCs are nonhematopoietic,
stromal cells, which also have the capacity to differentiate
into various types of osteocytic, chondrocytic, and adipocytic
lineages (De Cesaris et al., 2017).
In addition to bone marrow, other fibroblast-like stem
cells are found in other tissues including, circulating blood,
cord blood, placenta, amniotic fluid, heart, skeletal muscle,
adipose tissue, synovial tissue, and pancreas. In other words,
FIG. 4. (a–d) Ovarian sections of rats stained with H&E, 1 month after transplantation. H&E staining shows the follicular
atresia and evacuation in the negative control (a) and sham (c) groups, which indicates that recovery was not achieved after
1 month and 1 week after the injection of busulfan. While in the treatment group (d) follicular atresia has been improved
1 month after transplantation and follicular reserves are preserved. Positive group (b) (scale bars =200 lm). (e, f ) Com-
parison of ovarian weight and the number of follicles in four groups 1 month after injection. Data are expressed as
mean –SD (****p£0.0001). ns; SD, standard deviation.
254 NOORY ET AL.
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it can be hypothesized that all organs containing connective
tissue also contain MSCs (Kalervo Va
¨a
¨na
¨nen, 2005). Due to
the power of differentiation of MSCs into different tissues,
there is interest in using them to replace damaged tissues.
Given modern advances in gene therapy and tissue engi-
neering, they can be useful in improving the quality of life in
the future (Oreffo et al., 2005). These cells contribute to the
improvement of many diseases by the secretion of paracrine
agents, exosomes, secretomes, or even mitochondria (Bianco,
2014).
Previous studies have shown the therapeutic potential of
some MSCs on the ovary of laboratory animals in POF
model, including adipose tissue stem cells (Sun et al., 2013),
AFSCs (Ding et al., 2017; Pan et al., 2017; Xiao et al., 2016),
umbilical stem cells (Li et al., 2017; Song et al., 2016), and
bone marrow stem cells (Fu et al., 2008; Lee et al., 2007). All
of these have proven the improvement of ovarian function
after transplantation of cells in the POF model. Also, our
study showed improvement of some of the factors involved in
ovarian function after transplantation of mesenchyma-like
FIG. 5. TUNEL staining in ovary tissue sections after 1 month. TUNEL-positive cells labeled light, and nuclei labeled
dark (PI). (a–c) normal group, (d–f) negative control group, (g–i) sham group, and (j–l) treatment group. TUNEL-positive
cells are further restricted to proliferating granulosa and theca cells and lead to early depletion of ovarian follicles. TUNEL-
positive cells decreased in treatment group 1 month after transplantation of HuMenSCs (fluorescence microscope, scale
bar =10 lm). (m) There was no significant difference between the number of positive TUNELcells in the sham group and
the negative group. Apoptosis decreased in the treatment group, and showed a significant difference compared with three
groups: normal, negative control, and sham (***p<0.0001).
FIG. 6. Changes in the expression of Bax and Bcl2 genes in 4 groups 1 month after transplantation. In the negative control
and sham groups, the expression of Bax and Bcl2 genes was significantly different from that of the normal group. There was
no significant difference in the expression of these genes in the sham group compared with the negative control group. The
expression of Bax genes was significantly decreased, compared with the negative group and sham group and there was no
significant difference in Bax expression between treatment and normal groups (****p<0.0001). Bcl2, B cell lymphoma 2.
HUMENSC-DERIVED GRANULOSA CELLS PARTICIPATE IN PREMATURE OVARIAN FAILURE 255
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stem cells extracted from menstrual blood on rat POF model.
Moreover, It has been accepted that effective homing of in-
fused cells into the ovary is crucial in cell-based therapies.
Immunohistochemistry studies by Meirow et al. demon-
strated positive staining of apoptosis in pre GCs in patients
treated with cisplatin (Meirow et al., 1998). Chemotherapy
agents lead to apoptosis by stimulating p53 activity and its
downstream genes such as Bim and Casp9 (Happo et al.,
2010; Xiao et al., 2016). The present study proved the de-
generative changes busulfan induced in the ovarian tissue of
POF rat model as in previous studies (Lai et al., 2015; Tan
et al., 2010). Like previous studies, a significant reduction in
ovarian weight was also observed in POF (Chen et al., 2015;
Ding et al., 2017; Liu et al., 2014; Wang et al., 2017).
In 2008, Patel and Silva stated that menstrual blood stem
cells express the embryonic and multipotent markers, such
as Oct-4, SSEA-4, and c-kit, and markers of mesenchymal
cells, including CD90, CD166, and CD105, but, they do not
indicate the expression of hematopoietic cell markers, in-
cluding CD31 (endothelial), CD34 (hematopoietic and en-
dothelial stem cells), and CD45 (leukocyte) (Patel and Silva,
2008). The main advantage of these cells is easy, periodic,
and noninvasive access as compared with other MSCs.
Previous studies have shown that HuMenSCs have a low
level of immunogenic reactions and tumor formation (Azedi
et al., 2017; Borlongan et al., 2010). Our results indicate that
HuMenSCs express more than 85% of the specific mesen-
chymal markers, such as CD90 and CD44.
The therapeutic potential of HuMenSCs has been proven
in several disease models, such as Duchenne muscular
dystrophy (Cui et al., 2007), stroke (Borlongan et al., 2010;
Rodrigues et al., 2012), type 1 diabetes (Santamaria et al.,
2011; Wu et al., 2014), hepatic failure (Chen et al., 2017),
acute lung injury (Xiang et al., 2017), and myocardial in-
farction (Zhang et al., 2013). In the present study, we in-
vestigated the capacity of self-renewal and therapeutic
potentialofHuMenSCsontheovariantissueinPOF
model. Stem cells isolated from human menstrual blood
had fibroblast-like properties, adhesion to the flask, and high
proliferative potential, as demonstrated in previous studies
(Lai et al., 2015; Liu et al., 2014; Wang et al., 2017).
In 2017, Wang et al. (2017) showed that HuMenSCs la-
beled with GFP showed fluorescent signals in the interstitial
tissue 7 days after injection, but the fluorescence signal was
not observed 21 days after injection. HuMenSCs labeled
with DiO (green fluorescent) were observed 14 days after
injection in the ovary in POF mice (Liu et al., 2014).
Similarly, Dongmei Lai et al. examined the tracing of
GFP-positive cells by using live imaging, 6 hours to 14 days
after the HuMenSC injection in the POF model mice. They
observed that these cells first entered the pelvic organs 6 to 12
hours after the injection, and entered the chest 24 hours after
the injection and then, and observed weak signals 7 days after
injection in the pelvic organs. Immunofluorescence studies
after infusion of GFP-stained cells, confirmed the presence of
these cells 2 months after injection into the ovarian stromal
tissue (Lai et al., 2015).
Our findings indicate that the presence of DiI-labeled
HuMenSCs (red fluorescence) could be detected in the
ovary a month after the injection by using a fluorescence
microscope and 2 months after the HuMenSCs injection by
flow cytometry in the POF rat model. On the other hand, we
confirmed the presence of HuMenSCs at least 2 months after
transplantation into the ovaries of rat POF in vivo.
Also, the implantation assessment showed that these cells
are located in addition to interstitium of the ovary in GCs.
Thus, it can be said that these cells are likely to be differ-
entiated into GCs and, keeping in mind the process of re-
generation, evolution, and secretion of inhibitory hormones,
maintains FSH level low and prevents follicular evacuation
(based on the article accepted in the microscopy research
and techniques journal).
One month after injection of HuMenSCs, the weight of
the ovaries increased significantly, indicating implantation
and protective effect of these cells on ovarian tissue. Pre-
vious studies on HuMenSCs (Liu et al., 2014; Wang et al.,
2017), hAMSCs (human amniotic stem cells) (Ding et al.,
2017), have also noted this increase.
Our study shows a significant decrease in follicles at all
stages of development after POF induction compared with
the normal group. but, in the treatment group, the number of
these follicles increased in all stages compared with the
sham and negative groups and this increase was significant
for primordial and secondary follicles. Nonimprovement in
the sham group indicates that the ovarian function is not
improving spontaneously and is due to the therapeutic and
regenerative potential of these cells after migration to the
ovarian tissue.
Previous studies show an increase in the number of folli-
cles, 8 weeks and 21 days, respectively, after intravenous
injection of HuMenSCs into POF mice model (Lai et al.,
2015; Wang et al., 2017). This increase has also been re-
ported in other MSCs, including AFSCs (Ding et al., 2017),
umbilical MSCs (Li et al., 2017; Song et al., 2016), and bone
marrow MSCs (BMMSCs) (Fu et al., 2008) for in situ in-
jection. Sun et al. (2013) showed that adipose tissue stem
cells, both intravenous injection and in situ injection, increase
the number of follicles in all stages, 1 month after injection.
Perez et al. (1997) showed that chemotherapeutic agent-
exposed germ cells begin apoptosis due to the activation of
several death signaling pathways, including ceramide, Bax,
and caspase. Previous studies have identified the effect of
different alkylating agents on increased levels of GCs apo-
ptosis (Chen et al., 2015; Sun et al., 2013; Wang et al., 2017;
Yang et al., 2012). Our study also confirmed an increase
in apoptosis after induction of busulfan. However, after
transplantation of intravenous menstrual stem cells, apo-
ptosis significantly decreased. Also, in the negative control
and sham groups, the number of apoptotic cells in one level
was observed, indicating that the improvement in the
treatment group in the course of 1 month is caused by the
injection of stem cells and their therapeutic potential.
Guan-Yu Xiao et al. stated that exosomes derived from
AFSCs, through micro-RNAs (in which both miR-146a and
miR 10a are highly enriched) and their potential target genes
(involved in apoptosis), indicate antiapoptotic effects on
damaged GCs and also prevention of follicular atresia, es-
pecially the primordial follicles, in 72 hours after induction
of POF on mice (Xiao et al., 2016). Zhen Wang studies
showed that HuMenSCs and HuMenSC-derived conditioned
media (CM) exerted a protective effect and antiapoptotic
role on damaged ovaries through FGF2 secretion, which
also reduce the fibrosis in the ovarian interstitium and in-
crease follicle growth (Wang et al., 2017). Three recent
256 NOORY ET AL.
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studies have described the mechanisms of action of MSCs,
especially HuMenSCs, as well as their effect on ovarian
tissue and their protective effect.
We also examined the expression of Bax and Bcl2 genes
for further studies. Our study showed increased expression
of Bax and decreased expression of Bcl2, thereby increasing
the level of apoptosis in the POF model, as in the previous
study (Chen et al., 2015). Findings of Fu et al. regarding the
cytokines secretion from bone marrow stem cells in the
culture medium and the increased level of expression of
Bcl2 in vivo, showed that the reduction of apoptosis in GCs
after BMMSC transplantation in POF model, could be due
to upstream adjustment of Bcl2 genes (Fu et al., 2008). In
the present study, the reduction of the BAX gene expression
level significantly suggests that HuMenSCs can, by para-
crine factors, induce upregulation of Bax and Bcl2 genes
and antiapoptotic effects on GCs.
Conclusions
Our further research strongly proves the effectiveness of
stem cells in the treatment, regeneration, and improvement of
the function of busulfan-induced POF. Given the easy and
noninvasive access of these cells, they can be applied as a
new and effective approach for the treatment of POF patients.
Acknowledgment
The authors thank the staff of Tehran University of
Medical Sciences for animal care.
Ethics Approval and Consent to Participate
Animal experiments were approved by the Ethics Com-
mittee of Tehran University of Medical Sciences and all
procedures were performed in accordance with the univer-
sity’s guidelines. In the present work, we used animal model
considering all the rights based on the Ethics Committee of
Medical Faculty of Tehran University.
Author Disclosure Statement
The authors declare they have no conflicting financial
interests.
Funding Information
The authors are grateful to Tehran University of Medical
Sciences for their funding support.
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Address correspondence to:
Mehdi Abbasi
Department of Anatomy
School of Medicine
Tehran University of Medical Sciences
Tehran 0098
Iran
E-mail: abbasima@tums.ac.ir
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