BioMed Research International

Volume 2013 (2013), Article ID 690491, 12 pages

http://dx.doi.org/10.1155/2013/690491

Research Article

The Therapeutic Potential of Umbilical Cord Mesenchymal Stem Cells in Mice Premature Ovarian Failure

Shufang Wang,¹ Ling Yu,² Min Sun,³ Sha Mu,⁴ Changyong Wang,⁵ Deqing Wang,¹ and Yuanqing Yao⁴

¹Blood Transfusion Department, the General Hospital of the People’s Liberation Army, Beijing 100853, China

²Department of Obstetrics and Gynecology, First Affiliated Hospital of the General Hospital of the People’s Liberation Army, Beijing 100048, China

³Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China

⁴Department of Obstetrics and Gynecology, the General Hospital of the People’s Liberation Army, Beijing 100853, China

⁵Tissue Engineering Research Center of Academy of Military Medical Sciences, Beijing 100850, China

Received 26 April 2013; Revised 25 June 2013; Accepted 11 July 2013

Academic Editor: Elena Jones

Copyright © 2013 Shufang Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Mesenchymal stem cells, which are poorly immunogenic and have potent immunosuppressive activities, have emerged as promising cellular therapeutics for the treatment of several diseases. Mesenchymal-like cells derived from Wharton’s Jelly, called umbilical cord matrix stem cells (UCMSCs), reportedly secrete a variety of cytokines and growth factors, acting as trophic suppliers. Here, we used UCMSCs to treat premature ovarian failure (POF). Ovarian function was evaluated by ovulation and the number of follicles. Apoptosis of the granulosa cells (GC) was analyzed by TUNEL staining. We found that after transplantation of the UCMSCs, apoptosis of cumulus cells in the ovarian damage model was reduced and the function of the ovary had been recovered. The sex hormone level was significantly elevated in mice treated with UCMSCs. The number of follicles in the treated group was higher than in the control group. Our results demonstrate that UCMSCs can effectively restore ovary functionality and reduce apoptosis of granulosa cells. We compared the RNA expression of the UCMSCs treated group with the POF model and wild-type control group and found that the UCMSC group is most similar to the wild-type group. Our experiments provide new information regarding the treatment of ovarian function failure.

1. Introduction

Premature ovarian failure (POF), also referred to as hypergonadotropic hypogonadism, is classically defined as 4–6 months of amenorrhea in women under the age of 40 and is associated with menopausal levels of serum gonadotropins (FSH > 40 IU/L) and hypoestrogenism. POF prevents women from being able to conceive and may also be associated with other medical problems, such as blood clots, osteoporosis, and heart disease [1].

The cause of POF is complex. Some patients have abnormal ovary development because of genetic disorders, such as fragile X, Turner syndrome, and some autosomal genes mutation [2]. Some cases of POF are attributed to autoimmune disorders [3]. Also chemotherapy and radiation treatments for cancer can sometimes cause ovarian failure [4]. There are two basic categories of premature ovarian failure: one in which there are few to no remaining follicles, and in the second, there are an abundant number of follicles. In the first situation, the causes include genetic disorders [5], chemotherapy, radiation to the pelvic region, surgery, and so on; however, in most cases, the cause is unknown. In the second case, a frequent cause is autoimmune ovarian disease, which damages maturing follicles but leaves the primordial follicles intact. Currently, there are no proven treatments that restore normal functionality to a woman’s ovaries [6]. One of the most common treatments for women with premature ovarian failure is hormone replacement therapy (HRT), but HRT has been shown to increase the risk of blood clots in the veins, ovarian cancer, and breast cancer [7, 8].

Mesenchymal stem cells (MSCs) have been shown to differentiate into various cell types both in vivo and in vitro. They are found in embryonic and extraembryonic tissues, as well as in adult organs [9]. Several reports indicate that cells from Wharton’s Jelly (WJ), the main component of the umbilical cord extracellular matrix, are multipotent stem cells, expressing markers of bone marrow mesenchymal stem cells (BM-MSCs). The human umbilical cord was found to be a new source of stem cells and can be used for cell therapy [10]. UCMSCs express the MSC markers CD29, CD44, CD73, CD90, and CD105 and do not express CD31, CD45, and HLA-DR [11]. UCMSCs can differentiate into several mesodermal cell types. Some groups have started using stem cells to restore the functionality of failed neuron in animals [12, 13]. Additionally, UCMSCs result in almost no immune response [14] and can be used to treat graft versus host disease (GVHD) [15]. The low expression of human leukocyte antigen (HLA) major histocompatibility complex (MHC) Class I and the absence of MHC Class II molecules in UCB-derived MSCs indicate the presence of immune evasion, even in allogeneic transplantations [16–18]. There is considerable optimism that immune suppression and the anti-inflammatory properties of MSCs will be useful in many conditions, such as graft versus host disease, solid organ transplants, and pulmonary fibrosis [19–21].

Researchers have used mesenchymal stem cells to repair damaged ovaries in rats [22, 23]. This is further supported by clinical reports linking stem cell transplantations to the spontaneous and as-yet unexplained recovery of ovarian function and natural fertility in some women rendered prematurely menopausal by high-dose cyclophosphamide (CTX). However, whether human UCMSCs can help mice damaged ovaries recover and the optimal dose at which they should be administered have not yet been determined.

2. Methods

2.1. Animals

Specific pathogen-free (SPF-) grade CD1 (ICR) mice were purchased from Vital River. The mice were housed in the animal facility at the Academy of Military Medical Sciences and studied at 8–10 weeks of age. All studies followed procedures consistent with the Academy of Military Medical Sciences’ guide for the care and use of laboratory animals.

2.2. Cell Isolation and Culture

Full-term umbilical cords (UCs) were received following UC blood removal. We used the mechanical dissociation and explanted culture method. Whole UCs were manually dissected into smaller sections (5-6 grams UC tissue per isolation) and plated in polystyrene tissue culture flasks with L-DMEM supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) for 7 days in a 37°C humidified environment with 5% CO₂ [24]. After around 10 days the first colonies of UCMSCs, the UCMSCs can be seen. At confluence, the cells were harvested with 0.25% trypsin-EDTA.

2.3. Phenotype Analysis by Flow Cytometry

The UCMSCs surface marker expression was analyzed at passage six by flow cytometry, using fluorescein isothiocyanate (FITC)-conjugated human monoclonal antibodies against the following: CD105, CD90, CD29, CD44, human leukocyte antigen (HLA)-DR, and Phycoerythrin (PE-) conjugated human antibodies against CD34. For flow cytometry analysis, adherent cells were detached by treatment with 0.25% trypsin-EDTA, neutralized with FBS-containing culture medium, and disaggregated into single cell by pipetting. The cells were incubated with mAbs for 30 min at 4°C, washed twice with PBS, resuspended in 0.5 mL PBS, and immediately analyzed using an FACS Calibur flow cytometer (Becton Dickinson). At least 2 × 10⁵ cells were used for each sample, and Cell Quest software was used for data analysis.

2.4. POF Model

The mice were randomly divided into three groups of 15 mice each, namely, the WT group, the POF group, and the UCMSCs group. In the latter two groups, the mice received a daily dose of intraperitoneal CTX injection (50 mg/kg) for 15 consecutive days to establish POF models of chemotherapy-induced ovarian damage.

2.5. Cell Transplantation

Mice in the UCMSC group were injected intravenously with 1 × 10⁶ hUCMSCs in 100 μL PBS according to previous study [25–27], and those in the WT and POF group were injected with 100 μL PBS alone. The injections were repeated once on the following day. For cell tracking studies, the cells were washed with PBS, incubated with the fluorescent CM-Dil (Roche) for 5 min at 37°C and 15 min at 4°C, washed twice with PBS, and resuspended in PBS for a final volume of 10⁷ cells/mL. The recovery rate of this procedure was 80–90% of the viable cells.

2.6. Mice Superovulation

Female ICR mice were superovulated by an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin followed, 48 h later, by intraperitoneal injection of 5 IU of human chorionic gonadotropin (hCG). Fully grown GV oocytes were collected from 4- to 8-week-old ICR female mice 44–48 h after equine chorionic gonadotropin injection. The MII oocytes were collected from the oviducts of female mice, administered with equine chorionic gonadotropin and human chorionic gonadotropin. Parthenogenetic activation of MII oocytes was performed by culturing the oocytes in calcium-free CZB medium supplemented with 10 mM strontium and 1 mg/mL cytochalasin B (CB).

2.7. Hematoxylin and Eosin Staining of Ovary Slices

After one-week transplantation, the ovaries of three group mice were collected. The mouse ovaries were fixed in phosphate-buffered saline containing 4% formaldehyde, and routine paraffin-embedded sections were produced. The ovaries of three groups were collected five days after treatment. The 4 μm paraffin-embedded sections were rehydrated using xylene and a graded alcohol series and stained with hematoxylin and eosin.

2.8. Hormone Examination

Before the experiment, 1 mL of blood was collected in the diestrus phase and the serum was restored in a procoagulant tube (BD) for determination of the baseline levels of estradiol (E₂). The concentrations of estradiol in the ovarian homogenate were determined by chemiluminescence techniques.

2.9. Apoptosis Detection

In Situ Cell Death Detection Kit, POD (Roche) was used for detection of cell apoptosis in the mouse ovaries. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction. The In Situ Cell Death Detection Kit is designed to detect fragmented DNA histochemically by TUNEL (TdT-mediated dUTP Nick End Labeling). The fluorescein-labeled nucleotides are incorporated in situ onto the 3′ ends of DNA fragments, allowing histologic localization and detection of individual apoptotic cells.

Briefly, the tissue sections were washed in xylene and ethanol (absolute, 95%, 90%, 80%, and 70%, diluted in double distilled water), suspended in PBS, and incubated for 15–30 min at 21°C–37°C with proteinase K (10–20 μg/mL in 10 mM Tris/HCL, pH 7.4–8). The slides were rinsed twice in PBS, and the area around the sample was dried. Then, 50 μL of TUNEL reaction mixture was added to the samples, which were incubated for 60 min at 37°C in a humidified atmosphere in the dark. The slides were rinsed 3 times with PBS. We then dyed the cell nucleus with Hoechst 33324 for 15 min and washed 3 times with PBS.

2.10. RNA Microarray Analysis

RNA was isolated from mouse ovaries using Trizol (Invitrogen) using standard methods.

Labeling and hybridization were performed at the CapitalBio Company, according to protocols described in the 32 K mouse genome arrays user manual. The data were analyzed using LuxScan 3.0 Image analysis software (CapitalBio company).

2.11. Real-Time Quantitative PCR

Total RNA was extracted from WT, POF, and UCMSC group ovary using Trizol (Life Technologies). Superscript II reverse transcriptase (Life Technologies) was used to generate cDNA using 1 μg of RNA and oligo dT primer, according to the manufacturer’s instructions. The primers were designed using Primer Express version 2.0 (Applied Biosystems, Foster City, CA, USA) and are listed in Table 1. The primers used for this study were synthesized by Invitrogen. The qRT-PCR was performed in triplicate and was repeated in at least three separate experiments using the following conditions. Reaction mixtures contained 12.5 μL of SYBR Green I dye master mix (Applied Biosystems), 2 pmoles each of forward and reverse primers, and 5 μL of 100 times diluted cDNA. Thermocycle conditions included initial denaturation at 50°C and 95°C (10 min each), followed by 40 cycles at 95°C (15 s) and 60°C (1 min). Fluorescent data were acquired during each extension phase. After 40 cycles, a melting curve was generated by slowly increasing (0.1°C/s) the temperature from 60°C to 95°C, while the fluorescence was measured. The threshold cycle (CT) was calculated using the Sequence Detector Systems version 1.2.2 (Applied Biosystems) by determining the cycle number at which the change in the fluorescence of the reporter dye (ΔRn) crossed the threshold. To synchronize each experiment, the baseline was set automatically by the software. To rule out DNA contamination in the RNA preparations, the qRT-PCR controls were performed with RNA templates which did not show any amplification.

Table 1: The primers were used for the qRT-PCR.

Gene
GB.accession

Primer sequence

GAPDH

F
GTTGTCTCCTGCGACTTCA

R
TGGTCCAGGGTTTCTTACTC

Actin

F
GAGATTACTGCTCTGGCTCCTA

R
GGACTCATCGTACTCCTGCTTG

Bub1
NM_001005856.1
F
ATGAAGCCACAACGTACCCA

R
TCCTGCCGTCTACTCCTCTTG

Cdkn2b
NM_007670.4
F
TTTGGGTGGGTGCAGTCAG

R
TTCCTTGTCGAGCTGGAGGT

Chek1
NM_007691.5
F
TGGATGCGGACAAATCTTACC

R
ACCAAACCTTCTGGCTGCTC

Gadd45a
NM_007836.1
F
CTCATCCGTGCGTTCTGCT

R
TCCATTGTGATGAATGTGGGTT

Cdk4
NM_009870.3
F
CAGTCTACATACGCAACACCCG

R
CAAAGATTTTCCCCAACTGGTC

Cxcr4
NM_009911.3
F
GCTGAAAAGGCAGTCTATGTGG

R
ACAGGCTATCGGGGTAAAGG

Lhcgr
NM_013582.2
F
ACGCTGACCTACCCTAGCCA

R
CCCAGCCACTGAGTTCATTCT

Ly6a
NM_010738.2
F
CAATTCTTCCTGGAGCCCTCT

R
CATGAGCAGCAATCCACAACA

Smad2
NM_010754.5
F
CACCTTCATCACGCCTTGG

R
TGGGCATCTGCTTTCTTTGA

Nr5a2
NM_030676.3
F
TCAGTTCGATCAGCGGGAGT

R
TTCACCTGCTCTTGGACACCT

Akr1c18
NM_134066.2
F
CAAGTCCATCGGGGTGTCTAA

R
GCAGCTTGCTCTGGTTGAGAT

Cyp4f15
NM_134127.1
F
ATGACCCCTTCCGCTTTGA

R
CGCCACCTTCATCTCGTTC

3. Results

3.1. Characterizing the UCMSCs

The UCMSCs were harvested and cryopreserved 20–30 days after the initial processing, as time was required for the UCMSCs to migrate from the plated tissue (Figure 1(a)). A minimum of 10 days was required for the first colonies of UCMSCs to appear, and additional 5 days were needed to gain 70–80% confluency, at which point the cells were transferred to 75 cm² flasks. The proliferation rate decreased after 10 passages.

(a)

(b)

Figure 1: Isolation and identification of UCMSCs. (a) The UCMSCs were isolated from human umbilical cord and showed typical fibroblastic morphology. (b) FCM analysis of UCMSCs. CD90, CD44, CD29, and CD105 were positive and CD31, HLA-DR were negative. Scale bars: 100 μm.

The hMSCs from different sources displayed a homogenous spindle-shaped population and maintained this morphology during subsequent passages. FACS analysis was employed to identify the surface marker expression at passage 6. The UCMSC culture was shown to be devoid of HLA-DR and CD31. In contrast, a high expression of CD29, CD44, CD90, and CD105 markers were observed (Figure 1(b)). These markers were expressed in over 95% of the population.

3.2. UCMSCs Rescue the POF Ovulation Function

Females administered nonlethal doses of CTX (50 mg/kg) for 15 days exhibited premature ovary failure. There were fewer follicles in their ovaries than in the wild-type mice (Figures 2(a) and 2(b)). The wild-type mouse ovary possessed follicles, and the POF mouse ovary possessed follicles (Figure 2(g)) (POF versus WT ). The UCMSCs were delivered intravenously. The ovaries were collected one week after treatment, and the presence of normal follicles was determined. There was a slight increase in the weight of the ovaries in mice undergoing cell transplantation. The ovaries from female mice that were injected intravenously through the tail contained the similar number of follicles as the wild-type mice () (UCMSC versus WT ) (Figures 2(c) and 2(g)). And ovaries of mice receiving UCMSCs transplantation after chemotherapy possessed more oocyte-containing follicles at various stages of development than POF group (POF versus UCMSC ) (Figures 2(d), 2(e) and 2(f)).

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Figure 2: The effect of transplantation. (a, b, c) Hematoxylin eosin staining. (a) Follicles of the POF model. (b) Follicles of the WT group. (c) Follicles of the UCMSCs treated group. (d) Oocytes of the POF model. (e) Oocytes of the WT group. (f) Oocytes of the UCMSCs treated group. (g) Number of follicles of the three groups (POF, WT and UCMSCs treated). Data were means ± SD of numerous experiments. (POF versus WT , POF versus UCMSC , and UCMSC versus WT ). (h) The values of sexual hormone E₂ of the three groups (POF, WT and UCMSCs treated). Data were means ± SD of numerous experiments. (POF versus WT , POF versus UCMSC , and UCMSC versus WT ). Scale bars: 100 μm.

3.3. The UCMSCs Recover Ovary Function in Sex Hormone (E₂) Levels

Follicular development was assessed in all groups by monitoring E₂ levels in the serum. One week after MSC transplantation, the serum E₂ of the therapy group was significantly increased compared with the POF group () without treatment. There was no significant difference between the UCMSC treated group and WT groups (Figure 2(h)).

3.4. The UCMSCs Do Not Differentiate into Follicles

The cell-tracking technique may be generally suited to monitoring stem cell homing and engraftment. One week after transplantation, the intravenously injected labeled human UCMSCs could be traced in the ovaries. The labeled stem cells infused into the donor ovaries but did not develop into follicles, including cumulus cells or oocytes (data not show).

3.5. The UCMSCs Can Reduce Apoptosis of the Granulosa Cells

Following CTX, the granulosa cells and other cells in the ovaries underwent dramatic apoptosis. One week after UCMSCs transplantation, the percentage of TUNEL-positive GCs in the therapy group was significantly lower than that in the POF group (Figure 3). After the UCMSCs therapy, the apoptosis amount is almost the same as the wild-type group.

(a)

(b)

(c)

(d)

(e)

(f)

Figure 3: Apoptosis of granulosa cells around follicles. (a, b, c) Green fluorescence indicates the apoptosis of granulosa cells in the three groups (POF, WT, and UCMSCs treated). (d, e, f) Blue fluorescence indicates the nucleus of granulosa cells. Scale bars: 50 μm. The green stained by POD kit. The blue stained by Hoechst.

3.6. The UCMSC Therapy Group RNA Expression Pattern Is Closer to Wild-Type Than to That in the POF Model

The RNA expression pattern in the cell therapy and the WT groups was almost the same, only 31 genes different between the UCMSCs transplantation group and the WT group, 16 upregulated and 15 downregulated (Table 2). The POF group had 33 genes more highly expressed than the WT group and 58 genes with lower expression than the WT group (Figure 4 and Table 3).

Table 2: The different expression genes between the UCMSCs treated group and the WT group.

Name
Ratio = UCMSCs treated group/wt group
Oligo_id

Cfd
7.5935
M300002011

Car3
6.2399
M200000108

Adipoq
5.0866
M200001531

Tgtp
3.4595
M400008751

Fbxo39
2.7945
M300012693

Megf10
2.7605
M400013376

Nuf2
2.6878
M300005239

Rmcs2
2.6072
M200002417

Lbp
2.5563
M400000246

Ly6k
2.4823
M200014514

Saa1
2.3428
M400010894

Q4VCP1_MOUSE
2.3163
M400004214

U3
2.2201
M400015091

Chek1
2.1561
M300008080

Tspan11
2.1209
M200011768

D12Ertd647e
2.0849
M400009945

Col6a2
0.4936
M400000370

Lix1
0.4928
M300003775

Sfrs11
0.4562
M400009262

Cyp4f18
0.4557
M400011465

Akap2
0.4443
M400010322

Sectm1a
0.438
M200015271

St3gal5
0.4214
M400005399

Gdpd3
0.4141
M200005744

Cyp4f18
0.376
M400000072

Sectm1b
0.3312
M200011927

Gpnmb
0.2996
M300006890

4930422I07Rik
0.298
M300008255

Ptgds
0.2978
M200000369

Cxcr4
0.2072
M400014726

Cd7
0.1569
M200001594

The different expression genes between the UCMSCs treated group and the WT group. There are 31 genes, 16 genes upregulated and 15 genes downregulated. Bold: upregulated genes. Italic: downregulated genes.

Table 3: The different expression genes between the POF group and the WT group.

Name
Ratio = POF group/WTgroup
Oligo_id

Adipoq
9.7237
M200001531

Actg2
9.1892
M300000862

Fbxo39
8.1171
M300012693

Car3
7.243
M400010743

—
6.3766
M400011025

Fabp4
5.857
M300005685

Car3
5.6195
M200000108

—
5.4981
M400000320

H2-Ea
4.2115
M300010457

—
4.0834
M400009758

Tyrobp
3.3287
M200014463

H2-DMb2
2.9302
M300010992

Glo1
2.8122
M400009038

Tspan11
2.6785
M200011768

—
2.6672
M400006287

Aqp2
2.6298
M200003832

Bcl2a1c
2.5787
M400004604

Cdo1
2.5221
M200006769

Cd74
2.4419
M400010174

H2-T22
2.3833
M400006288

Hbb-b1
2.3606
M400000660

—
2.3277
M400008839

Ier3
2.324
M200005156

—
2.3114
M400003688

2210403B10Rik
2.2961
M300009216

Sars
2.237
M400011126

C3
2.224
M200003738

Drbp1
2.1762
M400012193

Hspb1
2.1179
M300017926

Spcs1
2.1146
M200006195

Folr1
2.097
M200000812

Sfrs5
2.0605
M400000441

Abcb1b
2.0557
M200016221

Slc16a9
0.4928
M200003760

Cp
0.4873
M300000454

—
0.4805
M400006370

Glipr1l2
0.4764
M200015469

S100a4
0.4727
M400011125

—
0.4643
M400009718

—
0.462
M400013641

—
0.46
M400010667

—
0.4533
M300016225

Phf20l1
0.4494
M300010640

Scarb1
0.4413
M300011232

—
0.4392
M400001328

—
0.4376
M400009125

Tnc
0.4353
M400001084

—
0.4329
M400013571

—
0.4168
M400008229

Atoh1
0.4148
M400010733

—
0.4115
M400008154

2610042L04Rik
0.4105
M400005704

—
0.41
M400008548

Onecut2
0.4098
M300016911

Nr5a2
0.4027
M300005083

—
0.4027
M400008507

—
0.4004
M300012538

LOC673685
0.3998
M400012723

Slc6a6
0.3969
M300007001

Procr
0.3923
M200001248

Ap4b1
0.3854
M200006396

—
0.3796
M400010333

4930534B04Rik
0.3795
M200007634

Ptgds
0.3744
M200000369

—
0.3691
M300005163

—
0.3686
M400006364

—
0.3684
M200002493

—
0.3677
M400015154

—
0.3655
M400007777

—
0.3613
M400005487

—
0.3525
M400011042

Decr1
0.3385
M200004880

Ly6a
0.3354
M300009411

—
0.3293
M400003416

—
0.329
M400005662

Slc25a30
0.3283
M200013505

Akr1c6
0.3158
M400000447

9930111J21Rik
0.3155
M400009499

Zfp277
0.3129
M400008100

—
0.3083
M400008356

—
0.3009
M400005940

Cyp4f18
0.2936
M400011465

—
0.2926
M400008231

—
0.2772
M400005702

Cyp4f18
0.2688
M400000072

Stard5
0.249
M200005178

—
0.2357
M400012678

Tap2
0.2245
M200003350

—
0.2197
M400008304

—
0.1969
M400005144

—
0.1619
M400012679

The different expression genes between the POF group and the WT group. There are 90 genes, 33 genes upregulated and 57 genes downregulated. Bold: upregulated genes. Italic: downregulated genes.

(a)

(b)

(c)

Figure 4: RNA array analyses. (a) UCMSC group has 16 genes higher expression than WT group and 15 genes lower expression than WT group. Red: gene upregulated. Green: gene downregulated. (b) POF group has 33 genes higher expression than WT group and 58 genes lower expression than WT group. Red: gene upregulated. Green: gene down-regulated. (c) Genes relative expression of the three groups.

Comparing the gene expression between the WT and POF groups, we found that a number of pathways were affected, including transcription regulation, the G-protein coupled receptor protein signaling pathway, the MAPK pathway, and the insulin pathway (Figure 5). These pathways are important for follicle and oocyte growth.

(a)

(b)

(c)

Figure 5: Genes correlation of three pathways. (a) UCMSC-WT gene Pathway Network. (b) POF-UCMSC gene Pathway Network. (c) POF-WT gene Pathway Network.

In addition to important pathways, there are also some important proteins that can be rescued by UCMSCs. Aldo-keto reductase family 1 members catalyze androgen, estrogen, and prostaglandin metabolism. AKR1C18 expression was very low in the POF model group mice. Existing as a monomer, AKR1C18 catalyzes the NADP+-dependent conversion of progesterone into 20-α-dihydroprogesterone, a biologically inactive metabolite [28]. This reaction is thought to be important for luteolysis (the degradation of the corpus luteum), as well as for overall reproductive health and newborn survival in mice. After stem cell therapy, the amount of AKR1C18 increased to normal levels.

LY6A, also named SCA-1, is a stem cell marker [29]. The mouse ovarian surface epithelium contains a population of LY6A (SCA-1) expressing progenitor cells that are regulated by ovulation-associated factors. The ovarian surface epithelium, a single layer of poorly differentiated epithelial cells, covers the surface of the ovary and is ruptured during ovulation. LY6A, also known as stem cell antigen-1 (SCA-1), has determined that the size of the LY6A-expressing (LY6A+) progenitor cell population is regulated by at least two ovulation-associated factors present in the follicular fluid, and LY6A has identified a population of LY6A+ MOSE progenitor cells on the surface of the ovary that may play a role in ovulatory wound healing.

The CDKN2B gene encodes a cyclin-dependent kinase inhibitor, also known as p15Ink4b protein, which forms a complex with CDK4 or CDK6 and prevents the activation of the CDK kinases by cyclin D. Thus, the encoded protein functions as a cell growth regulator that inhibits cell cycle G1 progression [30]. The expression of this gene was dramatically induced by TGF beta, which suggested its role in TGF beta-induced growth inhibition. p15INK4B is a potential effector of TGF beta-induced cell cycle arrest. When the ovary is destroyed by CTX, many cells undergo apoptosis. The ovary has to initiate more cell growth to supply follicle development, so the genes involved in cell cycle arrest are downregulated. After cell therapy, the expression of genes involved in cell cycle regulation returned to normal levels.

4. Discussion

Stem cells have emerged as a key element of regenerative medicine therapies due to their inherent ability to differentiate into variety of cell phenotypes, thereby providing numerous potential cell therapies to treat degenerative diseases. Although early menopause frequently occurs in female cancer patients after chemotherapy (CTX), bone marrow transplantation (BMT) has been linked to a recovery of ovarian function and fertility in some survivors. Stem cell transplantation has been found to restore the fertilization ability of patients [31], although the mechanism is not known.

UCMSCs have several properties that make them an interesting source of cells for therapeutic use. In our experiments, human UCMSCs could be isolated rapidly in large numbers from 90% of human cords. Thus, UCMSCs cells may be a robust source of MSC-like cells for therapeutic use because they can be frozen/thawed, clonally expanded, engineered to express exogenous proteins, and extensively expanded in culture.

We found that, after transplantation of UCMSCs, the function of the ovary recovered. Previous study has shown that UCMSCs transplanted by intravenous migrate into several organs after hours [32]. And the ovulation of mice needs 4 days, so we chose after one week to measure the UCMSC location and function. Although we found that human UCMSCs successfully recover mouse ovary function, we did not find that the cells differentiate into follicle components. Previous studies have shown that ES-conditioned media can improve oocyte maturation in vitro [33]. The UCMSCs are more likely to function through the paracrine pathway. We found that the UCMSC treatment group expressed more proteins involved in the DNA modification epigenetic pathway, the transcription pathway, the protein modification pathway, and cell signaling. UCMSCs quickly repair ovarian damage by granulosa cell growth stimulated by these pathways. The UCMSCs can also increase some ovarian stem cell function proteins, including AKR1C18 and Ly6a (Sca-1). Levels of the cell cycle control protein CDKN2B in the stem cell transplantation group returned to the levels normally observed in the wild-type group. UCMSCs can secrete potent combinations of trophic factors that modulate the molecular composition of the environment to evoke responses from resident cells [34–37].

In addition to accelerating the repair of damaged ovarian cells, UCMSCs dramatically reduce apoptosis of granulosa cells in the developing follicle. Most of the follicles progress to apoptosis after exiting from the arrested stage of the cell cycle. When CTX is administered to the mice, apoptosis increases to an even higher level. After UCMSC treatment, the rate of apoptosis is reduced. Previous studies have shown that stem cell can inhibit apoptosis through secretion of stanniocalcin-1, paracrine factors, improve the oxide response pathway, [38–40]. Therefore, we believe that UCMSCs function primarily by reactivating host oogenesis, which becomes impaired in a reversible manner after CTX (e.g., cell cycle arrest) or by indirect effects of the drug on the microenvironment (niches).

In summary, this study supports the idea that UCMSCs can rescue mouse ovary impairment, without causing immune rejection. In addition to BMT, we showed that hUCMSCs could rescue chemotherapy-induced POF in mice. hUCMSCs possess the immune properties of immune suppression and immune avoidance [41]. Following xenotransplantation of human UCMS cells into immune-competent rats, little to no host immune cell infiltration was observed, suggesting that UCMSCs possess low immunogenicity [42]. We provided evidence demonstrating that, after transplantation of UCMSCs, the functionality of the POF ovary is recovered and improved.

The potential clinical applications are immediately apparent. Each year millions of women undergo fertility treatments due to age-related loss of oocytes; these treatments offer only modest improvements in fertility. By the middle of the fifth decade, the therapies are largely futile. Furthermore, millions of young women are rendered sterile by chemotherapy-associated oocyte loss. If a viable source of oocyte production remains in these women, there is the potential to restore fertility. The identification of these stem cells gives hope to these women and leads us to ask if fertility restoration is possible. Further understanding and potential manipulation of the adult female germ stem cell niche may provide answers. In addition, at least for high-dose CTX-exposed females who exhibit reduced posttreatment lethality, UCMSCs might also rescue fertility as part of a more global beneficial effect on overall health.

Conflict of Interests

The authors have no conflict of interests to disclose.

Authors’ Contribution

Shufang Wang, Ling Yu, and Min Sun contributed equally for this paper.

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China 81100396 and China Postdoctoral Science Foundation.

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