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ISSN : 2671-4639(Print)
ISSN : 2671-4663(Online)
Journal of Animal Reproduciton and Biotechnology Vol.32 No.3 pp.209-220
DOI : https://doi.org/10.12750/JET.2017.32.3.209

17β-estradiol mediated effects on pluripotency transcription factors and differentiation capacity in mesenchymal stem cells derived porcine from newborns as steroid hormones non-functional donors

Won-Jae Leea, Ji-Sung Parkb, HyeonJeong Leeb, Seung-Chan Leec, Jeong-Hyun Leeb, Sun-A Ockc, Gyu-Jin Rhob,d, Sung-Lim Leeb,d
aCollege of Veterinary Medicine, Kyungpook National University, Daegu, 41566, Gyeongbuk, Republic of Korea.
bCollege of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Gyeongnam, Republic of Korea
cAnimal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, 1500 Kongjwipatjwi-ro, Isero-myeon, Wanju-gun, Jeollabuk-do 565-851, Republic of Korea
dResearch Institute of Life Sciences, Gyeongsang National University, Jinju, 52828, Gyeongnam, Republic of Korea

1 These authors contributed equally to this work.

Correspondence: Sung-Lim Lee +82-55-772-2349+82-55-772-2360sllee@gnu.ac.kr
20170804 20170913 20170917

Abstract

The estrogen-mediated effect of mesenchymal stem cells (MSCs) is a highly critical factor for the clinical application of MSCs. However, the present study is conducted on MSCs derived from adult donors, which have different physiological status with steroid hormonal changes. Therefore, we explores the important role of 17β-estradiol (E2) in MSCs derived from female and male newborn piglets (NF- and NM-pBMSCs), which are non-sexually matured donors with steroid hormones. The results revealed that in vitro treatment of MSCs with E2 improved cell proliferation, but the rates varied according to the gender of the newborn donors. Following in vitro treatment of newborn MSCs with E2, mRNA levels of Oct3/4 and Sox2 increased in both genders of MSCs and they may be correlated with both estrogen receptor α (ERα) and ERβ in NF-pBMSCs, but NM-pBMSCs were only correlated with ERα. Moreover, E2-treated NF-pBMSCs decreased in β-galactosidase activity but no influence on NM-pBMSCs. In E2-mediated differentiation capacity, E2 induced an increase in the osteogenic and chondrogenic abilities of both pBMSCs, but adipogenic ability may increased only in NF-pBMSCs. These results demonstrate that E2 could affect both genders of newborn donor-derived MSCs, but the regulatory role of E2 varies depending on gender-dependent characteristics even though the original newborn donors had not been affected by functional steroid hormones.


초록


    National Research Foundation of Korea
    NRF- 2015R1D1A1A01056639

    INTRODUCTION

    Mesenchymal stem cells (MSCs) can be obtained from a variety of sources, including bone marrow (Sekiya et al., 2002), which is the most popular MSC source and considered to be a potential source in stem-cell therapy due to its capacity for self-renewal and differentiation into cells of multiple lineages. Although bone marrow-MSCs (BMSCs) are present in small numbers in adults (Jones et al., 2002), for laboratory investigations and clinical applications, in vitro-expanded BMSCs are in great demand for cell therapy and tissue engineering.

    Several factors influence the properties of MSCs, so controlling these factors can increase the yield and proliferation rate of BMSCs. Estrogen is one of the known factors associated with cellular mitogenesis and proliferation of bone marrow stromal cells (Hong et al., 2009), as well as one of the key factors in the regulation of bone formation (Eriksen et al., 1988). The 17β-estradiol (E2)-pretreated BMSCs inhibit apoptosis and preserve the mitochondrial transmembrane potential via the mitochondrial death pathway, and reduce the donor cell damage (Chen et al., 2012). Most recently, it has been demonstrated that E2 treatment enhances the number of colony-forming units and osteogenic differentiation of MSCs in a dose-dependent manner (Qiu et al., 2014).

    However, estrogen is a primary female sex hormone, so its effects on BMSC proliferation and differentiation vary between males and females. Second, this hormone has multifunctional roles that differentially affect aging, development, and differentiation of various tissues in females and males. Moreover, gender differences in stem cells have been clearly determined (Tözüm et al., 2004; Corsi et al., 2007; Crisostomo et al., 2007; Deasy etal., 2007; Aksu et al., 2008; Chen et al., 2012). the understanding of the regulatory role of E2 in MSC is limited and unclear. We previously reported that estrogen has a gender-dependent role in BMSCs derived from sexually matured-adult minipigs. Anti-apoptotic activity and osteogenic ability were improved by E2 supplements administered only to female-derived BMSCs (Lee et al., 2016). However, other studies report that the osteogenic differentiation potential and bone regeneration of femalederived MSCs are lower than those of male-derived MSCs (Corsi et al., 2007; Aksu et al., 2008), notwithstanding the higher prevalence of osteoarthritis and degenerative joint diseases in women. The gender-dependent characterization of the MSCs that were used to study adult donor-derived BMSCs, such as adult females and males, or ovariectomized adult females, have certain physiological conditions (Calado et al., 2009; Jenei-Lanzl et al., 2010, Chen et al., 2012; Jenei-Lanzl et al., 2012). However, adult donors have been exposed to functionally active sex-steroid hormones as they are sexually mature and possess different endophysiological statuses.

    For this reason, the present study was conducted to find out the effects of estrogen on the proliferation and differentiation of newborn-derived BMSCs (male and female) that have not yet been exposed to active sex hormones. To the best of our knowledge, no studies to date have directly compared the influence of estrogen on the various characterizations and properties of female and male BMSCs derived from newborn donors.

    In this study, we investigated the effects of E2 on the cell proliferation of newborn-pBMSCs, and then inquired whether the cell proliferation effect of E2 could be associated with the regulatory effects of cellular characterization and differentiation capacity of BMSCs derived from male and female newborn donors since the sex-steroid hormones are not yet functionally activated at the infant stage.

    MATERIALS AND METHODS

    1.Chemicals and media

    All the chemicals and media used for cell culture were purchased from Gibco (Invitrogen Corporation, Grand Island, NY, USA) and Sigma-Aldrich Chemical Company (St. Louis, MO, USA), unless otherwise specified.

    2.Isolation and culture of pBMSCs derived from newborn donors

    All experiments were authorized by the Animal Center for Biomedical Experimentation at Gyeongsang National University. Bone marrow was extracted by processing tibia biopsies, three each of female and male newborn piglets. The isolation of cells and the establishment of MSCs derived from the bone marrow of newborn females (NF-pBMSCs) and males (NM-pBMSCs) were performed using the protocol described in a previous study (Patil et al., 2014). Briefly, bone marrow extracts were isolated with the Ficoll (Ficoll-PaqueTM PLUS, Amersham Biosciences, Uppsala, Sweden) density gradient procedure. Cells were cultured in the advanced Dulbecco’s modified Eagle medium (ADMEM), supplemented with 10% charcoal-stripped fetal bovine serum (FBS), 10 ng/mL basic fibroblast growth factor (bFGF), 1% GlutaMax (Gibco), and 1.0% penicillin–streptomycin (10,000 IU and 10,000 μg/mL, respectively, Gibco), under steroid-free conditions at 38.5°C in a humidified atmosphere of 5% CO2 in the air. Once confluent, the cells were trypsinized with 0.25% (w/v) trypsin–ethylenediamine tetra-acetic acid (EDTA) solution; the subculture and passage-3 cells were taken for further analysis. Following the initial primary culture, the NF- and NM-pBMSCs were grown with 0 M (E2 control, E2 Ctrl), 10-6 M, 10-8 M, 10-10 M, 10-12 M, and 10-14 M E2 (Sigma, UK) over the entire in vitro-cultivation and in vitro-differentiation periods. The first day of culture in the growth medium or differentiation (adipocytes, osteocytes, and chondrocytes) induction medium was defined as day 0, and the E2-treated medium was replaced every 3 days. Once the most influential concentration of E2 was determined by the Vybrant® MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] Cell Proliferation Assay (Molecular Probes, Invitrogen, Grand Island, NY, USA), this E2 concentration, compared with the E2-untreated control (0 M E2, E2 control), was used for further experiments.

    3.β-galactosidase activity

    The cellular senescence of both pBMSCs was evaluated, using the Mammalian β-Galactosidase (β-Gal) Assay Kit (Thermo ScientificTM PierceTM, Rockford, IL, USA). Briefly, the cells were cultured until they reached confluence, after being washed with Dulbecco's phosphate buffered saline (DPBS). Next, the cells were fixed for 5 min with 3% formaldehyde and incubated with the β-Gal assay reagent for 30 min. The time reaction was stopped when more than 80% of the cells were positively blue with the β-Gal stain, an indication of the onset of senescence. The optical density (OD) of positive staining was determined by using a VersaMaxTM microplate reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 480 nm.

    4.Induction of in vitro differentiation

    The ability of in vitro differentiation into adipocytes, osteocytes, and chondrocytes of NF- and NM-pBMSCs by E2 treatment was assayed with the protocols described in a previous study, with minor modifications (Lee et al., 2015). Briefly, both pBMSCs were cultured in ADMEM treated with E2 (10-12 M) and E2 control (0 M) until they reached about 70% confluence in a 35-mm dish under conditions conducive to adipogenic, osteogenic, and chondrogenic differentiation for 3 weeks. The medium was changed every 3 days. The adipogenic-differentiation medium consisted of ADMEM supplemented with 10% FBS, 1 μM dexamethasone, 10 μM insulin, 200 μM indomethacin, and 500 μM 3-isobutyl-1-methylxanthine (IBMX). The osteogenicdifferentiation medium consisted of ADMEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, and 10 mM β-glycerol phosphate. For the induction of chondrogenic differentiation, the cells were cultured in the STEMPRO Chondrogenesis Differentiation Kit media with 10% supplement (Gibco) for 21 days. To confirm adipogenic- and osteogenic-differentiation induction, the pBMSCs were stained with 0.5% Oil Red O solution to detect intracellular lipid droplets, and the matrix mineralization and calcium deposits were detected by Alizarin-Red S solution and 5% silver nitrate solution (Von Kossa) staining, respectively. The chondrogenic differentiation of induced pBMSCs was detected by staining with Alcian Blue 8 GX solution for the synthesis of glycosaminoglycans.

    5.Gene expression by quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

    The qRT-PCR analysis based on the multiplex method was conducted to quantify the mRNA levels of the genes involved in differentiation in NF- and NM-pBMSCs treated with E2 (0 M and 10-12 M). The mRNA levels of estrogen receptors, ERα, and ERβ were analyzed on control and E2 treated NF- and NM-pBMSCs. The mRNA levels of the genes related to adipocytes, osteocytes, and chondrocytes were analyzed in both differentiated and undifferentiated (control) cells. The total RNA was extracted from the cells, using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and measured by OPTIZEN 3220 UV BIO Spectrophotometer (Mecasys Co, Ltd, Korea). Reverse transcription was performed, using 1 μg of total RNA with the OmniscriptTM Reverse Transcription Kit (Qiagen, Valencia, CA, USA), with the oligo dT primer (Invitrogen, Carlsbad, CA, USA) at 60°C for 1 h for synthesis of cDNA, followed by relative qRT-PCR using LightCyclerTM with FastStart DNA Master SYBR Green I (Roche), consisting of 2 mM MgCl2, 2 μl SYBR Green, and 0.5 μM each of forward and reverse primers. The amplification program consisted of denaturation at 95°C for 1 min; 50 PCR cycles at 95°C for 10 sec, 60°C for 6 sec, and 72°C for 4 sec; melting curve analysis from 65°C to 95°C with an increase of 0.1°C per second; and cooling at 40°C for 30 sec. The mRNAs were normalized against reference genes, hydroxymethylbilane synthase (HMBS), and β-actin (ACTB). Table 1 lists all the primers for the analysis of in vitro differentiation.

    6.Statistical analysis

    For the analysis of the differences among the cells, this study used one-way analysis of variance (ANOVA) (via IBM SPSS Statistics, ver. 21) with Duncan’s and Turkey’s multiple-comparison tests. The mean values and standard deviation were calculated for the outcome variables, and the differences were considered significant when P-values were less than 0.05.

    RESULTS

    1.E2 treatment enhance cell proliferation ability in NF- and NMpBMSCs

    The NF- and NM-pBMSCs were established from the bone marrows of female and male newborn piglets with an isogenic background, respectively. The morphologies of the homogeneous adherent in both pBMSCs were similar, with a fibroblast-like shape and strongly stained alkaline phosphatase (AP) activity at passage 3 (not shown).

    The effects of E2 treatment on the cell proliferation rate of pBMSCs were evaluated by using the MTT assay (Fig. 1A). The proliferation rates of both pBMSCs with 10-12 M E2 was significantly (P < 0.05) higher than control.

    2.Different levels of estrogen receptors in NF- and NM-pBMSCs by E2 treatment

    The expression levels of ERα and ERβ following E2 treatment varied according to the gender of the pBMSC donors (Fig. 1B). The quantified mRNA level of ERα and ERβ was significantly (P < 0.05) up-regulated in NF-pBMSCs by E2 treatment. However, in the NM-pBMSCs, mRNA level of ERα was significantly (P < 0.05) up-regulated by E2 treatment, but mRNA level of ERβ did not differ between E2 non-treated and treated NM-pBMSCs.

    3.E2 induced increase in the transcriptional factors in the MSCs

    Both NF- and NF-pBMSCs expressed Oct4, Sox2, and Nanog, and the expression levels of those factors were similar between them (Figs. 2). Following E2 treatment, the expression levels of Oct4 and Sox2 were significantly (P < 0.05) increased in both pBMSCs, but Nanog expression was not affected by E2 treatment.

    4.E2 reduced cellular senescence in NF-pBMSCs

    The activity of senescence-associated β-Gal was analyzed between the E2 control and E2-treated NF- and NM-pBMSCs (Fig. 3). The quantified β-Gal activity was significantly (P < 0.05) decreased in only NF-pBMSCs treated with E2 compared with those in the E2 control, but had no difference in NM-pBMSCs.

    5.Differentially regulated differentiation ability between NF- and NM-pBMSCs by E2-mediate effects

    Under specific in vitro conditions for differentiation induction, the E2 control and E2-treated NF- and NM-pBMSCs were evaluated for their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation (Figs. 4-6). After 3 weeks of induction, Oil Red O (Figs. 4A-a and B-a), Von Kossa (Figs. 5A-a and B-a), and Alcian Blue 8 GX (Figs. 6A-a and B-a) staining verified that the E2 control and E2-treated NF- and NM-pBMSCs progressed toward differentiation into adipocytes, osteocytes, and chondrocytes, respectively. These three types of cytochemical staining were positively observed, not only in the E2 control but also in the E2-treated NF- and NM-pBMSCs, which were stained by the cytoplasmic accumulation of lipid droplets for adipocytes, the deposition of calcified extracellular matrix for osteocytes, and the synthesis of glycosaminoglycans for chondrocytes.

    After the complete induction period (3 weeks), the mRNA levels of genes related to adipocytes, osteocytes, and chondrocytes were comparatively quantified between the NF- and NM-pBMSCs that were cultured in the control (0 M) and E2-treated (10-12 M) NF- and NM-pBMSCs (Figs. 4-6). The qRT-PCR analysis revealed higher expressions of adipocyte-related genes, such as adipocyte protein 2 (aP2) and fatty acid-binding protein (FABP), in the differentiated NF- and NM-pBMSCs compared with the undifferentiated pBMSCs cultured in the E2 control and E2 treatment, respectively (Figs. 4A-b and B-b). In the NF-pBMSCs after adipogenic differentiation, the mRNA levels of aP2 and FABP were observed to be significantly (P < 0.05) higher in the E2 control than in its E2-treated counterpart, although the mRNA level of aP2 was only significantly (P < 0.05) higher in the E2 control than in the E2-treated NM-pBMSCs. The adipogenic-differentiation abilities of the NF- and NM-pBMSCs varied according to the gender of the pBMSC donors; the NF-pBMSCs possessed a greater ability compared with the NM-pBMSCs (Fig. 4B and D). This result was further confirmed by the significant differences in the mRNA levels of aP2, LPL, and FABP between the NF- and the NM-pBMSCs. The mRNA levels of these genes in the NF-pBMSCs were clearly higher than those in the NM-pBMSCs. For example, the quantified mRNA level of aP2 in the differentiation-induced NF-pBMSCs with the E2 treatment was 39.163 ± 0.421 (Fig. 4A-b), versus 0.095 ± 0.004 in the differentiation-induced NM-pBMSCs (Fig. 4B-b).

    Following the induction of osteogenic differentiation in NF and NM-pBMSCs, the mRNA levels of runt-related transcription factor 2 (RUNX2) and biglycan (BG) were significantly (P < 0.05) up-regulated in both pBMSCs compared with the undifferentiated cells (Figs. 5A-b and B-b). The mRNA levels of RUNX2 and BG in the osteogenic differentiation-induced NFand NM-pBMSCs were significantly (P < 0.05) higher in the cells cultured with the E2 treatment than those in the E2 control.

    The mRNA levels of the chondrocyte-related genes, collagen type 2 (COL2) and aggrecan (ACAN) were significantly (P < 0.05) up-regulated in both pBMSCs with the E2 control and E2 treatment after the induction of chondrogenic differentiation (Figs. 6A-b and B-b). Following the induction of chondrogenic differentiation in both pBMSCs, the mRNA levels of these three genes were significantly (P < 0.05) higher in the E2-treated pBMSCs than they were in the E2 control.

    6.Discussion

    As the sex-steroid hormone, E2 has recently been demonstrated to enhance various properties of MSCs (Eriksen et al., 1988; Hong et al., 2009; Chen et al., 2012), and further research on the regulatory effects of E2 on the MSCs is necessary to enhance our understanding of autogenic or allogenic transplantation in humans with different endophysiological statuses. However, most studies have been performed on adult donor-derived BMSCs; the effects of E2 from the newborn donor-derived MSCs have not yet been reported. Nonetheless, the regulatory effects of E2 from the newborn-derived BMSCs would be different from that of the adult-derived BMSCs. The reasons for the gender differences in the characterization and differentiation ability have been identified (Tözüm et al., 2004; Corsi et al., 2007; Crisostomo et al., 2007; Deasy etal., 2007; Aksu et al., 2008) in the adult donor-derived stem cells. On the other hand, the gender-dependent characteristics may exist in the newborn donor-derived stem cells with the regulation of the E2 supplementation, regardless of whether the donor is male or female, with no endophysiological change due to the lack of E2 functional secretion.

    We have investigated whether E2 influences the stemness property of the pBMSCs isolated from male and female newborn porcine since these piglets are not yet sexually mature, and their bodies are not yet actively exposed to sex-steroid hormones. Moreover, we have utilized the porcine-derived BMSCs with an isogenic background to compare the gender-dependent characteristics following E2 treatments, and those BMSCs make it possible to eliminate biological variations. Due to pigs’ physiological and anatomical similarities with humans, the porcine animal model is suitable for future human clinical therapies and xenotransplantation (Cooper et al., 2002). Moreover, porcine-derived MSCs have been found to be morphologically and immunophenotypically similar to human MSCs (Shukla et al., 2008).

    In this study, the investigation of the effect that depends on the E2 supplementation in the newborn piglet-derived MSCs reveals that the cell proliferation ability of pBMSCs treated with 10-12 M. Cell proliferation is E2 dose-dependent and regulated in the BMSCs derived from adult mice and rats, as previously reported (Hong et al., 2009, Jenei-Lanzl et al., 2010]. The proliferation rates in the BMSCs derived from adult female and male miniature pigs gradually increase with the decrease in E2 concentrations and peak at 10-12 M–10-14 M and 10-12 M in each gender, as shown in our previous study (Lee et al., 2016). These regulatory patterns, including the findings in the present study that suggest the strong mitogenic effect of E2, could be explained by the biphasic action of steroids, which lose their function and even inhibit the cell proliferation of BMSCs at high concentrations (Hong et al., 2009; 2011). In the adult donor-derived BMSCs, the effective doses of E2 for increasing cell proliferation also vary according to the species or gender of the donors (Hong et al., 2009; 2011), and their specific physiological status, such as ovariectomization (Jenei-Lanzl et al., 2010). The abovementioned reports and our results, overall, indicate that E2 seems to have a proliferative effect on the pBMSCs derived from both genders. Thus, we found that the supplementation of E2 can affect the cell proliferation of NF- and NM-pBMSCs, and these play an increasing role in the cell proliferation of E2, which is similar to adult donor-derived BMSCs (Lee et al., 2016) despite the varying effective doses of E2 (Hong et al., 2009; 2011). Moreover, E2 mediated activation in both newborn MSCs was confirmed to not only increase the proliferation capacity but also up-regulate stem cell transcription factors such as Oct3/4 and Sox2. Oct3/4 is an important stem cell transcriptional factor for maintaining pluripotency and it is significantly expressed with Sox2 and Nanog in MSCs, but Nanog cannot be affected by E2 supplement in both MSCs. To understand ER’s correlation, we measured the expression levels of mRNA of both ERs in both MSCs with predominantly higher mean levels of ERβ mRNA expression than ERα mRNA, and mRNA levels of both ERs were higher in NF-pBMSCs than they were in NM-pBMSCs. However, ERβ was not changed by E2 supplements in NM-pBMSCs, which means ERβ may play a dominant role in correlation with ERα but only with the E2-mediated effect on NF-pBMSCs.

    As shown in our findings, E2 has a regulatory effect on the cell proliferation of pBMSCs derived from newborn females and males; thus, it would be relevant to other important cellular processes in stem cells, such as cellular senescence (Fig. 3). The β-Gal activity has been used as a biomarker of cellular senescence, which is associated with replicative senescence in vitro (Dimri et al., 1995). Cellular senescence is unlike apoptosis, which eliminates damaged cells from tissues; senescent cells remain alive despite changes in morphology and derangement of differentiated functions (Itahana et al., 2004). The cellular senescence is similar between NF- and NM-pBMSCs at passage 3, but the quantified β-Gal activity is only significantly reduced in NF-pBMSCs by E2 treatment.

    Gao et al. (Gao et al., 2014) demonstrate the dose-dependent effect of E2 on osteo-adipogenic transdifferentiation and report that E2 treatment significantly increased AP activity, with the calcium deposition having a higher expression of osteogenicrelated genes, while it decreased the lipid droplet deposition during the osteo-adipogenic transdifferentiation of murine bone marrow-derived MSCs. To investigate the effect of E2 on the in vitro-differentiation ability, NF- and NM-pBMSCs were differentiated into adipocytes, osteocytes, and chondrocytes by providing specific conditioned media. Both pBMSCs possessed these differentiation abilities when the influential dose of E2 (10-12M) was provided and also in the absence of E2, according to the gross observation of specific staining, and when specific mRNA levels were increased after differentiation inductions. To the best of our knowledge, no other study has directly compared the effects of E2 on the in vitro-differentiation properties between female and male pBMSCs derived from newborn donors, which are MSC donors without any functional activity of sex-steroid hormones.

    In our study, following the adipogenic-differentiation induction with 10-12 M E2 treatment, the mRNA levels of aP2 and FABP are significantly (P < 0.05) up-regulated in NF-pBMSCs, while in NM-pBMSCs, only aP2 is up-regulated. The adipogenicdifferentiation ability of NF- and NM-pBMSCs varies according to the gender of the pBMSC donors, and NF-pBMSCs possess a greater ability compared with NM-pBMSCs. These results are further confirmed by the quantification of the mRNA levels of aP2 and FABP between NF- and NM-pBMSCs. The mRNA levels of these genes in NF-pBMSCs are clearly higher than those in NM-pBMSCs.

    The mRNA levels of RUNX2 and BG are significantly (P < 0.05) up-regulated in 10-12 M E2-treated osteogenic-induced NF- and NM-pBMSCs compared with the E2 control. In both females and males, estrogen is one of the key positive regulators of bone mass in vivo (Balasch et al., 2003; Syed et al., 2005). In the MSCs derived from mice, rodents, and humans, estrogen has been proven to improve the capacity for in vitro differentiation into osteocytes (Oreffo et al., 1995; Qu et al., 1998; Yeh et al., 1999); it also improves bone formation by inhibiting bone resorption (Verhaar et al., 1994; Qu et al., 1998). Moreover, the gender-dependent capacity for osteogenesis is distinctly established in the MSCs derived from adult donors (Tözüm et al., 2004; Corsi et al., 2007; Crisostomo et al., 2007). Previous reports about BMSCs derived from adult donors have revealed that estrogen plays a reciprocal function in adipogenesis and osteogenesis (Dang et al., 2004). Similarly, estrogen treatment has been reported to improve osteogenic ability by inhibiting adipogenesis in BMSCs derived from adult female mice and humans (Dang et al., 2004; Heim et al., 2004), as well as BMSCs derived from adult male human donors (Hong et al., 2006). In the present study, E2 does not play a reciprocal function in the adipogenic and osteogenic differentiation in newborn-derived pBMSCs. Although the adipogenic-differentiation ability might be gender-dependently affected by the E2 treatment in newborn-derived pBMSCs, our findings show that the osteogenic-differentiation ability is not gender-dependently influenced by E2 treatment.

    In the chondrogenic differentiation-induced NF- and NM-pBMSCs, the mRNA levels of COL2 and ACAN are significantly (P < 0.05) higher in the E2-treated NF- and NM-pBMSCs than in the E2-untreated control (Figs. 6A-b and B-b). Therefore, E2 treatment obviously increases the chondrogenic- differentiation ability of both pBMSCs without any gender differences. The cartilage is a sex hormone-sensitive tissue in the body, and estrogen affects the cartilage under certain physiological and pathological conditions; for instance, estrogen replacement therapy reduces the loss of articular cartilage in postmenopausal women (Wluka et al., 2001; Parker et al., 2003). However, many of the in vitro studies about E2 report the ineffective function or negative influence of chondrogenic differentiation; E2 does not significantly affect in vitro chondrogenic differentiation in rabbit MSCs (Ab-Rahim et al., 2008) and bovine articular chondrocytes (Claassen et al., 2006). Moreover, E2 supplementation shows a dose-dependent inhibition of 3D chondrogenesis, as well as reduced COL2 deposition in the BMSCs from young men (Calado et al., 2009). Our results suggest that E2 treatment could improve the chondrogenic ability of newborn-pBMSCs from both female and male donors, and this finding may be more similar to the effect of E2 on in vivo chondrogenesis.

    In conclusion, these findings are consistent with the idea that E2-mediated effects gender dependently regulate BMSCs derived from non-sexually matured donors with steroid hormones, such as female and male newborn porcine. E2 effectively improves or regulates the adipogenic-, osteogenic-, and chondrogenic-differentiation potential in newborn-pBMSCs derived from female minipig porcine, as well as improves the osteogenic and chondrogenic differentiation of newbornpBMSCs derived from male porcine. All the results indicate that the addition of E2 reduces cellular senescence with improvements in the cell proliferation of newborn-pBMSCs derived from female porcine, but these cellular regulations do not occur in newborn-pBMSCs derived from male porcine. Therefore, E2 supplementation mostly leads to regulating the osteogenic and chondrogenic differentiation capacity, along with cell proliferation, cellular senescence in newbornpBMSCs derived from females.

    Acknowledgment

    This work was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the government of the Republic of Korea (grant no. NRF- 2015R1D1A1A01056639).

    Figure

    JET-32-209_F1.gif

    Cell proliferation analysis and mRNA levels of estrogen receptors of E2-treated NF- and NM-pBMSCs. (A) Both pBMSCs were grown with steroid-free media with 10-12 E2 supplements for 14 days. The cell proliferation rates were quantified by measuring the absorbance at 540 nm by MTT assay. E2 control means 0 M E2 as E2-untreated cells. Bars with an * indicate significant (P < 0.05) differences in five replicates. (B) Both pBMSCs were cultured with E2 control and treated (10-12 M) conditions until confluent, and mRNA levels of estrogen receptor-α (ERα) and -β (ERβ) were quantified by qRT-PCR. Bars with an * indicate significant (P < 0.05) differences.

    JET-32-209_F2.gif

    Analysis of mRNA levels of stem cell transcription factors in E2-treated NF- and NM-pBMSCs at passage 3. The mRNA levels of Oct3/4, Sox2, and Nanog were quantified in E2 control and treated both pBMSCs by qRT-PCR. Bars with an * indicate significant (P < 0.05) differences.

    JET-32-209_F3.gif

    Senescence-associated β-galactosidase expression of E2-treated NF- and NM-pBMSCs. Both pBMSCs were cultured with E2 control (0M) and treated (10-12 M) conditions until confluent for the arrest of cell growth. Cellular senescence was quantified in NFand NM-pBMSCs by measuring β-galactosidase staining at the absorbance at 405 nm. Bars with an * indicate significant (P < 0.05) differences.

    JET-32-209_F4.gif

    Adipogenic differentiation abilities of E2-treated NF- (A) and NM-pBMSC (B). Following 3 weeks of induction, adipogenesis was demonstrated by the formation of lipid droplets and confirmed by Oil Red O staining (A-a and B-a). X100, scale bar = 100 μM. The mRNA levels of adipocytes-specific markers [adipocyte protein 2 (aP2) and fatty acid-binding protein (FABP)] were quantified in NF- (A-b) and NM-pBMSCs (B-b) by qRT-PCR. Five replicates were performed. Bars with an * indicate a significant (P < 0.05) difference between cells. Gray bars represent E2-untreated control, and black bars represent E2-treated (10-12 M) cells.

    JET-32-209_F5.gif

    Osteogenic differentiation abilities of E2-treated NF- and NM-pBMSC. After 3 weeks of osteogenic induction, mineralization of calcium nodules was indicated by Von Kossa staining in both pBMSCs (A-a and B-a). X 100, Bars 100 μm. The mRNA levels of osteogenic-specific markers [runt-related transcription factor 2 (RUNX2) and Biglycan (BG)] were quantified in NF- (A-b) and NM-pBMSCs (B-b) by qRT-PCR. Five replicates were performed. Bars with an * indicate a significant (P < 0.05) difference between cells. Gray bars represent E2-untreated control, and black bars represent E2-treated (10-12 M) cells.

    JET-32-209_F6.gif

    Chondrogenic differentiation abilities of E2-treated NF- and NM-pBMSC. Chondrogenic differentiation in the E2-untreated control and treated (10-12 M) NF- and NM-pBMSCs was induced for 3 weeks (A-a and B-a). Alcian Blue 8 GX solution staining positively determined the synthesis of glycosaminoglycans in both pBMSCs. X 100, Bars 100 μm. The mRNA levels of chondrocyte-specific markers [collagen type2 (COL2) and aggrecan (ACAN)] were quantified in NF- (A-b) and NM-pBMSCs (B-b) by qRT-PCR. Five replicates were performed. Bars with an * indicate a significant (P < 0.05) difference between cells. Gray bars represent E2-untreated control, and black bars represent E2-treated (10-12 M) cells.

    Table

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