Human Wharton’s jelly-derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT-mediated immunomodulation

Human Wharton’s jelly-derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT-mediated immunomodulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Human Wharton’s jelly-derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT-mediated immunomodulation Xiaoying Ding, Rongrong Wu, Tingting Yang, Beibei Jin, Chunyu Zhu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1698573/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Spontaneous abortion (SA) is a syndrome with many origins. Among them, immune imbalance is a major risk factor for SA. Wharton's jelly-mesenchymal stem cells (WJ-MSCs) are considered to be able to prevent abortion. However, the underlining molecular signaling pathways and regulatory mechanisms of WJ-MSCs in pregnancy maintenance are poorly understood. Here abortion mode is established by subcutaneous injection of bromocriptine in rat on day 9 and abortion prevention is achieved by WJ-MSCs injection via tail vein with or without JAK/STAT inhibitor. Here, we show that WJ-MSCs significantly lower the rate of embryo resorption of spontaneous abortion by reducing Th1-related cytokines while increasing Th2 and Th3-related cytokines in JAK/STAT-dependent manner. Together, our observation of WJ-MSCs in spontaneous abortion prevention will shed new light on potential therapeutic strategy development. JAK/STAT spontaneous abortion Th1 Th2 Th3 Wharton’s jelly- mesenchymal stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Spontaneous abortion (SA) is a common clinical disease that seriously affects the physical and mental health of women of childbearing age. Many risk factors, including genetic factors, endocrine disorders, infectious agents, immune dysfunction and genital anatomical abnormalities, contribute to this complex pathology[ 1 , 2 ]. So far, ≥ 50% of the causes are not clear. The immune imbalance has been reported to be an important factor associated with SA and infertility[ 3 ]. During pregnancy, the maternal immune system must be modified to accept the semi-allogeneic fetus. The immune relationship between embryonic and maternal tissue is extremely complex involving human leukocyte antigens, the complement pathway, cytokines, blood group antigens, trophoblast cell membrane antigen, and intercellular adhesion molecules[ 4 ]. As a result, the role of T helper cells (Th) and the balance of secreted cytokines in SA is an active area of research. Helper T cells are divided into Th1, Th2, Th3 and Th17 subtypes based on their cytokine production. Th1 cells secrete Interleukin-2 (IL-2), Interferon-γ (IFN-γ), Tumor necrosis factor-α (TNF-α), Tumor necrosis factor-β (TNF-β) and play a role in cellular immune responses. Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13, and are involved in humoral immunity. Th3 cells produce transforming growth factor-β (TGF-β) which inhibits the expression of Th1 cytokines. A normal pregnancy exhibits a shift towards Th2 cytokine production and inhibition of Th1 responses and disturbance of this balance may result in unexplained recurrent spontaneous abortion (URSA)[ 5 ]. Additionally, Th17 cells produce IL-17 which can cause an inflammatory response at the maternal interface that jeopardizes pregnancy[ 5 , 6 ]. The importance of T cells subtypes and the cytokine environment at the maternal-fetal interface suggests that immunotherapy may be a viable method of treating URSA in the clinic[ 7 , 8 ]. Current treatments for URSA include active immunotherapy, but their effectiveness is limited[ 9 , 10 ]. With progress in the understanding of reproductive immunology, the immune mechanism of SA and its immunological treatment has become an important topic of research[ 11 , 12 ]. It has been reported that the expression of related factors from Th1 / Th2 / Th3 / Th17 (IL-2, IL-3, IL-5, IL-6, IL-10, IL-17) mainly plays a biological function through JAK / STAT pathway, and these interleukins can activate JAK / STAT signaling pathway under specific conditions[ 13 ]. There are other pathways involved in interleukin mediating immune response, such as inhibiting the ability of nuclear factor-κB (NF-κB) to bind to DNA or inhibiting the activity of IκB kinase reversely to inhibit NF-κB activation. Mesenchymal stem cells(MSCs)are pluripotent stem cells, which have been studied in tissue engineering, hematopoietic stem cell transplantation and gene therapy. MSCs have been widely studied in the treatment of immune diseases because of their role in immune regulation[ 14 ]. It has been shown that Wharton's jelly (WJ) in the human umbilical cord is an important source of MSCs which secrets a large number of cytokines and possesses no tumorigenicity, lower infection rate of pathogenic microorganisms, fewer ethical problems, multidirectional differentiation potential and lower immunogenicity than bone marrow[ 15 , 16 ]. It has been shown [ 17 ] that the expression of IFN-γ secreted by Th1 decreased significantly, while the expression of IL-4, IL-13 and other factors secreted by Th2 increased after the co-culture of WJ-MSCs and immune cells. It has been also found that WJ-MSCs affect the differentiation and function of Th17 cells in the peripheral lymphocyte system and promote the proliferation and differentiation of Tregs cells to maintain the balance between Th17 cells and Tregs cells and improve the immune microenvironment of the body. These results above suggest that WJ-MSCs are important for local immune tolerance. Bromocriptine is a prolactin inhibitor to trigger abortion by disrupting the endocrine environment[ 18 ], such as low progesterone (P), low prolactin (PRL), and abnormal Th1 immune response. Human WJ-MSCs have previously been shown to prevent SA in this rat model by decreasing the expression of IFN-γ and IL-17 and up-regulating the expression of IL-10[ 18 ], but the detailed mechanism of the immunomodulatory effect remains elusive. Here we utilized bromocriptine-induced abortion in rats to investigate the therapeutic effects of WJ-MSCs on Th1/Th2/Th3 cytokines and to examine the roles of JAK/STAT and NF-κB signaling in immunomodulation of WJ-MSCs. Materials And Methods Isolation, culture and characterization of WJ-MSCs Human umbilical cords were obtained from normal full-term deliveries with the informed consent of the mother. The preparation of WJ-MSCs was approved by the Research Ethics Committee at the Affiliated Hospital of Nantong University. WJ-MSCs were isolated, cultured and characterized using the methods described in a previous study[ 18 ]. WJ-MSCs treated with JAK/STAT or NF-κB inhibitors P3 WJ-MSCs were cultured with DMEM/F12 medium (US, Hyclone) containing 10% fetal bovine serum (FBS; US, Hyclone) with/without the inhibitors of JAK/STAT (Germany, MERCK, AG490) or NF-κB (Solarbio, BAY-11-7085). The medium was removed 5 hours later, WJ-MSCs were washed twice with PBS and then cultured with DMEM/F12 medium containing 10% FBS. Cell viability test The viability of WJ-MSCs treated with JAK/STAT inhibitor or NF-κB inhibitor was measured by a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan). All experimental procedures were performed according to the manufacturer's instructions. Rat model of spontaneous abortion The protocol was approved by the Care and Use Committee of the Laboratory Animal Research Center of Nantong University. Age-matched virgin female (200–250 g; n = 48) and male (200–250g; n = 48) Sprague–Dawley (SD) rats were obtained from the Laboratory Animal Center of Nantong University. Day 1 of pregnancy was defined by the presence of a vaginal plug. Pregnant rats were randomly divided into 6 groups. Group A served as a normal pregnancy group and received a vehicle injection of 0.2ml of PBS by subcutaneous injection on gestational days 6–8 and 1ml of DMEM/F12 by tail vein injection on day 9 of gestation. Group B received a subcutaneous injection with 0.8 ml/kg of 75% ethanol on gestational days 6–8 and 1ml of DMEM/F12 via tail vein injection on day 9 of gestation. Group C served as the abortion model and received subcutaneous injections of 0.8 ml/kg of bromocriptine (0.5mg/ml) in 75% ethanol on gestational days 6–8 and 1ml of DMEM/F12 by tail vein injection on day 9 of gestation. Group D was similar to group C but with an additional injection of 107 WJ-MSCs in 1ml of DMEM/F12. Group D received JAK/STATor NF-κB signal pathway inhibitors in 1ml of DMEM/F12 served as Group E and F respectively. Tissues and serum collection Pregnant rats in each group were euthanized on day 14 of gestation and embryo resorption was assessed. The fetal/placental unit and decidua were removed from the uterus and rinsed with saline. A portion of the fresh tissues was stored at -80℃ for real-time polymerase chain reaction (RT-PCR) or Western blotting(WB). Whole blood was collected from the orbit and serum was obtained by centrifuging blood in serum separation tubes. Serum was stored at -80°C before being subjected to enzyme-linked immunosorbent assays (ELISA). Cytokines in serum Serum concentrations of cytokines (IL-2, IL-4, IL-5, IL-13, TNF-α, TNF-β, TGF-β) were measured by ELISA kit (eBioScience) following the manufacturer’s instructions. All samples were measured in triplicate. Cytokine in decidual/placental tissues Total protein was extracted from decidual and placental tissues as previously described 16 . Protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat milk powder and incubated with the following primary antibodies: anti-IL-2 (Abcam, ab25104), anti-IL-4 (Abcam, ab84278), anti-IL-5(Novus Biologicals, AF545), anti-IL-13(Abcam, ab271227), anti-TNF-α (Abcam, ab9755), anti-TNF-β (Abcam, ab106353) at 4°C overnight, washed and incubated with HRP-labeled secondary antibody (Abcam, ab6721) for 2h at room temperature. After three times washing, signals were detected by ECL (Millipore) according to the manufacturer’s instructions. Cytokine expressions were quantified using densitometry normalized by GAPDH. RT-PCR Analysis of Th-related cytokines Total RNAs were isolated from tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in RNA-free centrifuge tubes. According to the instructions of the cDNA synthesis Kit (Roche, Switzerland), 1µg total RNA was reverse transcribed in 5 × Reaction Buffer, 10 mmol/L dNTPs, 1µmol/L oligo-dT primer, 20 U RNase inhibitor, 200 U/µl RT enzyme, and an appropriate amount of DEPC-treated water. Quantitative RT-PCR (qRT-PCR) was performed in a total volume of 20 µl reaction containing 2 µL cDNA, 10 µl SYBR Green (Roche, Switzerland), indicated primer pairs and 6 µl ddH2O. Primer pairs used are shown in Table 1. Statistical methods Results were analyzed with SPSS 22.0 data analysis software. Data are expressed as mean ± standard error of the mean (mean ± SEM). p < 0.05 is considered to be statistically significant. Results Isolation, culture and characterization of WJ-MSCs The recent development of stem cell technology has revolutionized the treatment of various diseases and laid the foundation for immunomodulation[ 19 ]. To investigate WJ-MSCs’ function, we first cultured MSCs obtained from human umbilical cords[ 18 ]. Our results showed that a small number of WJ-MSCs appeared around the implanted blocks after seeding for 6–7 days (Fig. 1 A). After 9–10 days of culture, cells were gathered in pieces (Fig. 1 B). After 12–14 days of culture, cells proliferated rapidly to formed a single layer and exhibited a uniform spindle shape reminiscent of fibroblasts (Fig. 1 C). JAK/STAT or NF-κB inhibition did not cause a discernible difference in cell morphology and growth (Fig. 1 D-G). Flow cytometry of passage 3 cells showed that WJ-derived cells expressed the MSC markers CD105, CD29, CD73, CD166 and CD44, and were negative for the markers CD34, CD11b, CD14, CD45, and HLA-DR (Supplementary Fig. 1A), which was consistent with human mesenchymal stem cells[ 20 , 21 ]. Subsequently, the differentiation potentials of MSCs into osteoblasts and adipocytes were also confirmed (Supplementary Fig. 1B). The effects of WJ-MSCs transfusion in the rat spontaneous abortion model It was unclear how WJ-MSCs inhibited immunity in immune regulation. To determine whether WJ-MSCs would improve abortion and the potential mechanism, MSCs prepared from a human umbilical cord pretreated with or without JAK / STAT or NF- ĸ B inhibitors were injected into a pregnant rat in the presence of bromocriptine. The results showed that absorbing embryos were rarely detected in normal or ethanol pregnancies (Fig. 2 A, a & b), but were frequently detected in the bromocriptine-induced abortion model (Fig. 2 A, c). Treatment with WJ-MSC significantly reduced the visible occurrence of embryo absorption (Fig. 2 A, d). While JAK/STAT inhibitor but not NF-κB inhibitor primed WJ-MSC obviously compromised the effect of abortion prevention of WJ-MSC (Fig. 2 A, e and f). Quantification revealed that embryo absorption was significantly increased in the bromocriptine-treated group compared to the normal pregnancy or ethanol injection group (Fig. 2 B). WJ-MSC treatment significantly reduced the abortion rate in bromocriptine-treated pregnancy (Fig. 2 B). JAK/STAT inhibition abolished pregnancy-preserving effects of WJ-MSC but NF-κB inhibition did not (Fig. 2 B). Cytokine profiles during the abortion and the effects of WJ –MSC transfusion Serum cytokines by ELISA To further evaluate the local environmental immune profile changed by WJ-MSCs, the cytokines secreted from Th1, Th2 and Th3 cells were investigated in different groups. ELISA analysis showed that the levels of Th1-related cytokines IL-2, TNF-α and TNF-β in the bromocriptine-induced abortion group were significantly higher than in the normal pregnancy group or ethanol injection group (Fig. 3 A), while the levels of the Th2-related cytokines IL-4, IL-5 and IL-13 (Fig. 3 B), and Th3 cell-cytokine TGF-β were significantly decreased (Fig. 3 C). These results suggested that WJ-MSCs transfused into the abortion rat restored cytokine profiles to comparable levels of normal pregnancy group or ethanol injection group (Fig. 3 ). JAK/STAT pretreated WJ-MSC prevented their rescues effect on cytokine profiles, but not NF-κB inhibition (Fig. 3 ). Cytokines in decidual and placental tissues by Western Blot The results above showed that the mechanism of WJ-MSCs regulating abortion primarily depended on JAK/STAT signaling pathway. As the maternal-fetus interface was composed of both decidua and placenta, the expression of cytokines was detected in both parts. Western blot results showed that Th2 cytokines (IL-4, IL-5, IL-13) were significantly reduced in the bromocriptine-induced abortion group compared to the normal pregnancy group or ethanol group (Fig. 4 B), while the Th1 cytokines TNF-α and TNF-β significantly increased except IL-2 (Fig. 4 A). WJ-MSC injection significantly reversed these abortion-related effects on cytokine (Fig. 4 ). JAK/STAT inhibition of WJ-MSC abolished their ability to reverse Th2-related cytokine changes (Fig. 4 ) Cytokine mRNA in decidual and placental tissues To further validate our results, we extracted the mRNA from decidual and placental tissues. Our results suggested that Th2 (IL-4, IL-5, IL-13) and Th3 (TGF-β) cytokine mRNA levels were significantly reduced in the bromocriptine-induced abortion group compared to the normal pregnancy or ethanol group (Fig. 5 B, C), while the Th1 ( IL-2, TNF-α, TNF-β) cytokine mRNA levels were significantly increased (Fig. 5 A). WJ-MSC treatment of the abortion group significantly reversed these abortion-related effects on cytokine mRNA. JAK/STAT inhibition of WJ-MSC prevented them from reversing abortion-related cytokine mRNA changes (Fig. 5 ). Discussion The bromocriptine-induced abortion model examined here involved a shift from a Th2-dominated immune response that supports pregnancy to a detrimental Th1-dominated response. Human WJ-MSCs had immunomodulatory properties that prevented this shift and rescued pregnancy. There was a decrease of IL-2 in the WJ-MSCs group by ELISA and PCR in different groups but its expression detected by the Western Blot experiment showed less discrepancy which might be due to the secretory nature of IL-2. Human WJ-MSCs decreased the Th1 (IL-2, TNF-α and TNF-β) cytokines and increased the Th2 (IL-4, IL-5 and IL-13) cytokines in serum and decidual/placental tissue. Additionally, there was also evidence that WJ-MSCs promoted the expression of TGF-β, an anti-inflammatory cytokine produced by Th3 and regulatory T cells. We demonstrated that the immunomodulatory and pregnancy-preserving effects of WJ-MSCs were dependent on JAK/STAT signaling in WJ-MSCs, but not NF-κB signaling. The T cell cytokines profile presented here expanded our knowledge of the effects of WJ-MSCs in the rat bromocriptine-induced abortion group which only assessed the expression of IFN-γ, IL-10 and IL-17 in our previous study (REF). The observation that the changes of Th1/Th2 balance during the abortion and restoration by WJ-MSCs injection were detected in both peripheral blood and decidual/placental tissue which suggested that both systemic and local immune responses at the maternal/fetal interface contributed to this hemostasis maintenance and pregnancy success. Our results are consistent with reports that human endometrial MSCs prevented abortion in a mouse incompatible pregnancy abortion model[ 22 ], and that mouse bone marrow-derived MSCs prevented abortion in LPS-mediated and incompatible mating mouse models[ 23 ]. Our observation that WJ-MSCs tipped T cell cytokine responses from Th1 to Th2 was similar to the findings from the therapeutic application of MSCs to other models including mouse diabetes[ 24 ], rat corneal transplant[ 25 ], rat autoimmune uveoretinitis[ 26 ], rat renal injury[ 27 ] and mouse colitis[ 28 ]. Our results suggested that JAK/STAT signaling, an important mediator of responses to injury and inflammation, was required for WJ-MSCs to modify T cell responses. JAK/STAT signaling in human bone marrow MSCs had been implicated in the production of indoleamine 2,3-dioxygenase, a suppressor of T cell proliferation[ 29 ]. Other studies of human bone marrow MSCs indicated the requirement of JAK/STAT signaling for the release of modulators of innate immunity[ 30 ] and homing of MSCs to sites of injury/inflammation[ 31 ]. Taken together, these findings indicate the broad importance of JAK/STAT signaling in the therapeutic properties of MSC. Manipulating JAK/STAT signaling may allow the therapeutic effects of WJ-MSCs to be optimized. MSCs from various tissues had immunomodulatory properties making them potentially useful for treating a range of diseases and injuries. WJ-MSCs were an attractive source for therapy because they can be obtained in large numbers from umbilical cords that were usually discarded. The utilization of MSCs to prevent human recurrent spontaneous abortion might be practicable due to the pregnancy-preserving immunomodulation of these allogeneic cells rather than long-term engraftment. Our results presented here further corroborated that the administration of xenogeneic human MSCs had the desired effect of modulating a rat immune response. WJ-MSCs had already been shown to be beneficial for graft versus host disease in human patients undergoing hematopoietic stem cell transplantation, and clinical trials were underway for a range of other conditions[ 32 ]. Collectively, our study opened a new avenue to combat recurrent abortion and provide a promising strategy for clinical application. Declarations Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article. Acknowledgment Natural Science for Youth Foundation of China (Grant No.81401272) Research project of Jiangsu maternal and child health care association in 2021 (Grant No.FYX202103) Six Talent Peaks Project of Jiangsu province 2019 (WWS-067) Graduate Research innovation Project of Jiangsu Province（KYCX21-3122） Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. References Quenby S, Gallos ID, Dhillon-Smith RK et al (2021) Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss. Lancet 397(10285):1658–1667. DOI: 10.1016/s0140-6736(21)00682-6 Hajipour H, Nejabati HR, Latifi Z et al (2018) Lymphocytes immunotherapy for preserving pregnancy: Mechanisms and Challenges. Am J Reprod Immunol 80(3):e12853. 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Int J Stem Cells 12(2):218–226. DOI: 10.15283/ijsc18034 Tables Table 1 is available in the Supplementary Files section. Supplementary Files FigureS1.tif Figure S1. The Characterization of WJ-MSCs and the differentiation potential of WJ-MSCs toward the osteogenic and adipogenic lineages. (A) The surface antigens of WJ-MSCs in flow cytometry. WJ-MSCs were positive for CD105, CD29, CD73, CD166 and CD44, and were negative for the markers CD34, CD11b, CD14, CD45, and HLA-DR. (B) Osteogenic and adipogenic differentiation was assayed by the OriCell® procedure. Adipogenic differentiation was detected by oil red O staining and evidenced by the formation of lipid vacuoles after adipogenic induction. Osteogenic differentiation was evidenced by staining alizarin red in WJ-MSC after osteogenic induction. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1698573","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":137961784,"identity":"c4cbdc65-1d23-4257-be30-af2271836ff7","order_by":0,"name":"Xiaoying Ding","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Ding","suffix":""},{"id":137961785,"identity":"9a6c5bd6-6020-4b77-9eeb-8b3d180e775b","order_by":1,"name":"Rongrong Wu","email":"","orcid":"","institution":"Yancheng Third People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rongrong","middleName":"","lastName":"Wu","suffix":""},{"id":137961786,"identity":"6022863a-a29f-49cc-99ab-5de9719089ab","order_by":2,"name":"Tingting Yang","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Yang","suffix":""},{"id":137961787,"identity":"a5d0b2b1-4c8a-417c-94e8-6fa095171192","order_by":3,"name":"Beibei Jin","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Beibei","middleName":"","lastName":"Jin","suffix":""},{"id":137961788,"identity":"8d9d91ce-e283-4f3f-bede-835272286f68","order_by":4,"name":"Chunyu Zhu","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunyu","middleName":"","lastName":"Zhu","suffix":""},{"id":137961789,"identity":"a1a096aa-e9aa-4e51-b90e-09729ed9d1b0","order_by":5,"name":"Yuquan Zhang","email":"","orcid":"https://orcid.org/0000-0003-1660-5330","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuquan","middleName":"","lastName":"Zhang","suffix":""},{"id":137961790,"identity":"c37cf1e1-14ce-458d-947e-ed41caca7ec9","order_by":6,"name":"Xiaoqing yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYNACAxs5fmbmgw+IU80GIgrSjCXb2ZINSNDy4VDihvM8ZgJE6TCX7zGTeGNwIHHzYQYzBoYam2iCWizbeIwN5xjcMd52mCHtAcOxtNwGQloMjvEYPuYxeCYL1HLcgLHhMFFaDA4DEePmZsY2CWK1gGw5rLiBmZmNWC1pxUC/pBlLHGZjNkggyi+HD2+TePMHGJX95z8++FBjQ1gLAwOHAQMPjJ1AWDkIsD9AaBkFo2AUjIJRgA0AAPiWPs/6Wb7fAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5808-4380","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaoqing","middleName":"","lastName":"yang","suffix":""}],"badges":[],"createdAt":"2022-05-27 06:19:57","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1698573/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1698573/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26774253,"identity":"a548af40-75a7-4cb0-891c-d55a642fa9f4","added_by":"auto","created_at":"2022-09-21 16:07:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148468,"visible":true,"origin":"","legend":"\u003cp\u003eIsolated and cultured human WJ-MSCs. (A) 6-7 days after seeding, a small number of WJ-MSCs appeared around the implanted blocks. (B) After 9-10 days of culture, cells are gathered in pieces. (C) After 12 -14 days of culture, the majority of cells exhibited a uniform spindle shape similar to fibroblasts. (D-F) JAK/STAT or NF-ĸB inhibition did not alter the appearance of cultured WJ-MSC. (G) JAK/STAT or NF-ĸB inhibition significantly alters the growth and viability of WJ-MSC as measured by a CCK8 assay. A-F were taken using 100X magnification (n=8, *p\u0026lt;0.05, **p\u0026lt;0.01.).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/6a3102bb7e4648c650208d73.jpg"},{"id":26773672,"identity":"e8a0849c-7716-4153-9b78-047dedb600be","added_by":"auto","created_at":"2022-09-21 16:02:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117642,"visible":true,"origin":"","legend":"\u003cp\u003eRat bromocriptine model abortion and the effects of WJ-MSC. (A) Rat uteri at day 14 of gestation in a) normal pregnancy, b) ethanol vehicle control, c) bromocriptine-induced abortion, d) bromocriptine-induced abortion + WJ-MSC, e) bromocriptine-induced abortion + JAK/STAT inhibited WJ-MSC, f) bromocriptine-induced abortion + NF-ĸB inhibited WJ-MSC. Aborted absorbing embryos are shown with black arrows. (B) Quantification of the rate of abortion (fraction out of 1) for groups studied. (n=8, ***p\u0026lt;0.001.)\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/cdd3e44f4123fbf3eb32554f.jpg"},{"id":26773676,"identity":"f59a027e-6aa6-4872-977f-ee531e528417","added_by":"auto","created_at":"2022-09-21 16:02:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148382,"visible":true,"origin":"","legend":"\u003cp\u003eSerum cytokines during the abortion and after WJ – MSC treatment. ELISA was used to measure (A) the Th1 related cytokines, (B) Th2 related cytokines, and (C) the Th3 related cytokine TGF-β. JAK/STAT inhibition denotes JAK/STAT inhibitor-treated WJ-MSC. NF-ĸB inhibition denotes NF-ĸB inhibitor-treated WJ-MSC. （n=8, **p\u0026lt;0.05, ***p\u0026lt;0.001.）\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/682e38d2ff1ed68b9313dd08.jpg"},{"id":26773674,"identity":"7e5ed9df-cd57-4f83-ba6e-5c2860015780","added_by":"auto","created_at":"2022-09-21 16:02:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133606,"visible":true,"origin":"","legend":"\u003cp\u003eCytokines in decidual and placental tissues by Western Blot. Western blotting was used to detect (A, a1) Th1 related cytokines and (B, b1) Th2 related cytokines. GAPDH served as a loading control. (A, a2; B, b2 ) Cytokines were quantified relative to GAPDH. JAK/STAT inhibition denotes JAK/STAT inhibitor-treated WJ-MSC.（n=8, *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.001.）\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/ce699d49077e82642cf77f51.jpg"},{"id":26774254,"identity":"344ebfa0-4c00-4828-8a12-3b52d9ae4620","added_by":"auto","created_at":"2022-09-21 16:07:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138570,"visible":true,"origin":"","legend":"\u003cp\u003eCytokines in decidual and placental tissues by quantitative RT-PCR. Quantitative RT-PCR was used to measure mRNA of (A) Th1 related cytokines, (B) Th2 related cytokines, and (C) Th3 related cytokine TGF-β. GAPDH was used as a housekeeping control gene. The 2–∆∆Ct method was used to calculate relative mRNA levels. JAK/STAT inhibition denotes JAK/STAT inhibitor-treated WJ-MSC. (n=8, **p\u0026lt;0.05, ***p\u0026lt;0.001.）\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/21d4b6d8dde9b2f21c9f3ff2.jpg"},{"id":28660783,"identity":"6767190a-a1b4-4871-8b96-056d3958785d","added_by":"auto","created_at":"2022-11-04 11:00:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":886424,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/1494bbfb-268b-425a-8a8b-641d937875d1.pdf"},{"id":26773678,"identity":"89cc153c-7738-411c-b89b-151da0cd0d6b","added_by":"auto","created_at":"2022-09-21 16:02:15","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8318094,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1. The Characterization of WJ-MSCs and the differentiation potential of WJ-MSCs toward the osteogenic and adipogenic lineages. (A) The surface antigens of WJ-MSCs in flow cytometry. WJ-MSCs were positive for CD105, CD29, CD73, CD166 and CD44, and were negative for the markers CD34, CD11b, CD14, CD45, and HLA-DR. (B) Osteogenic and adipogenic differentiation was assayed by the OriCell® procedure. Adipogenic differentiation was detected by oil red O staining and evidenced by the formation of lipid vacuoles after adipogenic induction. Osteogenic differentiation was evidenced by staining alizarin red in WJ-MSC after osteogenic induction.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/6957b9dd72cb564dde5b2a4a.tif"},{"id":26773677,"identity":"628449d3-7a94-4afe-9a0f-34503bfeb768","added_by":"auto","created_at":"2022-09-21 16:02:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11121,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1698573/v2/c091d245060d2df9c50d9fb2.xlsx"}],"financialInterests":"","formattedTitle":"Human Wharton’s jelly-derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT-mediated immunomodulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpontaneous abortion (SA) is a common clinical disease that seriously affects the physical and mental health of women of childbearing age. Many risk factors, including genetic factors, endocrine disorders, infectious agents, immune dysfunction and genital anatomical abnormalities, contribute to this complex pathology[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. So far, \u0026ge; 50% of the causes are not clear. The immune imbalance has been reported to be an important factor associated with SA and infertility[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During pregnancy, the maternal immune system must be modified to accept the semi-allogeneic fetus. The immune relationship between embryonic and maternal tissue is extremely complex involving human leukocyte antigens, the complement pathway, cytokines, blood group antigens, trophoblast cell membrane antigen, and intercellular adhesion molecules[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As a result, the role of T helper cells (Th) and the balance of secreted cytokines in SA is an active area of research.\u003c/p\u003e \u003cp\u003eHelper T cells are divided into Th1, Th2, Th3 and Th17 subtypes based on their cytokine production. Th1 cells secrete Interleukin-2 (IL-2), Interferon-γ (IFN-γ), Tumor necrosis factor-α (TNF-α), Tumor necrosis factor-β (TNF-β) and play a role in cellular immune responses. Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13, and are involved in humoral immunity. Th3 cells produce transforming growth factor-β (TGF-β) which inhibits the expression of Th1 cytokines. A normal pregnancy exhibits a shift towards Th2 cytokine production and inhibition of Th1 responses and disturbance of this balance may result in unexplained recurrent spontaneous abortion (URSA)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Additionally, Th17 cells produce IL-17 which can cause an inflammatory response at the maternal interface that jeopardizes pregnancy[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The importance of T cells subtypes and the cytokine environment at the maternal-fetal interface suggests that immunotherapy may be a viable method of treating URSA in the clinic[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent treatments for URSA include active immunotherapy, but their effectiveness is limited[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. With progress in the understanding of reproductive immunology, the immune mechanism of SA and its immunological treatment has become an important topic of research[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been reported that the expression of related factors from Th1 / Th2 / Th3 / Th17 (IL-2, IL-3, IL-5, IL-6, IL-10, IL-17) mainly plays a biological function through JAK / STAT pathway, and these interleukins can activate JAK / STAT signaling pathway under specific conditions[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. There are other pathways involved in interleukin mediating immune response, such as inhibiting the ability of nuclear factor-κB (NF-κB) to bind to DNA or inhibiting the activity of IκB kinase reversely to inhibit NF-κB activation.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells(MSCs)are pluripotent stem cells, which have been studied in tissue engineering, hematopoietic stem cell transplantation and gene therapy. MSCs have been widely studied in the treatment of immune diseases because of their role in immune regulation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It has been shown that Wharton's jelly (WJ) in the human umbilical cord is an important source of MSCs which secrets a large number of cytokines and possesses no tumorigenicity, lower infection rate of pathogenic microorganisms, fewer ethical problems, multidirectional differentiation potential and lower immunogenicity than bone marrow[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has been shown [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] that the expression of IFN-γ secreted by Th1 decreased significantly, while the expression of IL-4, IL-13 and other factors secreted by Th2 increased after the co-culture of WJ-MSCs and immune cells. It has been also found that WJ-MSCs affect the differentiation and function of Th17 cells in the peripheral lymphocyte system and promote the proliferation and differentiation of Tregs cells to maintain the balance between Th17 cells and Tregs cells and improve the immune microenvironment of the body. These results above suggest that WJ-MSCs are important for local immune tolerance.\u003c/p\u003e \u003cp\u003eBromocriptine is a prolactin inhibitor to trigger abortion by disrupting the endocrine environment[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], such as low progesterone (P), low prolactin (PRL), and abnormal Th1 immune response. Human WJ-MSCs have previously been shown to prevent SA in this rat model by decreasing the expression of IFN-γ and IL-17 and up-regulating the expression of IL-10[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], but the detailed mechanism of the immunomodulatory effect remains elusive. Here we utilized bromocriptine-induced abortion in rats to investigate the therapeutic effects of WJ-MSCs on Th1/Th2/Th3 cytokines and to examine the roles of JAK/STAT and NF-κB signaling in immunomodulation of WJ-MSCs.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation, culture and characterization of WJ-MSCs\u003c/h2\u003e \u003cp\u003eHuman umbilical cords were obtained from normal full-term deliveries with the informed consent of the mother. The preparation of WJ-MSCs was approved by the Research Ethics Committee at the Affiliated Hospital of Nantong University. WJ-MSCs were isolated, cultured and characterized using the methods described in a previous study[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWJ-MSCs treated with JAK/STAT or NF-κB inhibitors\u003c/h2\u003e \u003cp\u003eP3 WJ-MSCs were cultured with DMEM/F12 medium (US, Hyclone) containing 10% fetal bovine serum (FBS; US, Hyclone) with/without the inhibitors of JAK/STAT (Germany, MERCK, AG490) or NF-κB (Solarbio, BAY-11-7085). The medium was removed 5 hours later, WJ-MSCs were washed twice with PBS and then cultured with DMEM/F12 medium containing 10% FBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability test\u003c/h2\u003e \u003cp\u003eThe viability of WJ-MSCs treated with JAK/STAT inhibitor or NF-κB inhibitor was measured by a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan). All experimental procedures were performed according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRat model of spontaneous abortion\u003c/h2\u003e \u003cp\u003e The protocol was approved by the Care and Use Committee of the Laboratory Animal Research Center of Nantong University. Age-matched virgin female (200\u0026ndash;250 g; n\u0026thinsp;=\u0026thinsp;48) and male (200\u0026ndash;250g; n\u0026thinsp;=\u0026thinsp;48) Sprague\u0026ndash;Dawley (SD) rats were obtained from the Laboratory Animal Center of Nantong University. Day 1 of pregnancy was defined by the presence of a vaginal plug. Pregnant rats were randomly divided into 6 groups. Group A served as a normal pregnancy group and received a vehicle injection of 0.2ml of PBS by subcutaneous injection on gestational days 6\u0026ndash;8 and 1ml of DMEM/F12 by tail vein injection on day 9 of gestation. Group B received a subcutaneous injection with 0.8 ml/kg of 75% ethanol on gestational days 6\u0026ndash;8 and 1ml of DMEM/F12 via tail vein injection on day 9 of gestation. Group C served as the abortion model and received subcutaneous injections of 0.8 ml/kg of bromocriptine (0.5mg/ml) in 75% ethanol on gestational days 6\u0026ndash;8 and 1ml of DMEM/F12 by tail vein injection on day 9 of gestation. Group D was similar to group C but with an additional injection of 107 WJ-MSCs in 1ml of DMEM/F12. Group D received JAK/STATor NF-κB signal pathway inhibitors in 1ml of DMEM/F12 served as Group E and F respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTissues and serum collection\u003c/h2\u003e \u003cp\u003ePregnant rats in each group were euthanized on day 14 of gestation and embryo resorption was assessed. The fetal/placental unit and decidua were removed from the uterus and rinsed with saline. A portion of the fresh tissues was stored at -80℃ for real-time polymerase chain reaction (RT-PCR) or Western blotting(WB). Whole blood was collected from the orbit and serum was obtained by centrifuging blood in serum separation tubes. Serum was stored at -80\u0026deg;C before being subjected to enzyme-linked immunosorbent assays (ELISA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytokines in serum\u003c/h2\u003e \u003cp\u003eSerum concentrations of cytokines (IL-2, IL-4, IL-5, IL-13, TNF-α, TNF-β, TGF-β) were measured by ELISA kit (eBioScience) following the manufacturer\u0026rsquo;s instructions. All samples were measured in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCytokine in decidual/placental tissues\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from decidual and placental tissues as previously described\u003csup\u003e16\u003c/sup\u003e. Protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat milk powder and incubated with the following primary antibodies: anti-IL-2 (Abcam, ab25104), anti-IL-4 (Abcam, ab84278), anti-IL-5(Novus Biologicals, AF545), anti-IL-13(Abcam, ab271227), anti-TNF-α (Abcam, ab9755), anti-TNF-β (Abcam, ab106353) at 4\u0026deg;C overnight, washed and incubated with HRP-labeled secondary antibody (Abcam, ab6721) for 2h at room temperature. After three times washing, signals were detected by ECL (Millipore) according to the manufacturer\u0026rsquo;s instructions. Cytokine expressions were quantified using densitometry normalized by GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRT-PCR Analysis of Th-related cytokines\u003c/h2\u003e \u003cp\u003eTotal RNAs were isolated from tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in RNA-free centrifuge tubes. According to the instructions of the cDNA synthesis Kit (Roche, Switzerland), 1\u0026micro;g total RNA was reverse transcribed in 5 \u0026times; Reaction Buffer, 10 mmol/L dNTPs, 1\u0026micro;mol/L oligo-dT primer, 20 U RNase inhibitor, 200 U/\u0026micro;l RT enzyme, and an appropriate amount of DEPC-treated water. Quantitative RT-PCR (qRT-PCR) was performed in a total volume of 20 \u0026micro;l reaction containing 2 \u0026micro;L cDNA, 10 \u0026micro;l SYBR Green (Roche, Switzerland), indicated primer pairs and 6 \u0026micro;l ddH2O. Primer pairs used are shown in Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical methods\u003c/h2\u003e \u003cp\u003eResults were analyzed with SPSS 22.0 data analysis software. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003eIsolation, culture and characterization of WJ-MSCs\u003c/h2\u003e\n \u003cp\u003eThe recent development of stem cell technology has revolutionized the treatment of various diseases and laid the foundation for immunomodulation[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. To investigate WJ-MSCs\u0026rsquo; function, we first cultured MSCs obtained from human umbilical cords[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our results showed that a small number of WJ-MSCs appeared around the implanted blocks after seeding for 6\u0026ndash;7 days (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). After 9\u0026ndash;10 days of culture, cells were gathered in pieces (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). After 12\u0026ndash;14 days of culture, cells proliferated rapidly to formed a single layer and exhibited a uniform spindle shape reminiscent of fibroblasts (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). JAK/STAT or NF-\u0026kappa;B inhibition did not cause a discernible difference in cell morphology and growth (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD-G).\u003c/p\u003e\n \u003cp\u003eFlow cytometry of passage 3 cells showed that WJ-derived cells expressed the MSC markers CD105, CD29, CD73, CD166 and CD44, and were negative for the markers CD34, CD11b, CD14, CD45, and HLA-DR (Supplementary Fig.\u0026nbsp;1A), which was consistent with human mesenchymal stem cells[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Subsequently, the differentiation potentials of MSCs into osteoblasts and adipocytes were also confirmed (Supplementary Fig.\u0026nbsp;1B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eThe effects of WJ-MSCs transfusion in the rat spontaneous abortion model\u003c/h2\u003e\n \u003cp\u003eIt was unclear how WJ-MSCs inhibited immunity in immune regulation. To determine whether WJ-MSCs would improve abortion and the potential mechanism, MSCs prepared from a human umbilical cord pretreated with or without JAK / STAT or NF- ĸ B inhibitors were injected into a pregnant rat in the presence of bromocriptine. The results showed that absorbing embryos were rarely detected in normal or ethanol pregnancies (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, a \u0026amp; b), but were frequently detected in the bromocriptine-induced abortion model (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, c). Treatment with WJ-MSC significantly reduced the visible occurrence of embryo absorption (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, d). While JAK/STAT inhibitor but not NF-\u0026kappa;B inhibitor primed WJ-MSC obviously compromised the effect of abortion prevention of WJ-MSC (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, e and f). Quantification revealed that embryo absorption was significantly increased in the bromocriptine-treated group compared to the normal pregnancy or ethanol injection group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). WJ-MSC treatment significantly reduced the abortion rate in bromocriptine-treated pregnancy (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). JAK/STAT inhibition abolished pregnancy-preserving effects of WJ-MSC but NF-\u0026kappa;B inhibition did not (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n \u003ch2\u003eCytokine profiles during the abortion and the effects of WJ \u0026ndash;MSC transfusion Serum cytokines by ELISA\u003c/h2\u003e\n \u003cp\u003eTo further evaluate the local environmental immune profile changed by WJ-MSCs, the cytokines secreted from Th1, Th2 and Th3 cells were investigated in different groups. ELISA analysis showed that the levels of Th1-related cytokines IL-2, TNF-\u0026alpha; and TNF-\u0026beta; in the bromocriptine-induced abortion group were significantly higher than in the normal pregnancy group or ethanol injection group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), while the levels of the Th2-related cytokines IL-4, IL-5 and IL-13 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), and Th3 cell-cytokine TGF-\u0026beta; were significantly decreased (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results suggested that WJ-MSCs transfused into the abortion rat restored cytokine profiles to comparable levels of normal pregnancy group or ethanol injection group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). JAK/STAT pretreated WJ-MSC prevented their rescues effect on cytokine profiles, but not NF-\u0026kappa;B inhibition (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eCytokines in decidual and placental tissues by Western Blot\u003c/h2\u003e\n \u003cp\u003eThe results above showed that the mechanism of WJ-MSCs regulating abortion primarily depended on JAK/STAT signaling pathway. As the maternal-fetus interface was composed of both decidua and placenta, the expression of cytokines was detected in both parts. Western blot results showed that Th2 cytokines (IL-4, IL-5, IL-13) were significantly reduced in the bromocriptine-induced abortion group compared to the normal pregnancy group or ethanol group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), while the Th1 cytokines TNF-\u0026alpha; and TNF-\u0026beta; significantly increased except IL-2 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). WJ-MSC injection significantly reversed these abortion-related effects on cytokine (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). JAK/STAT inhibition of WJ-MSC abolished their ability to reverse Th2-related cytokine changes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003eCytokine mRNA in decidual and placental tissues\u003c/h2\u003e\n \u003cp\u003eTo further validate our results, we extracted the mRNA from decidual and placental tissues. Our results suggested that Th2 (IL-4, IL-5, IL-13) and Th3 (TGF-\u0026beta;) cytokine mRNA levels were significantly reduced in the bromocriptine-induced abortion group compared to the normal pregnancy or ethanol group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, C), while the Th1 ( IL-2, TNF-\u0026alpha;, TNF-\u0026beta;) cytokine mRNA levels were significantly increased (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). WJ-MSC treatment of the abortion group significantly reversed these abortion-related effects on cytokine mRNA. JAK/STAT inhibition of WJ-MSC prevented them from reversing abortion-related cytokine mRNA changes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe bromocriptine-induced abortion model examined here involved a shift from a Th2-dominated immune response that supports pregnancy to a detrimental Th1-dominated response. Human WJ-MSCs had immunomodulatory properties that prevented this shift and rescued pregnancy. There was a decrease of IL-2 in the WJ-MSCs group by ELISA and PCR in different groups but its expression detected by the Western Blot experiment showed less discrepancy which might be due to the secretory nature of IL-2. Human WJ-MSCs decreased the Th1 (IL-2, TNF-α and TNF-β) cytokines and increased the Th2 (IL-4, IL-5 and IL-13) cytokines in serum and decidual/placental tissue. Additionally, there was also evidence that WJ-MSCs promoted the expression of TGF-β, an anti-inflammatory cytokine produced by Th3 and regulatory T cells. We demonstrated that the immunomodulatory and pregnancy-preserving effects of WJ-MSCs were dependent on JAK/STAT signaling in WJ-MSCs, but not NF-κB signaling.\u003c/p\u003e \u003cp\u003eThe T cell cytokines profile presented here expanded our knowledge of the effects of WJ-MSCs in the rat bromocriptine-induced abortion group which only assessed the expression of IFN-γ, IL-10 and IL-17 in our previous study (REF). The observation that the changes of Th1/Th2 balance during the abortion and restoration by WJ-MSCs injection were detected in both peripheral blood and decidual/placental tissue which suggested that both systemic and local immune responses at the maternal/fetal interface contributed to this hemostasis maintenance and pregnancy success. Our results are consistent with reports that human endometrial MSCs prevented abortion in a mouse incompatible pregnancy abortion model[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and that mouse bone marrow-derived MSCs prevented abortion in LPS-mediated and incompatible mating mouse models[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our observation that WJ-MSCs tipped T cell cytokine responses from Th1 to Th2 was similar to the findings from the therapeutic application of MSCs to other models including mouse diabetes[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], rat corneal transplant[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], rat autoimmune uveoretinitis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], rat renal injury[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and mouse colitis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results suggested that JAK/STAT signaling, an important mediator of responses to injury and inflammation, was required for WJ-MSCs to modify T cell responses. JAK/STAT signaling in human bone marrow MSCs had been implicated in the production of indoleamine 2,3-dioxygenase, a suppressor of T cell proliferation[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Other studies of human bone marrow MSCs indicated the requirement of JAK/STAT signaling for the release of modulators of innate immunity[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and homing of MSCs to sites of injury/inflammation[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Taken together, these findings indicate the broad importance of JAK/STAT signaling in the therapeutic properties of MSC. Manipulating JAK/STAT signaling may allow the therapeutic effects of WJ-MSCs to be optimized.\u003c/p\u003e \u003cp\u003eMSCs from various tissues had immunomodulatory properties making them potentially useful for treating a range of diseases and injuries. WJ-MSCs were an attractive source for therapy because they can be obtained in large numbers from umbilical cords that were usually discarded. The utilization of MSCs to prevent human recurrent spontaneous abortion might be practicable due to the pregnancy-preserving immunomodulation of these allogeneic cells rather than long-term engraftment. Our results presented here further corroborated that the administration of xenogeneic human MSCs had the desired effect of modulating a rat immune response. WJ-MSCs had already been shown to be beneficial for graft versus host disease in human patients undergoing hematopoietic stem cell transplantation, and clinical trials were underway for a range of other conditions[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Collectively, our study opened a new avenue to combat recurrent abortion and provide a promising strategy for clinical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declared no potential conflicts of interest concerning\u0026nbsp;the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNatural Science for Youth Foundation of China (Grant No.81401272)\u003c/p\u003e\n\u003cp\u003eResearch project of Jiangsu maternal and child health care association in 2021 (Grant No.FYX202103)\u003c/p\u003e\n\u003cp\u003eSix Talent Peaks Project of\u0026nbsp;Jiangsu province\u0026nbsp;2019 (WWS-067)\u003c/p\u003e\n\u003cp\u003eGraduate Research innovation Project of Jiangsu Province（KYCX21-3122）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQuenby S, Gallos ID, Dhillon-Smith RK et al (2021) Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss. 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DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15283/ijsc18034\u003c/span\u003e\u003cspan address=\"10.15283/ijsc18034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"JAK/STAT, spontaneous abortion, Th1, Th2, Th3, Wharton’s jelly- mesenchymal stem cells","lastPublishedDoi":"10.21203/rs.3.rs-1698573/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1698573/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Spontaneous abortion (SA) is a syndrome with many origins. Among them, immune imbalance is a major risk factor for SA. Wharton's jelly-mesenchymal stem cells (WJ-MSCs) are considered to be able to prevent abortion. However, the underlining molecular signaling pathways and regulatory mechanisms of WJ-MSCs in pregnancy maintenance are poorly understood. Here abortion mode is established by subcutaneous injection of bromocriptine in rat on day 9 and abortion prevention is achieved by WJ-MSCs injection via tail vein with or without JAK/STAT inhibitor. Here, we show that WJ-MSCs significantly lower the rate of embryo resorption of spontaneous abortion by reducing Th1-related cytokines while increasing Th2 and Th3-related cytokines in JAK/STAT-dependent manner. Together, our observation of WJ-MSCs in spontaneous abortion prevention will shed new light on potential therapeutic strategy development.","manuscriptTitle":"Human Wharton’s jelly-derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT-mediated immunomodulation","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-09-21 16:02:13","doi":"10.21203/rs.3.rs-1698573/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2022-06-01 18:53:41","doi":"10.21203/rs.3.rs-1698573/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6d34668f-d62c-4ac6-8622-bc3e6c43b905","owner":[],"postedDate":"September 21st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-04T11:00:17+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-21 16:02:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1698573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1698573","identity":"rs-1698573","version":["v2"]},"buildId":"re_ckhLnmML6MCF96OHNJ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}