Fibrin Facilitates Mesenchymal Stem Cells to Ameliorate Rats with Polycystic Ovary Syndrome

applied sciences Article Fibrin Facilitates Mesenchymal Stem Cells to Ameliorate Rats with Polycystic Ovary Syndrome 1,2,3, 1,2,3, 4,5, 1,2,3 2,6, Yuanyuan Li y , Jia Guo y, Shoulong Deng y, Zili Gao , Yixun Liu * and Qi Gu 1,2,3,* 1 State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (Y.L.); [email protected] (J.G.); [email protected] (Z.G.) 2 Institute of Zoology, University of Chinese Academy of Sciences, Beijing 100049, China 3 Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China 4 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; [email protected] 5 Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing 100021, China 6 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China * Correspondence: [email protected] (Y.L.); [email protected] (Q.G.) These authors contributed equally to this work. y Received: 20 April 2020; Accepted: 14 May 2020; Published: 22 May 2020 Abstract: Polycystic ovarian syndrome (PCOS) is a ubiquitous hormonal disorder and induces female infertility and heterogeneous syndromes, for which there is still no effective treatment. Thanks to the properties of immunomodulatory and endocrine regulation, mesenchymal stem cells (MSCs) have been widely used in various disease types. There were few reports for MSCs injected to ovaries due to the size limitation and complicated vascular network. Here, we develop one simple and efficient approach to deliver and stabilize MSCs in the outside of the ovary without blood leaking through the fibrin gelation, which also possesses excellent biocompatibility to support MSC survival. Notably, the transplantation of MSCs, encapsulated in fibrin hydrogel, could rescue ovarian function more efficiently compared to only MSC control in terms of elevated estradiol (E2) and progesterone (P) levels, diminished gonadotropins (LH/FSH), testosterone (T), and transforming growth factor-β1 (TGF-β1) levels, regular estrous cycles, enhanced number of granulosa cells, and reduced number of immature cystic follicles. The size and weight of the ovary increased for MSCs both within and without fibrin in PCOS rat models in two weeks. Moreover, we have shown the versatility of fibrin hydrogel as a cell-compatible platform for advanced stem cell translation, including identifying novel mechanisms of cell survival support, tissue development, and regenerative medicine. Keywords: PCOS; MSCs; fibrin; ovarian structure; TGF-β1 1. Introduction Polycystic ovarian syndrome (PCOS) is a complicated endocrine disease occurring in 6–7% of reproductive-age women [1], which affects patients’ health by increasing the risk of cardiac disease, obesity, insulin resistance, and infertility [2]. The pathogenesis of PCOS has not been well elucidated, although it has been reported to be a multifactorial disease that involves genetic and environmental factors [1,3,4]. Excessive gonadotropin (LH/FSH) and androgen (T) levels affect ovarian function along with increasing transforming growth factor-β1 (TGF-β1) levels and enhancing apoptosis of granulosa cells, in which case, many immature cystic follicles form in the ovaries of PCOS patients [5–7]. Currently, different kinds of therapies are used for the treatment of PCOS such as weight management, medicines targeting insulin resistance, or hormonal drugs [8]. Especially, inositol therapy was demonstrated Appl. Sci. 2020, 10, 3598; doi:10.3390/app10103598 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 3598 2 of 14 effective and beneficial to improve ovulation as a supplementation of clomiphene citrate [9,10]; it was reported there was no sufficient evidence to demonstrate the availability for the administration of myo-inositol in PCOS patients experiencing ovulation induction [11]. Moreover, hydrolaparoscopic ovarian drilling is a unique treatment for anovulatory women with PCOS, but this surgery causes tissue adhesion and other complications [12]. Therefore, due to such limitations, there is a need to identify an ideal treatment for PCOS. Human mesenchymal stem cells (hMSCs) have become a research focus of clinical therapeutics owing to their properties of stemness, anti-inflammatory activity, the secretion of active factors, and differentiation into various types of somatic cells. MSCs can secrete numerous active factors such as vascular endothelial growth factor (VEGF), IL-10, and TNF-α [13–15] that are anti-inflammatory, angiogenic, and antifibrotic [15–17], which therefore can be helpful to impaired lungs, liver, kidney, endometrium, and ovary [18,19]. Moreover, MSCs can secrete estrogen, which, in consequence, may be potentially used as an effective treatment for patients with estrogen deficiency [20]. A previous study has demonstrated that MSCs administered to mice via tail vein injection had inhibited inflammation in PCOS [21]. Furthermore, MSCs improve the fertility of women with premature ovarian failure through intraovarian injection, which can minimize MSC diffusion to other tissues [22,23]. In particular, umbilical cord MSCs (UC-MSCs) entrapped in fibrin gels have been used in in vitro/in vivo construction of bone [24,25], cartilage [26], and the microvascular network [27]. Fibrin has been widely used as a desirable scaffold and carrier for MSCs, which can help MSCs reach the specific organ or tissue and subsequently decrease/stop bleeding after cell transplantation [28,29]. It has been used to construct artificial ovaries to preserve the fertility of a patient. In adults, fibrin clots can recruit hMSCs; however, the strong fibrinolytic activity of hMSCs plays a similar role as fibrin in matrix remodeling and wound healing [30]. This study aims to investigate whether intraovarian injection of UC-MSCs alone or within fibrin hydrogels can restore ovarian function in a PCOS rat model, to elucidate its underlying mechanism. This study also identifies therapeutic targets for assisted reproduction technology. 2. Result 2.1. Fibrin Hydrogel Enhances UC-MSCs Growth The minimal phenotypic criteria for the cultured MSCs include positive expression of CD90, CD29, and CD105, and negative expression of HLA-DR, CD34, and CD45 [31]. Our results indicated that the UC-MSCs cultured in the study expressed CD90, CD29, and CD105, but not HLA-DR, CD34, or CD45 surface markers (Figure1A). These results were confirmed by immunofluorescence histochemistry (Figure1B). Moreover, MSCs have the ability to undergo extensive di fferentiation into multiple cell lineages such as cartilaginous and adipose cells (Figure1C,D). Appl. Sci. 2020, 10, 3598 3 of 14 Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 13 FigureFigure 1. 1. CharacterizationCharacterization of of umbilical umbilical cord cord mesenchymal mesenchymal stem stem cells cells ( (UC-MSCs).UC-MSCs). ( (AA)) Flow Flow cytometry cytometry showingshowing the percentage ofof cellscells expressing expressing HLA-DR, HLA-DR, CD-34, CD-34, CD-45, CD-45, CD90, CD90, CD29, CD29, and and CD105. CD105. The The cells cellsalso expressedalso expressed CD90, CD90, CD29, CD29, and CD105, and CD105, but not but HLA-DR, not HL CD34,A-DR, or CD34, CD45. or The CD45. red curve The red represents curve representsthe blank, andthe blank, the blue and curve the indicatesblue curve positive indicates expression. positive (expression.B) UC-MSCs (B on) UC glass-MSCs slides on were glass detected slides wereby fluorescence detected microscopyby fluorescence after hybridizationmicroscopy withafter anti-CDhybridization antibodies with (red: anti phycoreythrin-CD antibodies (PE) (red: label, phycoreythringreen: fluorescein (PE) isothiocyanate label, green: (FITC)fluorescein label). isothiocyanate (C) Cartilage cells(FITC) that label). differentiated (C) Cartilage from UC-MSCscells that differentiatedwere stained darkfrom blue.UC-MSCs (D) Adipocyte were stained cells dark that blue. differentiated (D) Adipocyte from UC-MSCscells that differentiated were shown asfrom red UCoil- droplets.MSCs were shown as red oil droplets. TheThe cross-linkedcross-linked fibrin fibrin formed formed the three-dimensional the three-dimensio meshnal for MSCmesh encapsulation for MSC encapsulation (Supplemental (SupplementalFigure S1). The Figure UC-MSCs S1). exhibitedThe UC-MSCs unfolding exhibited in a three-dimensional unfolding in a three sca-dimensionalffold of fibrin scaffold and were of tightlyfibrin andlinked were with tightly fibrin linked fibers with (Figure fibrin2A, fib B right),ers (Fig buture UC-MSCs 2A, B right), on thebut surfaceUC-MSCs of fibrin on the were surface similar of fibrin with werethat onsimilar the culture with that dish, on which the culture was tightly dish, which arranged was and tightly had arranged no more roomand had to stretchno more out room further to stretch(Figure out2B left).further The (Figure proliferation 2B left). e ffiTheciency proliferation of UC-MSCs efficiency inside of the UC fibrin-MSCs gel inside was higher the fibrin than gel that was on higherthe surface than ofthat the on fibrin the surface gel (Figure of the2C). fibrin gel (Figure 2C). Appl. Sci. 2020, 10, 3598 4 of 14 Appl. Sci. 2020, 10, x FOR PEER REVIEW 4 of 13 Figure 2. The status of growth and proliferation of UC-MSCsUC-MSCs cultured with fibrinfibrin gel.gel. ( A) Frozen scanning electron micrographs. Right, magnified magnified form of the cell indicated by black dotted bordered rectrectangle;angle;

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