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In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions.

ABSTRACT: BACKGROUND:Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. RESULTS:Testicular tissue samples from azoospermic patients were obtained and then these were freeze-thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P?

SUBMITTER: Mohaqiq M

PROVIDER: S-EPMC6438003 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions.

Mohaqiq Mahdi M Movahedin Mansoureh M Mazaheri Zohreh Z Amirjannati Naser N

Biological research 20190327 1

<h4>Background</h4>Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture.<h4>Results</h4>Testicular tiss ...[more]

PMID: 30917866

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Project description:ObjectiveApplications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells.Materials and methodsIn this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson's trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC.ResultsHaematoxylin-eosin (H and E) staining showed that the cells were successfully removed by SDS and Triton X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This conﬁrmed that our decellularisation protocol was efﬁcient. Masson's trichrome and alcian blue staining respectively showed that glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous tubule. Analyses of gene expression using real-time PCR indicated that expression of the Plzf gene was unchanged over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture, suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules.ConclusionSpermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.

Project description:BackgroundOvarian tissue cryopreservation and transplantation are novel therapeutic approaches for fertility preservation. However, follicle loss caused by ischemic and hypoxic damage is one of the issues after frozen-thawed ovarian tissue transplantation. Promoting angiogenesis in grafts is the key to restore cryopreserved ovarian function. Mesenchymal stem cells (MSCs) have been reported to facilitate angiogenesis in the cryopreserved ovarian tissue transplantation. However, the risk of embolization, immunogenic effect and tumorigenesis hinders the clinical application of MSCs to human organ transplantation. In this study, we established an in vitro ovarian culture system to restore frozen-thawed ovarian function before transplantation with the application of umbilical cord mesenchymal stem cells (UC-MSCs), and explored the effects of UC-MSCs on frozen-thawed ovaries in vitro ovarian culture system and the mechanisms of UC-MSCs on the angiogenesis of frozen-thawed ovaries.MethodsA simple in vitro three dimensional (3D) ovarian culture system using Matrigel was established to support to an ideal niche, and ovary was alone cultured in the 24-well plate as a control. We also evaluated the effects of UC-MSCs treatment on ovarian function with or without Matrigel support. All thawed ovaries were randomly divided into control group (Matrigel-/UC-MSCs-), Matrigel group (Matrigel+/UC-MSCs-), UC-MSCs group (Matrigel-/UC-MSCs+) and UC-MSCs + Matrigel group (Matrigel+/UC-MSCs+). HE staining was used to detect the histological structure of follicles and TUNEL staining was used to detect cell apoptosis. The number of microvessels was counted to evaluate neovascularization. The mRNA expression of VEGFA, IGF1 and ANGPT2 were detected by RT-PCR. Western blotting was used to measure the expression of GSK-3β, β-catenin and p-β-catenin.ResultsIn the absence of UC-MSCs, 3D culture system supported by Matrigel showed significantly improved follicular development and microvascular number. Additionally, UC-MSCs were also found to effectively improve follicular development and microvascular number regardless of the culture condition used. However, alleviated follicular apoptosis, increased mRNA expression of angiogenesis-related gene and activated Wnt/β-catenin pathway occurred only in the UC-MSCs + Matrigel group. Besides, with the application of IWP-2 in UC-MSCs + Matrigel group, Wnt//β-catenin pathway could be blocked by IWP-2 serving as one of Wnt/β-catenin pathway inhibitors.ConclusionsThis in vitro study showed the beneficial effects of UC-MSCs on thawed ovaries and explored a potential mechanism inducing angiogenesis. In particular, 3D ovarian culture system supported by Matrigel further improved UC-MSCs treatment. The in vitro culture system using Matrigel and UC-MSCs may provide a potential treatment strategy for improving the success rate of thawed ovaries transplantation.

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In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions.

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In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions.

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