In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin.

Spermatogenesis refers to the differentiation of the spermatogonial stem cells (SSCs) located in the base seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix (ECM), paracrine factors, and testicular somatic cells which play a supporting role for SSCs. Thus, the present study evaluated the potential of co-culturing Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin, the main components of the ECM. Following the three-week conventional culture of human testicular cells, the cells were cultured in agarose hydrogel or agarose/laminin one (hybrid) for 74 days. Then, immunocytochemistry, real-time PCR, electron microscopy, and morphological staining methods were applied to analyze the presence of SSCs, as well as the other cells of the different stages of spermatogenesis. Based on the results, the colonies with positive spermatogenesis markers were observed in both culture systems. The existence of the cells of all three phases of spermatogenesis (spermatogonia, meiosis, and spermiogenesis) was confirmed in the two groups, while morphological spermatozoa were detected only in the hybrid hydrogel group. Finally, a biologically improved 3D matrix can support all the physiological activities of SSCs such as survival, proliferation, and differentiation.

Application of Tissue-Specific Extracellular Matrix in Tissue Engineering: Focus on Male Fertility Preservation.

In vitro spermatogenesis and xenotransplantation of the immature testicular tissues (ITT) are the experimental approaches that have been developed for creating seminiferous tubules-like functional structures in vitro and keeping the integrity of the ITTs in vivo, respectively. These strategies are rapidly developing in response to the growing prevalence of infertility in adolescent boys undergoing cancer treatment, by the logic that there is no sperm cryopreservation option for them. Recently, with the advances made in the field of tissue engineering and biomaterials, these methods have achieved promising results for fertility preservation. Due to the importance of extracellular matrix for the formation of vascular bed around the grafted ITTs and also the creation of spatial arrangements between Sertoli cells and germ cells, today it is clear that the scaffold plays a very important role in the success of these methods. Decellularized extracellular matrix (dECM) as a biocompatible, functionally graded, and biodegradable scaffold with having tissue-specific components and growth factors can support reorganization and physiologic processes of originated cells. This review discusses the common protocols for the tissue decellularization, sterilization, and hydrogel formation of the decellularized and lyophilized tissues as well as in vitro and in vivo studies on the use of the testis-derived dECM for testicular organoids.

The effect of mesenchymal stem cells-derived exosomes on the prostate, bladder, and renal cancer cell lines.

We aimed to explain the role of mesenchymal stem cells (MSC-exosomes) on gene expressions of epithelial to mesenchymal transition (EMT), angiogenesis, and apoptosis. Four different cell lines were employed, including ACHN, 5637, LNCaP, and PC3, as well-known representatives for renal, bladder, hormone-sensitive, and hormone-refractory prostate cancers, respectively. Cell lines were exposed to diverse concentrations of mesenchymal stem cells-derived exosomes to find IC50 values. Percentages of apoptotic cells were evaluated by Annexin/P.I. staining. Micro Culture Tetrazolium Test assessed proliferative inhibitory effect; and prostate biomarker (KLK2), EMT (E-cadherin and Snail), angiogenesis genes (VEGF-A/VEGF-C), apoptosis genes (BAX/BCL2, P53) and Osteopontin variants (OPNa/b, and c) mRNA levels were studied by realtime PCR method. All 5637, LNCaP, and PC3 following treatment with exosomes illustrated specific responses with changes in expression of different genes. The increased TP53 and decreased BCL2 expressions were seen in 5637, LNCaP, and PC3. In PC3, OPNb and OPNc have raised more than P53; in LNCap, the increase was in VEGF-c. In 5637 cells, more than TP53 and BCL2 changes, two other genes, VEGFa and B.A.X., have decreased, suggesting exosomes' anti-apoptotic and anti-angiogenic effects. The kidney tumor cell line saw no significant gene expression change in ten targeted genes. MSC-exosomes therapy has augmented some interesting antitumor effects on prostate, bladder, and kidney cancer cell lines. This effect which originates from exosomes' potency to persuade apoptosis and prevent the proliferation of cancer cells simultaneously, was more substantial in bladder cancer, moderate in prostate cancer, and mild in renal cancer.

The importance of personalized medicine in urological cancers.

A better understanding of key regulatory pathways involved in cancers has led to the development of molecularly targeted therapies. Molecular profiling based on genomics, proteomics, and metabolomics in tumors provides clinicians with the necessary information to maintain a personalized therapeutic regimen according to the patient's needs. for example, androgen deprivation therapy (ADT) for advanced prostate cancer is one of the earliest forms of targeted therapy and has remained a choice of treatment by physicians. Unfortunately, most patients will eventually become non-responsive to ADT and succumb to the disease. Since the emergence of ADT, the understanding of androgen receptor (AR) signaling and mechanisms driving the resistance to ADT has been significantly improved. Inactivation of the PTEN gene is a common occurrence in prostate cancers and is associated with metastatic potential, androgen independence, and poor prognosis. Several studies over personalized medicine for muscle-invasive and metastatic bladder cancer discussed potential molecular biomarkers which are currently under investigation and based on the excision repair cross-complementing group 1 (ERCC1) gene and its role in tumor development and therapeutic resistance to cytotoxic DNA-damaging chemotherapy and ionizing radiation. In this review, we consider personalized medicine for four urological cancers.

Mixture of fibroblast, epithelial and endothelial cells conditioned media induce monocyte-derived dendritic cell maturation.

Fully matured DCs with large amount cytoplasm and copious dendritic projections were visible at the end of culturing period in the presence of MCM, TNF-α and poly (I:C), with or without FEECM. Thus, DCs generated with these maturation factors are nonadherent and have typical satellite morphology. Flow cytometric analysis using anti-CD14, -CD80, -CD86, -HLA-DR and -CD83 revealed that expression of CD14 is decreased in particular in FEECM treated DCs, on day 5 and expression of CD80, CD86 and HLA-DR was the higher when FEECM are added to maturation factor. Functionally, when DCs matured in the presence of FEECM elicited stronger MLR, reduced phagocytic activity. These results support the use of the FEECM with MCM, TNF-α and poly (I-C) as maturation factor in DC generation that could result in functionally mature monocyte-derived DCs in comparison to either alone.

The air-liquid interface culture of the mechanically isolated seminiferous tubules embedded in agarose or alginate improves in vitro spermatogenesis at the expense of attenuating their integrity.

Optimization of tissue culture systems able to complete male germ cell maturation to post-meiotic stages is considered as an important matter in reproductive biology. Considering that hypoxia is one of the factors limiting the efficiency of organ culture, the aim of this study was to use isolated seminiferous tubules (STs), having more surface and less thickness, in an organotypic culture system in order to improve oxygen diffusion and reduce hypoxia. The mechanically separated STs embedded in agarose or alginate and 1-3-mm3 testicular tissue fragments of 3 adult mice were separately placed on the flat surface of agarose gel that was half-soaked in the medium. Survival and differentiation of germ cells using PLZF and SCP3 markers, identity of Sertoli cell using GATA4, cell proliferation with the Ki67 marker, and ST integrity using a ST scoring were evaluated up to 36 d at different culture times, each corresponding to the duration of one spermatogenic cycle. We observed a significantly reduced ST integrity in STs embedded in agarose or alginate on day 9 (versus tissue fragments p ≤ 0.05). There was no difference in the number of PLZF-positive cells between groups, but the number of SCP3 (in all-time points) and GATA4-positive cells was significantly higher in the culture of embedded STs. Although embedding STs can be useful for the progress of in vitro spermatogenesis, it makes them sensitive to degeneration. Further improvements are required to modify the air-liquid interface method to maintain ST integrity.

Isolation, identification and differentiation of human spermatogonial cells on three-dimensional decellularized sheep testis.

Improvement of in vitro culture methods of Spermatogonial Stem Cells (SSCs) is known to be an effective procedure for further study of the process of spermatogenesis and can offer effective therapeutic modality for male infertility. Tissue decellularization by providing natural 3D and extracellular matrix (ECM) conditions for cell growth can be an alternative procedure to enhance in vitro culture conditions. In the present study, the testicular tissues were taken from brain death donors. After enzymatic digestion, the tissue cells were isolated and cultured for four weeks. Then the identity of the SSCs was confirmed using anti-GFRα1 and anti-PLZF antibodies via immunocytochemistry (ICC). The differentiation capacity of SSCs were evaluated by culture of them on a layer of decellularized testicular matrix (DTM) prepared from sheep testis, as well as under two-dimensional (2D) culture with differentiation medium. After four and six weeks of the initiation of differentiation culture, the pre-meiotic, meiotic and post- meiotic genes at the mRNA and protein levels was examined via qPCR and ICC methods, respectively. The results showed that pre-meiotic, meiotic and post-meiotic genes expressions were significantly higher in the cells cultured in DTM substrate (P ≤ 0.01).The present study indicated that, the natural structure of ECM prepare the suitable conditions for further study of the spermatogenesis process in the in vitro and contributes to the maintenance and treatment of male infertility.

Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin.

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

Human Menstrual Blood Stem Cell-Derived Granulosa Cells Participate in Ovarian Follicle Formation in a Rat Model of Premature Ovarian Failure In Vivo.

We recently reported the application of human menstrual blood stem cells' (HuMenSCs) transplantation as a treatment modality in a rat model of premature ovarian failure (POF). We continued to investigate further in this respect. Female rats were injected intraperitoneally with 36 mg/kg busulfan. HuMenSCs were obtained, grown, and analyzed for immunophenotypic features at passage three. The cells were labeled with CM-Dil and infused into the rats. There were four groups: normal, negative control, treatment, and Sham. One month after treatment, the ovaries were collected and weighed. Histological sections were prepared from the ovary and HuMenSCs were tracking. Subsequently, we examined the changes of expression of Bax and B cell lymphoma 2 (Bcl2) genes by real-time polymerase chain reaction assay. One month after HuMenSCs transplantation, these cells were located in the ovarian interstitium and granulosa cells (GCs). The number of TUNEL-positive cells significantly decreased in the treatment group. Also the expression level of Bax genes, unlike Bcl2 gene, significantly decreased compared with negative and sham groups. In our study, HuMenSCs were tracked in ovarian tissues within 2 months after transplantation, and they differentiated into GCs. Therefore, the use of these cells can be a practical and low-cost method for the treatment of POF patients.

Organ culture of seminiferous tubules using a modified soft agar culture system.

BACKGROUND: In-vitro spermatogenesis in mammalian species is considered an important topic in reproductive biology. New strategies for achieving a complete version of spermatogenesis ex vivo have been conducted using an organ culture method or culture of testicular cells in a three-dimensional soft agar culture system (SACS). The aim of this study was to develop a new method that supports spermatogenesis to the meiotic phase and morphologically mature spermatozoa through the culture of testicular cells and seminiferous tubules (STs) in a modified SACS, respectively. METHODS: First, enzymatically dissociated testicular cells and mechanically dissociated STs of neonatal mice were separately embedded in agarose and then placed on the flat surface of agarose gel half-soaked in the medium to continue culture with a gas-liquid interphase method. RESULTS: Following 40 days of culture, the meiotic (Scp3) and post-meiotic (Acr) gene expression in aggregates and STs was confirmed by real-time polymerase chain reaction. These results were complemented by immunohistochemistry. The presence of morphologically mature spermatozoa in the frozen sections of STs was demonstrated with hematoxylin and eosin staining. We observed Plzf- or Integrin α6-positive spermatogonia in both cultures after 40 days, indicating the potency of the culture system for both self-renewal and differentiation. CONCLUSIONS: This technique can be used as a valuable approach for performing research on spermatogenesis and translating it into the human clinical setting.

Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems.

Optimization of in vitro culture system for the expansion and the maturation of male germ cells to post meiotic stages is a valuable tool for studies exploring spermatogenesis regulation and the management of male infertility. Several studies have reported promising results of mouse spermatogonial stem cells culture in three-dimensional (3D) culture systems and a subsequent production of sperm. In the present study, we investigated the capacity of a three-dimensional soft agar culture system (SACS) supplemented with Knockout Serum Replacement (KSR) in colony formation and inducing human germ cells to reach post-meiotic stages. Testicular cells from testes of brain -dead donors were first cultured for three weeks in proliferation medium. The cells were subsequently cultured in the upper layer of the SACS (3D group) in a medium supplemented with KSR and hormones, and the results were compared with that of a two-dimensional (2D) culture system. We found that the number and diameter of colonies and the levels of expression of Scp3 and Integrin α6 in the 3D culture group were significantly higher than in the 2D group. Our findings indicate that SACS can reconstruct a microenvironment capable of regulating both proliferation and differentiation of cell colonies.

In vitro effects of melatonin on colonization of neonate mouse spermatogonial stem cells.

We have recently reported that antioxidant supplements enhance the efficacy of cryopreserved spermatogonial stem cells. Melatonin is considered a free radical scavenger which has direct and indirect antioxidant effects in in vitro and in vivo microenvironments. Due to the anti-apoptotic properties of melatonin, researchers have proposed that melatonin may improve the efficiency of spermatogonial stem cell (SSC) transplantation. However, the appropriate methodology which facilitates SSC proliferation remains to be determined. Identification of a proper propagation system is essential for the future application of SSCs in the field of infertility. The aim of the present study was to investigate the effects of melatonin on the colonization of SSCs. SSCs were isolated from the testes of three to six day old mice, and their purities were assessed by cytometry using Plzf antibody. Isolated testicular cells were cultured in the absence or presence of melatonin extract for two weeks. Suppression of differentiation and maintenance of spermatogonial stem cells was confirmed by alkaline phosphatase staining and immunocytochemistry using Plzf antibody. The number and diameter of the colonies of SSCs were assessed during the 7th and 14th days of culture, and the expression of Id4, Plzf, and C-kit were evaluated using real-time PCR at the end of the culture period. The survival rate of the cultured cells in the presence of melatonin was significantly higher than the control group. The number and diameter of colonies also increased in the cells treated with melatonin. The results of our study suggest that culture of SSCs with 100 µM melatonin supplementation can increase SSCs proliferation significantly.