Therapeutic potential of exosomes in spermatogenesis regulation and male infertility.

BACKGROUND: Spermatogenesis is a fundamental process crucial for male reproductive health and fertility. Exosomes, small membranous vesicles released by various cell types, have recently garnered attention for their role in intercellular communication. OBJECTIVE: This review aims to comprehensively explore the role of exosomes in regulating spermatogenesis, focusing on their involvement in testicular development and cell-to-cell communication. METHODS: A systematic examination of literature was conducted to gather relevant studies elucidating the biogenesis, composition, and functions of exosomes in the context of spermatogenesis. RESULTS: Exosomes play a pivotal role in orchestrating the complex signaling networks required for proper spermatogenesis. They facilitate the transfer of key regulatory molecules between different cell populations within the testes, including Sertoli cells, Leydig cells, and germ cells. CONCLUSION: The emerging understanding of exosome-mediated communication sheds light on novel mechanisms underlying spermatogenesis regulation. Further research in this area holds promise for insights into male reproductive health and potential therapeutic interventions.

Testicular niche repair after gonadotoxic treatments: Current knowledge and future directions.

The testicular niche, which includes the germ cells, somatic cells, and extracellular matrix, plays a crucial role in maintaining the proper functions of the testis. Gonadotoxic treatments, such as chemotherapy and radiation therapy, have significantly improved the survival rates of cancer patients but have also been shown to have adverse effects on the testicular microenvironment. Therefore, repairing the testicular niche after gonadotoxic treatments is essential to restore its function. In recent years, several approaches, such as stem cell transplantation, gene therapy, growth factor therapy, and pharmacological interventions have been proposed as potential therapeutic strategies to repair the testicular niche. This comprehensive review aims to provide an overview of the current understanding of testis damage and repair mechanisms. We will cover a range of topics, including the mechanism of gonadotoxic action, repair mechanisms, and treatment approaches. Overall, this review highlights the importance of repairing the testicular niche after gonadotoxic treatments and identifies potential avenues for future research to improve the outcomes for cancer survivors.

In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration.

Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.

Polycaprolactone/Testicular Extracellular Matrix/Graphene Oxide-Based Electrospun Tubular Scaffolds for Reproductive Medicine: Biomimetic Architecture of Seminiferous Tubules.

Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4',6-diamidino-2-phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive evaluations are performed to assess the properties of the synthetic scaffolds. These evaluations encompass Fourier-transform infrared spectroscopy, scanning electron microscopy imaging, scaffold degradation testing, mechanical behavior analysis, methylthiazolyldiphenyl-tetrazolium bromide assay, and in vivo biocompatibility assessment. The PCL/decellularized extracellular matrix with 0.5% GO formulation exhibits superior fiber morphology and enhanced mechanical properties, and outperforms other groups in terms of in vitro biocompatibility. Consequently, these scaffolds present a viable option for implementation in "in vitro spermatogenesis" procedures, holding promise for future sperm production from spermatogonial cells.

Ultrastructural study: in vitro and in vivo differentiation of mice spermatogonial stem cells.

Mouse testicular tissue is composed of seminiferous tubules and interstitial tissue. Mammalian spermatogenesis is divided into three stages: spermatocytogenesis (mitotic divisions) in which spermatogonial stem cells (SSCs) turn into spermatocytes, followed by two consecutive meiotic divisions in which spermatocytes form spermatids. Spermatids differentiate into spermatozoa during spermiogenesis. Various factors affect the process of spermatogenesis and the organization of cells in the testis. Any disorder in different stages of spermatogenesis will have negative effects on male fertility. The aim of the current study was to compare the in vitro and in vivo spermatogenesis processes before and after transplantation to azoospermic mice using ultrastructural techniques. In this study, mice were irradiated with single doses of 14 Gy 60Co radiation. SSCs isolated from neonatal mice were cultured in vitro for 1 week and were injected into the seminiferous tubule recipient's mice. Testicular cells of neonatal mice were cultured in the four groups on extracellular matrix-based 3D printing scaffolds. The transplanted testes (8 weeks after transplantation) and cultured testicular cells in vitro (after 3 weeks) were then processed for transmission electron microscopy studies. Our study's findings revealed that the morphology and ultrastructure of testicular cells after transplantation and in vitro culture are similar to those of in vivo spermatogenesis, indicating that spermatogenic cell nature is unaltered in vitro.

The Effect of Chitosan/Alginate/Graphene Oxide Nanocomposites on Proliferation of Mouse Spermatogonial Stem Cells.

Male survivors of childhood cancer have been known to be afflicted with azoospermia. To combat this, the isolation and purification of spermatogonial stem cells (SSCs) are crucial. Implementing scaffolds that emulate the extracellular matrix environment is vital for promoting the regeneration and proliferation of SSCs. This research aimed to evaluate the efficiency of nanocomposite scaffolds based on alginate, chitosan, and graphene oxide (GO) in facilitating SSCs proliferation. To analyze the cytotoxicity of the scaffolds, an MTT assay was conducted at 1, 3, and 7 days, and the sample containing 30 µg/mL of GO (ALGCS/GO30) exhibited the most favorable results, indicating its optimal performance. The identity of the cells was confirmed using flow cytometry with C-Kit and GFRα1 markers. The scaffolds were subjected to various analyses to characterize their properties. FTIR was employed to assess the chemical structure, XRD to examine crystallinity, and SEM to visualize the morphology of the scaffolds. To evaluate the proliferation of SSCs, qRT-PCR was used. The study's results demonstrated that the ALGCS/GO30 nanocomposite scaffold exhibited biocompatibility and facilitated the attachment and proliferation of SSCs. Notably, the scaffold displayed a significant increase in proliferation markers compared to the control group, indicating its ability to support SSC growth. The expression level of the PLZF protein was assessed using the Immunocytochemistry method. The observations confirmed the qRT-PCR results, which indicated that the nanocomposite scaffolds had higher levels of PLZF protein expression than scaffolds without GO. The biocompatible ALGCS/GO30 is a promising alternative for promoting SSC proliferation in in vitro applications.

In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility.

Men's reproductive health exclusively depends on the appropriate maturation of certain germ cells known as sperm. Certain illnesses, such as Klinefelter syndrome, cryptorchidism, and syndrome of androgen insensitivity or absence of testis maturation in men, resulting in the loss of germ cells and the removal of essential genes on the Y chromosome, can cause non-obstructive azoospermia. According to laboratory research, preserving, proliferating, differentiating, and transplanting spermatogonial stem cells or testicular tissue could be future methods for preserving the fertility of children with cancer and men with azoospermia. Therefore, new advances in stem cell research may lead to promising therapies for treating male infertility. The rate of progression and breakthrough in the area of in vitro spermatogenesis is lower than that of SSC transplantation, but newer methods are also being developed. In this regard, tissue and cell culture, supplements, and 3D scaffolds have opened new horizons in the differentiation of stem cells in vitro, which could improve the outcomes of male infertility. Various 3D methods have been developed to produce cellular aggregates and mimic the organization and function of the testis. The production of an artificial reproductive organ that supports SSCs differentiation will certainly be a main step in male infertility treatment.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

PURPOSE: Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. METHODS: The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). RESULTS: Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. CONCLUSION: Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

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.

Intra-Accumbal D1- But not D2-Like Dopamine Receptor Antagonism Reverses the Inhibitory Effects of Cannabidiol on Extinction and Reinstatement of Methamphetamine Seeking Behavior in Rats.

Introduction: Methamphetamine (METH) is an addictive psychostimulant that facilitates dopamine transmission to the nucleus accumbens (NAc), resulting in alterations in the mesocorticolimbic brain regions. Cannabidiol (CBD) is considered the second most abundant component of cannabis and is believed to decrease the METH effects. Reversing psychostimulant-induced abnormalities in the mesolimbic dopamine system is the main mechanism for this effect. Various other mechanisms have been proposed: increasing endocannabinoid system activity and modulating gamma-aminobutyric acid (GABA) and glutamate neurons in NAc. However, the exact CBD action mechanisms in reducing drug addiction and relapse vulnerability remain unclear. Methods and Results: The present study aimed to investigate the effects of intracerebroventricular (ICV) administrating 5, 10, and 50 µg/5 µL CBD solutions on the extinction period and reinstatement phase of a METH-induced conditioned place preference. This research also aimed to examine the NAc D1-like dopamine receptor (D1R) and D2-like dopamine receptor (D2R) roles in the effects of CBD on these phases, as mentioned earlier, using SCH23390 and sulpiride microinjections as an antagonist of D1R and D2R. The obtained results showed that microinjection of CBD (10 and 50 µg/5 µL, ICV) suppressed the METH-induced reinstatement and significantly decreased mean extinction latency in treated groups compared to both vehicles and/or untreated control groups. In addition, the results demonstrated that administrating intra-accumbal SCH23390 (1 and 4 µg/0.5 µL saline) reversed the inhibitory effects of CBD on extinction and reinstatement phases while different doses of sulpiride (0.25, 1, and 4 µg/0.5 µL; dimethyl sulfoxide 12%) could not alter the CBD effects. Conclusions: In summary, this study showed that CBD made shorter extinction latencies and suppressed the METH reinstatement, in part, by interacting with D1R but not D2R in the NAc.

Genetic and Epigenetic Evaluation of Human Spermatogonial Stem Cells Isolated by MACS in Different Two and Three-Dimensional Culture Systems.

Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of this
study is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy in
propagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS).
Materials and Methods: In this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin ß1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse.
Results: The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm.
Conclusion: Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection of
spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.

In vitro production of mouse morphological sperm in artificial testis bioengineered by 3D printing of extracellular matrix.

Since autologous stem cell transplantation is prone to cancer recurrence, in vitro sperm production is regarded a safer approach to fertility preservation. In this study, the spermatogenesis process on testicular tissue extracellular matrix (T-ECM)-derived printing structure was evaluated. Ram testicular tissue was decellularized using a hypertonic solution containing triton and the extracted ECM was used as a bio-ink to print an artificial testis. Following cell adhesion and viability examination, pre-meiotic and post-meiotic cells in the study groups (as testicular suspension and co-culture with Sertoli cells) were confirmed by real-time PCR, flow-cytometry and immunocytochemistry methods. Morphology of differentiated cells was evaluated using transmission electron microscopy (TEM), toluidine blue, Giemsa, and hematoxylin and eosin (H&E) staining. The functionality of Leydig and Sertoli cells was determined by their ability for hormone secretion. The decellularization of testicular tissue fragments was successful and had efficiently removed the cellular debris and preserved the ECM compounds. High cell viability, colonization, and increased expression of pre-meiotic markers in cultured testicular cells on T-ECM-enriched scaffolds confirmed their proliferation. Furthermore, the inoculation of neonatal mouse testicular cells onto T-ECM-enriched scaffolds resulted in the generation of sperm. Morphology evaluation showed that the structure of these cells was quite similar to mature sperm with a specialized tail structure. The hormonal analysis also confirmed production and secretion of testosterone and inhibin B by Leydig and Sertoli cells. T-ECM printed artificial testis is a future milestone that promises for enhancing germ cell maintenance and differentiation, toxicology studies, and fertility restoration to pave the way for new human infertility treatments in the future.

In vitro elimination of EL4 cancer cells from spermatogonia stem cells by miRNA-143- and 206-loaded folic acid-conjugated PLGA nanoparticles.

Aim: MiRNA's-143 and -206 are powerful apoptotic regulators in cancer cells. This study aimed to use miRNA-143- and 206-loaded poly(lactic-co-glycolic) acid (PLGA) nanoparticles conjugated with folic acid to induce apoptosis in the EL4 cancer cells. Materials & methods: The therapy was conducted in six groups: treatment with both miRNAs simultaneously (mixed miRNAs), miRNA-206 treatment, miRNA-143 treatment, blank PLGA, blank polyethylenimine (PEI) and complex PEI-miRNAs. Results: In terms of viability, in mixed miRNAs no synergistic effect was observed on EL4 cell elimination. However, in the single miRNA-206 group, a stronger apoptotic effect was observed than the mixed miRNAs group and single miRNA-143 group alone. Conclusion: MiRNAs' apoptotic induction effects in cancer cells were found to be remarkable.

Astaxanthin Relieves Busulfan-Induced Oxidative Apoptosis in Cultured Human Spermatogonial Stem Cells by Activating the Nrf-2/HO-1 pathway.

Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10µM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways.

The therapeutic potential of adipose tissue-derived mesenchymal stromal cells in the treatment of busulfan-induced azoospermic mice.

PURPOSE: The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes. METHODS: Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice. RESULTS: In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules. CONCLUSION: In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.

The Role of MicroRNA 143 and MicroRNA 206 in The Regulation of Apoptosis in Mouse Lukemia Cancer Cells and Spermatogonial Cells.

OBJECTIVE: In cancer treatments, smart gene delivery via nanoparticles (NPs) can be targeted for cancer cells, while concurrently minimizing damage to healthy cells. This study assessed the efficiency of poly lactic-co-glycolic acid (PLGA)-miR 143/206 transfection on apoptosis in mouse leukemia cancer cells (El4) and spermatogonial stem cells (SSCs). MATERIALS AND METHODS: In this experimental study, neonatal mouse spermatogonia cells and EL4 cancer cell lines were used. MicroRNA-PLGA NPs were prepared, characterized, and targeted with folate. Several doses were evaluated to obtain a suitable miR dose that can induce appropriate apoptosis in EL4 cells, while not harming SSCs. Cells were treated separately at 3 doses of each miR (for miR 143, doses of 25, 50 and 75 nmol and for miR 206, doses of 50, 100 and 150 nmol). The experiments were performed at 24, 48 and 72 hours. Viability and apoptosis were investigated by MTT and Annexin Kits. RESULTS: Based on MTT assay results, the optimal dose of miR 143 was 75 nmol (59.87 ± 2.85 % SSC and 35.3 ± 0.78 % EL4) (P≤0.05), and for miR 206, the optimal dose was 150 nmol (54.82 ± 6.7 % SSC and 33.92 ± 3.01% EL4) (P≤0.05). The optimal time was 48 hours. At these doses, the survival rate of the EL4 cells was below the half maximal inhibitory concentration (IC50) and SSC survival was above 50%. Annexin V staining also confirmed the selected doses (for miR 143 total apoptosis was 6.62% ± 1.8 SSC and 37.4% ± 4.2 EL4 (P≤0.05), and miR 206 was (10.98% ± 1.5 SSC and 36.4% ± 3.7 EL4, P≤0.05). CONCLUSION: Using intelligent transfection by NPs, we were able to induce apoptosis on EL4 cells and maintain acceptable SSC survival rates.

Human Umbilical Cord Mesenchymal Stem Cells Differentiated into Neuron-Like Cells via Laminin and Schwann Cells.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUMSCs) have been considered to repair damaged tissues and cells. This study aims to investigate the differentiation efficiency affected by Schwann cells (SCs) and laminin and also compare them to other strategies using chemicals or growth factors. METHODS: SCs and hUMSCs were separated from dorsal root ganglion of rats and newborn human umbilical cords (hUCs), respectively, and then cultured. The marker expressions of mesenchymal stem cells (MSCs), hematopoietic and endothelial for hUMSCs were confirmed by flow cytometry. The hUMSCs were cultured in four groups: 1) control, 2) co-culture with SCs (C), 3) laminin (L), and 4) co-culture with SCs treated by laminin (CL). The expression of protein and gene-related differentiation NSE, MAP2 and ß-tubulin were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry after 12 days. RESULTS: The flow cytometry analysis revealed high expression of mesenchymal and low expression of hematopoietic and endothelial markers, where the SCs expressed S100 at a high level (97.4%±2.25). The expression of NSE, MAP2 and ß-tubulin increased significantly in the C, L and CL groups compared to the control group (P<0.001), where the CL group had the highest expression among the groups [7.59±0.126, 7.87±0.191, 6.36±0.420, respectively, (P<0.01)]. Also, the expression of neural proteins was significantly increased in tested groups in comparison to the control group. CONCLUSION: Combined laminin and SCs co-culturing with hUMCSs could be the most effective strategy for neural differentiation.

Artificial testis: a testicular tissue extracellular matrix as a potential bio-ink for 3D printing.

Testicular scaffolds may be an option for fertility preservation. The aim was to develop various procedures for the decellularization of testicular tissue and to design a bio-ink to construct a bioartificial testis. Ram testicular tissue fragments were decellularized using NaCl buffer, NaCl buffer-Triton, SDS and SDS-Triton. The removal of the cells from the tissues was confirmed by DAPI and H & E staining, as well as the evaluation of the DNA content. Alcian blue, Orcein and Masson's trichrome staining methods were also used to confirm that T-ECM was preserved intact. Then, the optimal decellularization protocol was selected to determine the parameters of the bio-ink and printing of the scaffold. The extracted T-ECM was used to print the hydrogel scaffold in combination with alginate-gelatin. The printability, morphological, mechanical and biological properties of the printed hydrogels were characterized. Decellularization of testicular tissue fragments using the NaCl buffer-Triton protocol was significantly more efficient than other decellularization methods in removing the cellular debris and preserving the T-ECM compounds. The 3D printed scaffold with 5% T-ECM showed a uniform surface morphology with high cell attachment and cyto-biocompatibility properties for spermatogonia stem cells in vitro and in vivo compared to other groups. It is concluded that T-ECM can be used as a biomimetic material to make an artificial testis with possible in vitro sperm production.

Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing.

Decellularized scaffolds have been found to be excellent platforms for tissue engineering applications. The attempts are still being made to optimize a decellularization protocol with successful removal of the cells with minimal damages to extracellular matrix components. We examined twelve decellularization procedures using different concentrations of Sodium dodecyl sulfate and Triton X-100 (alone or in combination), and incubation time points of 15 or 30 min. Then, the potential of the decellularized scaffold as a three-dimensional substrate for colony formation capacity of mouse spermatogonial stem cells was determined. The morphological, degradation, biocompatibility, and swelling properties of the samples were fully characterized. The 0.5%/30 SDS/Triton showed optimal decellularization with minimal negative effects on ECM (P ≤ 0.05). The swelling ratios increased with the increase of SDS and Triton concentration and incubation time. Only 0.5%/15 and 30 SDS showed a significant decrease in the SSCs viability compared with other groups (P < 0.05). The SSCs colony formation was clearly observed under SEM and H&E stained slides. The cells infiltrated into the subcutaneously implanted scaffold at days 7 and 30 post-implantation with no sign of graft rejection. Our data suggest the %0.5/30 SDS/Triton as an excellent platform for tissue engineering and reproductive biology applications.