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.

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.

Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in vitro.

BACKGROUND: Some children who have survived cancer will be azoospermic in the future. Performing isolation and purification procedures for spermatogonial stem cells (SSC) is very critical. In this regard, performing the process of decontamination of cancerous cells is the initial step. The major objective of the present study is to separate the malignant EL4 cell line in mice and spermatogonial stem cells in vitro. METHODS: The spermatogonial stem cells of sixty neonatal mice were isolated, and the procedure of co-culturing was carried out by EL4 which were classified into 2 major groups: (1) the control group (co-culture in a growth medium) and (2) the group of co-cultured cells which were separated using the microfluidic device. The percentage of cells was assessed using flow cytometry technique and common laboratory technique of immunocytochemistry and finally was confirmed through the laboratory technique of reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The actual percentage of EL4 and SSC after isolation was collected at two outlets: the outputs for the smaller outlet were 0.12% for SSC and 42.14% for EL4, while in the larger outlet, the outputs were 80.38% for SSC and 0.32% for EL4; in the control group, the percentages of cells were 21.44% for SSC and 23.28% for EL4 (based on t test (p ≤ 0.05)). CONCLUSIONS: The present study demonstrates that the use of the microfluidic device is effective in separating cancer cells from spermatogonial stem cells.

Future of Spermatogonial Stem Cell Culture: Application of Nanofiber Scaffolds.

BACKGROUND: Spermatogonial stem cells (SSCs) are unique in mammals because they can transmit genetic information from generation to generation and it is of significant importance. In testes, Sertoli cells, peritubular myoid cells, Leydig cells and other interstitial cells contribute to the spermatogonial stem cell "niche". So, creation of niche in an in vitro condition that mimics the in vivo environment is essential to maintain functional characteristic of SSCs. OBJECTIVE: In this review, we describe the impact of nanofiber scaffolds on the culture of SSCs derived from human-to-mouse. RESULTS: Nanofiber Matrices mimic the architecture and size scale of the natural extracellular matrix (ECM). The scaffold provides more three-dimensional (3D), biomimicking and topographical signals to the cells and results in a more physiologically relevant cellular phenotype. Several investigators use different nanofiber scaffold-like carbon nanotubes (CNTs) scaffold, poly-L-lactic acid (PLLA) nanofiber scaffold, 3D soft agar culture system, human serum albumin (HSA)/tri calcium phosphate nanoparticles (TCPNPs) and electrospun polyamide nanofiber for proliferation and maintenance of self-renewal activity of the SSCs. CONCLUSION: Application of nanofiber scaffolds for in vitro culture of the SSCs may produce spermatogonial stem cells that can be used in regenerative medicine, tissue engineering, assisted reproductive technology and in the treatment of infertility in pre-pubertal cancer patients.