Cell Journal (Yakhteh)
Volume:24 Issue: 8, Aug 2022

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may adversely affect male reproductive tissues and malefertility. This concern is elicited by the higher susceptibility and mortality rate of men to the SARS-CoV-2 mediated coronavirus disease-19 (COVID-19), compared to the women. SARS-CoV-2 enters host cells after binding to a functional receptor named angiotensin-converting enzyme-2 (ACE2) and then replicates in the host cells and gets released into the plasma. SARS-CoVs use the endoplasmic reticulum (ER) as a site for viral protein synthesis and processing, as well as glucose-regulated protein 78 (Grp78) is a key ER chaperone involved in protein folding by preventing newly synthesized proteins from aggregation.Therefore, we analyzed Grp78 expression in various human organs, particularly male reproductive organs, using BroadInstitute Cancer Cell Line Encyclopedia (CCLE), the Genotype-Tissue Expression (GTEx), and Human Protein Atlas onlinedatasets. Grp78 is expressed in male reproductive tissues such as the testis, epididymis, prostate, and seminal vesicle. It can facilitate the coronavirus entry into the male reproductive tract, providing an opportunity for its replication. This link between the SARS-CoV-2 and the Grp78 protein could become a therapeutic target to mitigate its harmful effects on male fertility.

Primordial germ cells develop into oocytes and sperm cells. These cells are useful resources in reproductive biology and regenerative medicine. The mesenchymal stem cells (MSCs) have been examined for in vitro production of primordial germ cell-like cells. This study aimed to summarize the existing protocols for MSCs differentiation into primordial germ cell-like cells (PGLCs). In the limited identified studies, various models of mesenchymal stem cells, including those derived from adipose tissue, bone marrow, and Wharton's jelly, have been successfully differentiated into primordial germ cell-like cells. Although the protocols of specification induction are basically very similar, they have been adjusted to the mesenchymal cell type and the species of origin. The availability of MSCs has made it possible to customize conditions for their differentiation into primordial germ cell-like cells in several models, including humans. Refining germ cell-related signaling pathways during induced differentiation of MSCs will help define extension to the protocols for primordial germ cell-like cells production.

According to the mounting data, microRNAs (miRNAs) may play a key role in reprogramming. miR-106bis considered as an enhancer in reprogramming efficiency. Based on induced pluripotent stem cells (iPSCs), cell treatments have a huge amount of potential. One of the main concerns about using iPSCs in therapeutic settings is the possibility of tumor formation. It is hypothesized that a procedure that can reprogram cells with less genetic manipulation reduces the possibility of tumorigenicity. In this experimental study, miR-106b-5p transduced by pLV-miRNA vector into mice isolated spermatogonial stem cells (SSCs) to achieve iPS-like cells. Then the transduced cells were cultured in specific conditions to study the formation of three germ layers. The tumorigenicity of these iPS-like cells was investigated by transplantation into male BALB/C mice. We show that SSCs can be successfully reprogrammed into induced iPS-like cells by pLV-miRNA vector to transduce the hsa-mir-106b-5p into SSCs and generating osteogenic, neural and hepatoblast lineage cells in vitro as a result of pluripotency. Although these iPS-like cells are pluripotent, they cannot form palpable tumors in vivo. These results demonstrate that infection of hsa-mir-106b-5p into SSCs can reprogram them into iPSCsand advanced germ cell lineages without tumorigenicity. Also, a novel approach for studying the generation of iPSCsand the application of iPS or iPS-like cells in regenerative medicine is presented.

Insulin insufficiency due to the reduced pancreatic beta cell number is the hallmark of diabetes, resulting inan intense focus on the development of beta-cell replacement options. One approach to overcome the problem is tosearch for alternative sources to induce insulin-producing cells (IPCs), the advent of mesenchymal stem cells (MSCs)holds great promise for producing ample IPCs. Encapsulate the MSCs with alginate improved anti-inflammatory effectsof MSC treatment. This study aimed to evaluate the differentiation of wharton jelly-derived MScs into insulin producingcells using alginate encapsulation. In this experimental study, we established an efficient IPCs differentiation strategy of humanMSCs derived from the umbilical cord’s Wharton jelly with lentiviral transduction of Pancreas/duodenum homeoboxprotein 1 (PDX1) in a 21-day period using alginate encapsulation by poly-L-lysine (PLL) and poly-L-ornithine (PLO)outer layer. During differentiation, the expression level of PDX1 and secretion of insulin proteins were increased. Results showed that during time, the cell viability remained high at 87% at day 7. After 21 days, the differentiated beta-like cells in microcapsules were morphologically similar to primary beta cells. Evaluation of the expression of PDX1 and INS by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on days 7, 14 and 21 of differentiation exhibited the highest expression on day 14. At the protein level, the expression of these two pancreatic markers was observed after PDX1 transduction. Results showed that the intracellular and extracellular insulin levels in the cells receiving PDX1 is higher than the control group. The current data showed that encapsulation with alginate by PLL and PLO outer layer permitted to increase the microcapsules’ beta cell differentiation. Encapsulate the transduced-MSCs with alginate can be applied in an in vivo model in order to do the further analysis.

Primordial germ cell (PGCs) lines are a source of a highly specialized type of cells, characteristically oocytes,during female germline development in vivo. The oocyte growth begins in the transition from the primary follicle. It isassociated with dynamic changes in gene expression, but the gene-regulating signals and transcription factors that control oocyte growth remain unknown. We aim to investigate the differentiation potential of mouse bone marrow mesenchymal stem cells (mMSCs) into female germ-like cells by testing several signals and transcription factors. In this experimental study, mMSCs were extracted from mice femur bone using the flushingtechnique. The cluster-differentiation (CD) of superficial mesenchymal markers was determined with flow cytometric analysis. We applied a set of transcription factors including retinoic acid (RA), titanium nanotubes (TNTs), and fibrin such as TNT-coated fibrin (F+TNT) formation and (RA+F+TNT) induction, and investigated the changes in gene, MVH/ DDX4, expression and functional screening using an in vitro mouse oocyte development condition. Germ cell markers expression, (MVH / DDX4), was analyzed with Immunocytochemistry staining, quantitative transcription-polymerase chain reaction (RT-qPCR) analysis, and Western blots. The expression of CD was confirmed by flow cytometry. The phase determination of the TNTs and F+TNT were confirmed using x-ray diffraction (XRD) and scanning electron microscope (SEM), respectively. Remarkably, applying these transcription factors quickly induced pluripotent stem cells into oocyte-like cells that were sufficient to generate female germlike cells, growth, and maturation from mMSCs differentiation. These transcription factors formed oocyte-like cells specification of stem cells, epigenetic reprogramming, or meiosis and indicate that oocyte meiosis initiation and oocyte growth are not separable from the previous epigenetic reprogramming in stem cells in vitro. Results suggested several transcription factors may apply for arranging oocyte-like cell growth and supplies an alternative source of in vitro maturation (IVM).

In addition to the carboxy region, Smad2 transcription factor can be phosphorylated in the linker region aswell. Phosphorylation of Smad2 linker region (Smad2L) promotes the expression of plasminogen activator inhibitor type1 (PAI-1) which leads to cardiovascular disorders such as atherosclerosis. The purpose of this study was to evaluate the role of dual transactivation of EGF and TGF-β receptors in phosphorylation of Smad2L and protein expression of PAI-1 induced by endothelin-1 (ET-1) in bovine aortic endothelial cells (BAECs). In addition, as an intermediary of G protein-coupled receptor (GPCR) signaling, the functions of ROCK and PLC were investigated in dual transactivation pathways. The experimental study is an in vitro study performed on BAECs. Proteins were investigatedby western blotting using protein-specific antibodies against phospho-Smad2 linker region residues (Ser245/250/255),phospho-Smad2 carboxy residues (465/467), ERK1/(Thr202/Thr204), and PAI-1. TGF (2 ng/ml), EGF (100 ng/ml) and ET-1 (100 nM) induced the phosphorylation of Smad2L. This response wasblocked in the presence of AG1478 (EGFR antagonists), SB431542 (TGFR inhibitor), and Y27632 (Rho-associated protein kinase (ROCK antagonist). Moreover, ET-1-increased protein expression of PAI-1 was decreased in the presence of bosentan (ET receptor inhibitor), AG1478, SB431542, and Y27632. The results indicated that ET-1 increases the phosphorylation of Smad2L and protein expression of PAI-1via induced the transactivation pathways of EGFR and TGFR. This study is the first attempt to scrutinize the significant role of ROCK in the protein expression of PAI-1.

Chronic lymphoid leukemia (CLL) is the most common type of leukemia among adults. Increased levels of Mcl-1 and Bcl-xL is linked to resistance to Bcl-2 inhibitors including ABT-199. In this study, we investigated the effect of miRNA-16-1 on apoptosis and sensitivity of the CLL cells to ABT-199. In this experimental study, the Mcl-1 and Bcl-2 expression were measured using qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The effect of treatments on cell survival and growth were explored with MTT assay and Trypan blue assay, respectively. The drug interaction was evaluated using combination index analysis. Apoptosis was assessed by ELISA cell death and caspase-3 activity assays. MiRNA-16-1 markedly inhibited the expression of Mcl-1 and Bcl-2 in a time dependent manner (P<0.05, relativeto blank control). Pretreatment with miRNA-16-1 synergistically suppressed the cell growth and survival and reduced the half-maximal inhibitory concentration (IC50) value of ABT-199. Moreover, miRNA-16-1 markedly augmented the apoptotic effect of ABT-199 in CLL cells (P<0.05). Our findings propose that miRNA-16-1 act in concert with ABT-199 to exert synergistic anticancer efficacy against CLL, which is attributed to the inhibition of Bcl-2 and Mcl-1. This may propose a promising strategy for CLL resistant patients.

Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of thisstudy is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy inpropagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS). 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 β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse. 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. Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection ofspermatogonial 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.