Overcoming Chemotherapy Resistance in Metastatic Cancer: A Comprehensive Review.

The management of metastatic cancer is complicated by chemotherapy resistance. This manuscript provides a comprehensive academic review of strategies to overcome chemotherapy resistance in metastatic cancer. The manuscript presents background information on chemotherapy resistance in metastatic cancer cells, highlighting its clinical significance and the current challenges associated with using chemotherapy to treat metastatic cancer. The manuscript delves into the molecular mechanisms underlying chemotherapy resistance in subsequent sections. It discusses the genetic alterations, mutations, and epigenetic modifications that contribute to the development of resistance. Additionally, the role of altered drug metabolism and efflux mechanisms, as well as the activation of survival pathways and evasion of cell death, are explored in detail. The strategies to overcome chemotherapy resistance are thoroughly examined, covering various approaches that have shown promise. These include combination therapy approaches, targeted therapies, immunotherapeutic strategies, and the repurposing of existing drugs. Each strategy is discussed in terms of its rationale and potential effectiveness. Strategies for early detection and monitoring of chemotherapy drug resistance, rational drug design vis-a-vis personalized medicine approaches, the role of predictive biomarkers in guiding treatment decisions, and the importance of lifestyle modifications and supportive therapies in improving treatment outcomes are discussed. Lastly, the manuscript outlines the clinical implications of the discussed strategies. It provides insights into ongoing clinical trials and emerging therapies that address chemotherapy resistance in metastatic cancer cells. The manuscript also explores the challenges and opportunities in translating laboratory findings into clinical practice and identifies potential future directions and novel therapeutic avenues. This comprehensive review provides a detailed analysis of strategies to overcome chemotherapy resistance in metastatic cancer. It emphasizes the importance of understanding the molecular mechanisms underlying resistance and presents a range of approaches for addressing this critical issue in treating metastatic cancer.

Single-Cell Transcriptomics for Unlocking Personalized Cancer Immunotherapy: Toward Targeting the Origin of Tumor Development Immunogenicity.

Cancer immunotherapy is a promising approach for treating malignancies through the activation of anti-tumor immunity. However, the effectiveness and safety of immunotherapy can be limited by tumor complexity and heterogeneity, caused by the diverse molecular and cellular features of tumors and their microenvironments. Undifferentiated tumor cell niches, which we refer to as the "Origin of Tumor Development" (OTD) cellular population, are believed to be the source of these variations and cellular heterogeneity. From our perspective, the existence of distinct features within the OTD is expected to play a significant role in shaping the unique tumor characteristics observed in each patient. Single-cell transcriptomics is a high-resolution and high-throughput technique that provides insights into the genetic signatures of individual tumor cells, revealing mechanisms of tumor development, progression, and immune evasion. In this review, we explain how single-cell transcriptomics can be used to develop personalized cancer immunotherapy by identifying potential biomarkers and targets specific to each patient, such as immune checkpoint and tumor-infiltrating lymphocyte function, for targeting the OTD. Furthermore, in addition to offering a possible workflow, we discuss the future directions of, and perspectives on, single-cell transcriptomics, such as the development of powerful analytical tools and databases, that will aid in unlocking personalized cancer immunotherapy through the targeting of the patient's cellular OTD.

Risk of secondary tumours in patients with non-metastatic and metastatic human retinoblastoma.

BACKGROUND: Retinoblastoma is an intraocular cancer in children and infants. Despite all the available treatment options and high survival rates in children with retinoblastoma, exposure to secondary tumours in adulthood is one of the concerns that physicians face. In many cases, dysfunction of the RB1 gene is the main cause of secondary tumours due to retinoblastoma. Therefore, the aim of this study was to evaluate the incidence of other secondary tumours in children with retinoblastoma. METHODS: In this regard, we performed continuous and integrated bioinformatics analyses to find genes, protein products, and signal pathways involved in other cancers. RESULTS: 1170 high-expression genes and 960 low-expression genes between non-invasive and invasive retinoblastoma were isolated. After examining the signal pathways, we observed bladder cancer and small cell lung cancer in the overexpressed genes. We also observed 5 cancers of endometriosis, prostate, non-small cell lung cancer, glioblastoma and renal cell carcinoma in low-expression genes. Based on the P-value index, non-small cell lung cancer, prostate and bladder cancers had the highest risk, and endometriosis cancer showed a lower probability of developing a secondary tumour in patients with retinoblastoma. In addition, the network between proteins also showed us that TP53, CDK2, SRC, MAPK1 proteins with high expression and JUN, HSP90AA1, and UBC proteins with low-expression play a significant role in candidate cancers. CONCLUSION: Lastly, we used continuous bioinformatics analysis to show that seven cancers are strongly linked to retinoblastoma cancer. Of course, more research is needed to find the best way to care for children who have been treated for retinoblastoma.

Ectopic expression of OCT4B1 Decreases Fertility Rate and Changes Sperm Parameters in Transgenic Mice.

Background: The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. Objectives: The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). Material and Methods: The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. Results: The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. Conclusion: In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Investigation of key signaling pathways and appropriate diagnostic biomarkers selection between non-invasive to invasive stages in pancreatic cancer: a computational observation.

Pancreatic cancer is the seventh most lethal cancer in the world. Despite its moderate prevalence, the 5-year survival rate of patients with pancreatic cancer is about 10%. Despite different therapeutic and diagnostic strategies for pancreatic cancer, this cancer is still uncontrollable in the invasive stage and can invade various body organs and cause death. Early detection for pancreatic cancer can be an excellent solution to manage treatment better and increase patients' survival rates. This study aimed to find diagnostic biomarkers between non-invasive to invasive stages of pancreatic cancer in the extracellular matrix to facilitate the early diagnosis of this cancer. Using bioinformatics analysis, we selected the appropriate datasets between non-invasive and invasive pancreatic cancer stages and categorized their genes. Then, we charted and confirmed the signaling pathways, gene ontology, protein relationships, and protein expression levels in the human samples using bioinformatics databases. Cell adhesion and hypoxia signaling pathways were observed in up-regulated genes, different phases of the cell cycle, and metabolic signaling pathways with down-regulated genes between non-invasive and invasive pancreatic cancer stages. For proper diagnostic biomarkers selection, the overexpressed genes that released protein into the extracellular matrix were examined in more detail, with 62 proteins selected and SPARC, THBS2, COL11A1, COL1A1, COL1A2, COL3A1, SERPINH1, PLAU proteins chosen. Bioinformatics analysis can more accurately assess the relationship between molecular mechanisms and key actors in pancreatic cancer invasion and metastasis to facilitate early detection and improve treatment management for patients with pancreatic cancer.

Stem Cell Therapy in Limb Ischemia: State-of-Art, Perspective, and Possible Impacts of Endometrial-Derived Stem Cells.

As an evidence-based performance, the rising incidence of various ischemic disorders has been observed across many nations. As a result, there is a growing need for the development of more effective regenerative approaches that could serve as main therapeutic strategies for the treatment of these diseases. From a cellular perspective, promoted complex inflammatory mechanisms, after inhibition of organ blood flow, can lead to cell death in all tissue types. In this case, using the stem cell technology provides a safe and regenerative approach for ischemic tissue revascularization and functional cell formation. Limb ischemia (LI) is one of the most frequent ischemic disease types and has been shown to have a promising regenerative response through stem cell therapy based on several clinical trials. Bone marrow-derived mononuclear cells (BM-MNCs), peripheral blood CD34-positive mononuclear cells (CD34+ PB-MNCs), mesenchymal stem cells (MSCs), and endothelial stem/progenitor cells (ESPCs) are the main, well-examined stem cell types in these studies. Additionally, our investigations reveal that endometrial tissue can be considered a suitable candidate for isolating new safe, effective, and feasible multipotent stem cells for limb regeneration. In addition to other teams' results, our in-depth studies on endometrial-derived stem cells (EnSCs) have shown that these cells have translational potential for limb ischemia treatment. The EnSCs are able to generate diverse types of cells which are essential for limb reconstruction, including endothelial cells, smooth muscle cells, muscle cells, and even peripheral nervous system populations. Hence, the main object of this review is to present stem cell technology and evaluate its method of regeneration in ischemic limb tissue.

Stem cells-derived natural killer cells for cancer immunotherapy: current protocols, feasibility, and benefits of ex vivo generated natural killer cells in treatment of advanced solid tumors.

Nowadays, natural killer (NK) cell-based immunotherapy provides a practical therapeutic strategy for patients with advanced solid tumors (STs). This approach is adaptively conducted by the autologous and identical NK cells after in vitro expansion and overnight activation. However, the NK cell-based cancer immunotherapy has been faced with some fundamental and technical limitations. Moreover, the desirable outcomes of the NK cell therapy may not be achieved due to the complex tumor microenvironment by inhibition of intra-tumoral polarization and cytotoxicity of implanted NK cells. Currently, stem cells (SCs) technology provides a powerful opportunity to generate more effective and universal sources of the NK cells. Till now, several strategies have been developed to differentiate types of the pluripotent and adult SCs into the mature NK cells, with both feeder layer-dependent and/or feeder laye-free strategies. Higher cytokine production and intra-tumoral polarization capabilities as well as stronger anti-tumor properties are the main features of these SCs-derived NK cells. The present review article focuses on the principal barriers through the conventional NK cell immunotherapies for patients with advanced STs. It also provides a comprehensive resource of protocols regarding the generation of SCs-derived NK cells in an ex vivo condition.

A Case Report of Genetic Cascade Screening in Dilated Cardiomyopathy: A Perspective for Preventive Cardiology.

Cardiomyopathies are heterogeneous and critical disorders of cardiovascular diseases. One of the most common inherited cardiomyopathies is DCM (dilated cardiomyopathy). Genetic disorders are found in approximately 50% of DCM cases. We aimed to describe a case of DCM in a 42-year-old woman in 2018 at Farhud Genetic Clinic, Tehran, Iran. To detect genetic involvement, Next-generation sequencing (NGS) was performed and the data were evaluated carefully. Variations in different genes coding crucial proteins in cardiac muscle structure (i.e. Titin, Obscurin, MYH6, and LAMA4) and proteins involved in channels (i.e. CAVNA1C, SCN1B and SCN5A) were detected by whole-exome sequencing (WES). In agreement with the clinical manifestations and molecular analysis, DCM was confirmed. This study provides further evidence on the diagnostic role of NGS in borderline DCM cases. It also shows the recently developed high throughput sequencing can provide clinicians with this approach to diagnosis, treatment, and prevention of such hard-to-diagnose disorders. Furthermore, this study highlights the basis of personalized medicine, namely detection of high-risk individuals by revealing some genetic variants as predictive risk factors, and initial prevention of DCM.

Contradictory Effect of Notch1 and Notch2 on Phosphatase and Tensin Homolog and its Influence on Glioblastoma Angiogenesis.

Many genes induce angiogenesis in tumors, and among them, Notch family genes have received particular attention due to their extensive network of connections with other genes active in this function. Suppression of angiogenic signaling has been studied in various cancers, confirming Notch's fundamental and extensive role. According to studies, four Notch genes work independently with many genes such as vascular endothelial growth factor, phosphatase and tensin homolog, Phosphoinositide 3-kinase/Akt, and matrix metalloproteinases, and so many other genes, as well as proteins (such as hypoxia-inducible factor-1 alpha) significantly affect tumor angiogenesis. Notch1 regular activity in a healthy person causes angiogenesis in body tissues, controlled by normal Notch2 activity. However, in many cases of glioblastoma, whether on patients or tumor xenografts or in vivo models, a mutation in one of these two essential genes or at least one of the genes and proteins that affected by them can cause better angiogenesis in hypoxic conditions and lead to become an invasive tumor. In this review, we examined the contrasting activity of Notch1 and Notch2 and the signaling cascade that each generates in the angiogenesis of glioblastoma, the most invasive cancer of the central nervous system.

Inflammatory Microenvironment of Acute Myocardial Infarction Prevents Regeneration of Heart with Stem Cells Therapy.

Over the past years, the benefits of stem cell therapy approach for treatment of the cardiovascular diseases have been shown through the rebuilding of new cardiomyocytes and blood vessels. while a successful regeneration of the myocardium has been proven on the animal models of acute myocardial injuries resulted from the stem cells transplantation, no significant long-term regenerative with autologous stem cell therapy in patients with acute myocardial infarction have been reported based on recent meta-analyses. It seems that the inflammatory microenvironment of acute myocardial infarction has an inhibitory effect on the stem cells potential for regenerating the injured myocardium. Secretion of critical cytokines with pro-inflammatory properties including tumor necrosis factor-α, interleukin-1ß, and interleukin-6 as well as induction of hypoxic condition and finally formation of cytotoxic elements cause the cellular death and hinder the stem cells proliferation and differentiation. Based on the evidence, application of some approaches like co-delivery of mesenchymal stem cells with the other useful cells, using the stem cells derived productions, administration of preconditioned and modified cells, and also using the anti-inflammatory agents besides the cell therapy are hypothesized as the primary developed safe and practical approaches for decreasing destructive effects of the inflammation on the implanted stem/progenitor cells. In this review, we critically discuss the quiddity of the inflammatory microenvironment and its promoted mechanisms as the main elements to hinder the efficacy of stem cell therapy in the cases of acute myocardial infarction. Also, we finally propose some applied solutions to the problem of cardiac regeneration with stem cells therapy.

Application of Stem Cell Technologies to Regenerate Injured Myocardium and Improve Cardiac Function.

In the recent decades, cardiovascular diseases emerged as the major leading cause of human mortality. However, current clinical approaches still do not encompass a thorough therapeutic solution for improving heart function of the patients who suffered an extensive myocardial injury. Based on this status quo, stem cells could become a novel option, as a natural source of the new myocardium lineage cells, being capable of paracrine factors secretion, protection or even regeneration of the damaged heart muscle. Efficient stem cell-based therapy of the heart should lead to repair or/and replacement of the degenerated tissue with functional myocardial and endothelial cells. Hereon, various types of pluripotent and multipotent stem cells have been already studied in the pre-clinical and clinical settings, demonstrating their cardiomyogenic and regenerative potential. In this context, as a type of male adult stem/ progenitors, spermatogonial stem cells feature a remarkable ability for a formation of cardiovascular lineages, based on our own observations. Presented data supports the presumption, that spermatogonial stem cells not only have a suitable capacity to generate functional heart cells but can also potentially improve the function of an injured myocardium. In this review article, we first describe the essential molecular and pathophysiological mechanisms involved in the heart tissue injury. Afterwards, based on our ongoing study, we review the impact of the stem cell technologies on the regeneration therapy in cardiovascular and myocardial diseases. Particular emphasis is being put on the usability of spermatogonial stem cells in cardiac therapy.

Differentiation of bone marrow-derived stage-specific embryonic antigen 1 positive pluripotent stem cells into male germ cells.

Studies published in recent years have changed the outlook on sterility and germ cell development by producing gametes from stem cells. In present study, a novel approach on differentiation of bone marrow-derived stage-specific embryonic antigen 1 positive (SSEA-1+ ) pluripotent stem cells into male germ cells has been addressed. SSEA-1+ stem cells were separated from murine bone marrow using magnetic-activated cell sorting (MACS) system and propagated on a feeder layer cells. To evaluate the pluripotency characteristic of the purified cells, they were differentiated toward cells of three germ layers. Later the SSEA-1+ stem cells were induced to differentiate along male germ cell lineage with retinoic acid. Flowcytometric analysis of SSEA-1+ stem cells revealed purity of about 62% which increased to 91% after cultivation over feeder cells. Expression of specific transcripts of Oct4, SSEA-1, Nanog, Dppa3, fragilis, Rex-1, SOX-2, and alkaline-phosphatase and immunofluorescence evaluation of Oct4 and SSEA-1 expression showed the differentiation of purified stem cells toward the cells of three germ layers. Differentiation potential of purified cells was positively evidenced by expression markers specific for primordial germ cells, spermatogonial stem cells and spermatogonia including Mvh, fragilis, Dppa3, Stra8, DAZL, Piwil2, ß1, and α6-integrins as well as meiotic-specific marker SYCP3. Our results showed that SSEA-1+ pluripotent stem cells are able to differentiate into male germ cells. The results of the present study are encouraging enough to merit further investigation, provide a new hope for those suffering from infertility and introduce a novel platform for research on germ cell development.

In vitro differentiation process of human Wharton's jelly mesenchymal stem cells to male germ cells in the presence of gonadal and non-gonadal conditioned media with retinoic acid.

Human umbilical Wharton's jelly-derived mesenchymal stem cells (HWJMSCs) are the best candidate to get plentiful stem cells and differentiate them to germ cells under appropriate conditions to treat infertility. We sought to determine under which conditions HWJMSCs could form male germ cells in vitro. So, HWJMSCs were differentiated to male germ cells under a mixture of bone morphogenetic protein-4 (BMP-4) and testicular and placental culture condition (TCC and PCC) medium followed by retinoic acid for 21 d. In the present study, the HWJMSCs were obtained from Wharton's jelly of umbilical cords of male neonates delivered by cesarean section. At the third passage, mesenchymal stem cell markers and differentiation to osteocytes and adipocytes were investigated. Then, HWJMSCs were induced to differentiate into male germ cells in the presence of BMP-4, all-trans retinoic acid, PCC, and TCC for 21 d. The profile of c-Kit, DDX4, Piwil2, and Dazl gene expression was evaluated by qPCR and ICC. Data was analyzed by ANOVA test. After 3 wk of treatment with different reagents, the morphology of these spindle-like cells changed to shiny clusters and germ cell-specific markers in mRNA were upregulated in both TCC + retinoic acid (RA) and BMP-4 + RA. Induction of HWJMSCs with TCC in the presence of RA resulted in significant upregulation (P ≤ 0.05) of all germ cell-specific genes (c-Kit 2.6795 ± 0.75, DDX4 4.3188 ± 1.18, Piwil2 4.9962 ± 1.55, Dazl 6.1199 ± 0.78) compared to control and PCC + RA. Our results indicated that TCC and RA are involved in human germ cell development. Moreover, BMP signaling also induced differentiation. Our findings provide a novel effective approach for generation of germ cells in vitro and studying the interaction of germ cells with their niche. Our work represents an essential step toward gaining knowledge of the molecular properties of HWJMSCs in the field of cell therapy. We demonstrated that under a suitable situation, HWJMSCs have the ability to differentiate into germ cells and this provides an excellent pattern to study infertility cause and cure.

A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells.

OBJECTIVES: To culture the in vitro mouse embryonic stem cells (mESCs) and to direct their differentiation to germ-line cells; in present study we used a vector backbone containing the fusion construct Stra8-EGFP to select differentiated ES cells that entered meiosis. Retinoic acid was used to differentiate embryonic stem cells to germ cells. MATERIALS AND METHODS: A fragment of Stra8 gene promoter (-1400 to +7) was inserted in ScaI/HindIII multiple cloning site of pEGFP-1 vector. The electroporation was done on embryonic stem cells and positive colonies were selected as puromycin-resistant after three weeks of treatment with puromycin. All-trans retinoic acid (RA) was used for differentiation of mESCs at final concentration of 10(-)5M. The expression of protamine 1 (Prm1) gene was checked as post meiotic marker in differentiated mESCs after 5, 10, 15, 21 and 30 days after RA induction. RESULTS: The PCR amplification by specific primers for Stra8-EGFP fusion gene was detected in DNA sample from mESCs after electroporation and puromycin treatment. GFP-positive mESC colonies were observed after 72 hr RA induction. The protamine 1 gene was expressed after 21 days of RA induction. CONCLUSION: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

Evaluation of in vitro spermatogenesis system effectiveness to study genes behavior: monitoring the expression of the testis specific 10 (Tsga10) gene as a model.

BACKGROUND: In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (mESC) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. METHODS: mESCs were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th, and 25th days of the culture and were subjected to expression analysis of a panel of germ cell specific genes. Expression of Tsga10 in RNA and protein levels was then analyzed. RESULTS: Transition from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This finding is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed forms of the related protein was similar to those in vivo as well. CONCLUSIONS: Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.

Retinoic acid induces mouse bone marrow-derived CD15⁺, Oct4⁺ and CXCR4⁺ stem cells into male germ-like cells in a two-dimensional cell culture system.

We have examined the effect of retinoic acid (RA) on differentiation of bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells. Bone marrow stem cells (BMSCs) were isolated from the femur of 3-4-week-old male C57BL/6 mice. Magnetic-activated cell sorting (MACS) system was used to sort CD15(+) , Oct4(+) and CXCR4(+) cells. RT-PCR was used to follow the expression of pluripotency markers. Sorted CD15(+) , Oct4(+) and CXCR4(+) cells were cultured in an undifferentiated condition on a feeder layer of mitomycin C-inactivated C2C12. The embryoid-like bodies were differentiated into male germ cells by retinoic acid. To identify the expression of male germ specific markers, differentiated cells were analysed by means of reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence staining. RT-PCR and immunofluorescence show that bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells express pluripotency markers, Oct4, Nanog, Rex-1, SOX-2 and AP. The purified CD15(+) , Oct4(+) and CXCR4(+) formed structures like embryoid bodies when plated over a feeder layer; these bodies were alkaline phosphatase positive. When cells were induced by RA, bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) were positive for Mvh, Dazl, Piwil2, Dppa3 and Stra8, that known molecular markers of male germ cells. Thus RA can induce differentiation of mouse bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells in vitro. Negative results for the gene expression analysis of female germ cells markers, GDF9 and ZP3, confirmed this conclusion.

A novel in vitro model for cancer stem cell culture using ectopically expressed piwil2 stable cell line.

OBJECTIVE: Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells (CSCs), and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model. MATERIALS AND METHODS: In this experimental study, mouse embryonic fibroblasts (MEFs) were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter (CMV). Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions. RESULTS: The results of reverse transcriptase-polymerase chain reaction (RT-PCR), western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells (ANOVA, p< 0.05). Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells (ANOVA, p< 0.05). The MEF-Piwil2 cells tolerated hypoxia mimetic conditions (CoCl2 ) with more than 95% viability. CONCLUSION: According to the molecular and functional studies, it has been realized that Piwil2 plays a key role(s) in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse.

Co-culture of Mouse Embryonic Stem Cells with Sertoli Cells Promote in vitro Generation of Germ Cells.

OBJECTIVE(S): Sertoli cells support in vivo germ cell production; but, its exact mechanism has not been well understood. The present study was designed to analyze the effect of Sertoli cells in differentiation of mouse embryonic stem cells (mESCs) to germ cells. MATERIALS AND METHODS: A fusion construct composed of a Stra8 gene promoter and the coding region of enhanced green fluorescence protein was produced to select differentiated mESCs. To analyze sertoli cells' effect in differentiation process, mESCs were separated into two groups: the first group was cultured on gelatin with retinoic acid treatment and the second group was co-cultured with sertoli cell feeder without retinoic acid induction. Expressions of pre-meiotic (Stra8), meiotic (Dazl and Sycp3) and post-meiotic (Prm1) genes were evaluated at different differentiation stages (+7, +12 and +18 days of culture). RESULTS: In the first group, expressions of meiotic and post-meiotic genes started 12 and 18 days after induction with retinoic acid, respectively. In the second group, 7 days after co-culturing with Sertoli cells, expression of meiotic and post-meiotic genes was observed. CONCLUSION: These results show that differentiation process to germ cells is supported by Sertoli cells. Our findings provide a novel effective approach for generation of germ cell in vitro and studying the interaction of germ cells with their niche.

In vitro-derived gametes from stem cells.

Sperm and eggs are essential cells for reproduction and fertility in mammals. Lack of sperm production is one of the leading causes of infertility, a major and growing problem in the developed world affecting 13 to 18% of reproductive-age couples. The birth of the first test tube baby by in vitro fertilization marked an advance in infertility treatment. Later on, several important new techniques called assisted reproductive technologies were developed to help couples who experience infertility. One limiting factor is the requirement of reproductive cells (gametes) for use in in vitro fertilization. For azoospermic men lacking sperm cells, producing gametes in vitro could be a new window to overcome infertility. In the past few years, several reports have been published on generating germ cells from stem cells, one of the epitomes of which was the report on functional in vitro-derived (IVD) germ cells. These mature haploid sperm cells from mouse embryonic stem cells were capable of egg fertilization and producing live offspring. In tandem with previous advancements in germ cell research, development of new technologies based on IVD gametes will change the future of infertility and provide a new basis for the establishment of novel therapeutic approaches to cure more complicated conditions of infertility. In addition, IVD gametogenesis provides an accessible system for studying the specification and differentiation of sperm cells and related processes such as meiosis, morphogenesis, and motility.

BMP4 can generate primordial germ cells from bone-marrow-derived pluripotent stem cells.

Evidence of germ cell derivation from embryonic and somatic stem cells provides an in vitro model for the study of germ cell development, associated epigenetic modification and mammalian gametogenesis. More importantly, in vitro derived gametes also represent a potential strategy for treating infertility. In mammals, male and female gametes, oocyte and sperm, are derived from a specific cell population, PGCs (primordial germ cells) that segregate early in embryogenesis. We have isolated pluripotent SSEA-1+ (stage-specific embryonic antigen-1+) cells from mice bone marrow using a MACS (magnetic-activated cell sorting) system. SSEA-1+ cells were directly separated from the suspension of MMCs (murine mononuclear cells) harvested from bone marrow of 2-4-week-old mice. Flow-cytometry assay immediately after sorting and culturing under undifferentiated condition showed 55±7% and 87±4% purity respectively. RT-PCR (reverse transcription-PCR) analysis after differentiation of SSEA-1+ cells into derivations of three germ layers showed the pluripotency properties of isolated cells. SSEA-1+ cells were induced to differentiate along germ cell lineage by adding BMP4 (bone morphogenic factor-4) to the medium. Regarding the expression of germ cell markers (PGCs, male and female germ cell lineage), it was found that adding exogenous BMP4 to culture medium could differentiate pluripotent SSEA-1+ cells isolated from an adult tissue into gamete precursors, PGCs. Differentiated cells expressed specific molecular markers of PGCs, including Oct4, fragilis, Stella and Mvh (mouse vasa homologue). Therefore BMP4 is insufficient to induce SSEA-1+ cells derived from PGCs to develop further into late germ cells in vitro.