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.

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.

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.

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.

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.

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.

Pluripotency of spermatogonial stem cells from adult mouse testis.

Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.

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.

In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice.

Male gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed to adult mice. This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.

PSCDGs of mouse multipotent adult germline stem cells can enter and progress through meiosis to form haploid male germ cells in vitro.

Spermatogonial stem cells (SSCs) provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and differentiation into sperm. SSC-derived cell lines called multipotent adult germline stem cells (maGSCs) were recently shown to be pluripotent and to have the same potential as embryonic stem cells (ESCs). In a differentiation protocol using retinoic acid (RA) and based on a double selection strategy, we have shown that ESCs are able to undergo meiosis and produce haploid male germ cells in vitro. Using this differentiation protocol we have now succeeded to generate haploid male germ cells from maGSCs in vitro. maGSCs derived from a Stra8-EGFP transgenic mouse line were differentiated into stable spermatogonial stages and further cultured. These cells were transfected with a postmeiotic specific promoter construct Prm1-DsRed to monitor retinoic acid (RA) induced differentiation into haploid male gametes. Our protocol is another approach for the production of pluripotent stem cell derived gametes (PSCDGs) and is an alternative for the investigation of mammalian spermatogenesis, germ line gene modification and epigenetic reprogramming. If reproducible with pluripotent cell lines derived from human SSCs, it could also be used as a therapeutic approach for the treatment of male infertility.

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.

Haploinsufficiency of the germ cell-specific nuclear RNA binding protein hnRNP G-T prevents functional spermatogenesis in the mouse.

Human HNRNPGT, encoding the protein hnRNP G-T, is one of several autosomal retrogenes derived from RBMX. It has been suggested that HNRNPGT functionally replaces the sex-linked RBMX and RBMY genes during male meiosis. We show here that during normal mouse germ cell development, hnRNP G-T protein is strongly expressed during and after meiosis when proteins expressed from Rbmx or Rbmx-like genes are absent. Amongst these Rbmx-like genes, DNA sequence analyses indicate that two other mouse autosomal Rbmx-derived retrogenes have evolved recently in rodents and one already shows signs of degenerating into a non-expressed pseudogene. In contrast, orthologues of Hnrnpgt are present in all four major groups of placental mammals. The sequence of Hnrnpgt is under considerable positive selection suggesting it performs an important germ cell function in eutherians. To test this, we inactivated Hnrnpgt in ES cells and studied its function during spermatogenesis in chimaeric mice. Although germ cells heterozygous for this targeted allele could produce sperm, they did not contribute to the next generation. Chimaeric mice with a high level of mutant germ cells were infertile with low sperm counts and a high frequency of degenerate seminiferous tubules and abnormal sperm. Chimaeras made from a 1:1 mix of targeted and wild-type ES cell clones transmitted wild-type germ cells only. Our data show that haploinsufficiency of Hnrnpgt results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of HNRNPGT could, therefore, be a cause of male infertility in humans.

Adult germ line stem cells as a source of functional neurons and glia.

The derivation of autologous pluripotent cells has become a central goal in translational stem cell research. Although somatic cell nuclear transfer and transcription factor-based reprogramming enable the generation of pluripotent cells from adult tissue, both methodologies depend on complex epigenetic alterations. Recent data suggest that the adult germ line may represent an alternative and natural source of pluripotent stem cells. Multipotent adult germ line stem cells (maGSCs) with properties similar to those of embryonic stem cells have been derived from mouse spermatogonial stem cells. These cells exhibit extensive self-renewal, expression of pluripotency markers, and differentiation into derivatives of all three germ layers. Here we report the derivation of multipotent neural and glial precursors as well as adherently proliferating neural stem cells from maGSCs. Characterization of maGSC-derived neurons revealed segregation into GABAergic, glutamatergic, serotonergic, and tyrosine hydroxylase-positive phenotypes. On a functional level, maGSC-derived neurons generate spontaneously active functional networks, which use both glutamatergic and GABAergic synaptic transmission and engage in synchronized oscillatory activity. maGSC-derived oligodendrocytes undergo full maturation and ensheathe host axons in myelin-deficient tissue. Our data suggest that neural stem and precursor cells derived from maGSCs could provide a versatile and potentially autologous source of functional neurons and glia.

Generation of functional cardiomyocytes from adult mouse spermatogonial stem cells.

Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into cells from all 3 germ layers. In the present study, we used the hanging drop method to differentiate maGSCs into cardiomyocytes and analyzed their functional properties. Differentiation efficiency of beating cardiomyocytes from maGSCs was similar to that from ESCs. The maGSC-derived cardiomyocytes expressed cardiac-specific L-type Ca(2+) channels and responded to Ca(2+) channel-modulating drugs. Cx43 was expressed at cell-to-cell contacts in cardiac clusters, and fluorescence recovery after photobleaching assay showed the presence of functional gap junctions among cardiomyocytes. Action potential analyses demonstrated the presence of pacemaker-, ventricle-, atrial-, and Purkinje-like cardiomyocytes. Stimulation with isoproterenol resulted in a significant increase in beating frequency, whereas the addition of cadmium chloride abolished spontaneous electrical activity. Confocal microscopy analysis of intracellular Ca(2+) in maGSC-derived cardiomyocytes showed that calcium increased periodically throughout the cell in a homogenous fashion, pointing to a fine regulated Ca(2+) release from intracellular Ca(2+) stores. By using line-scan mode, we found rhythmic Ca(2+) transients. Furthermore, we transplanted maGSCs into normal hearts of mice and found that maGSCs were able to proliferate and differentiate. No tumor formation was found up to 1 month after cell transplantation. Taken together, we believe that maGSCs provide a new source of distinct types of cardiomyocytes for basic research and potential therapeutic application.

Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles.

Spermatogonial stem cells (SSCs) isolated from the adult mouse testis and cultured have been shown to respond to culture conditions and become pluripotent, so called multipotent adult germline stem cells (maGSCs). microRNAs (miRNAs) belonging to the 290 and 302 miRNA clusters have been previously classified as embryonic stem cell (ESC) specific. Here, we show that these miRNAs generally characterize pluripotent cells. They are expressed not only in ESCs but also in maGSCs as well as in the F9 embryonic carcinoma cell (ECC) line. In addition, we tested the time-dependent influence of different factors that promote loss of pluripotency on levels of these miRNAs in all three pluripotent cell types. Despite the differences regarding time and extent of differentiation observed between ESCs and maGSCs, expression profiles of both miRNA families showed similarities between these two cell types, suggesting similar underlying mechanisms in maintenance of pluripotency and differentiation. Our results indicate that the 290-miRNA family is connected with Oct-4 and maintenance of the pluripotent state. In contrast, members of the 302-miRNA family are induced during first stages of in vitro differentiation in all cell types tested. Therefore, detection of miRNAs of miR-302 family in pluripotent cells can be attributed to the proportion of spontaneously differentiating cells in cultures of pluripotent cells. These results are consistent with ESC-like nature of maGSCs and their potential as an alternative source of pluripotent cells from non-embryonic tissues.