Pelota mediates gonocyte maturation and maintenance of spermatogonial stem cells in mouse testes.

Pelota (Pelo) is an evolutionarily conserved gene, and its deficiency in Drosophila affects both male and female fertility. In mice, genetic ablation of Pelo leads to embryonic lethality at the early implantation stage as a result of the impaired development of extra-embryonic endoderm (ExEn). To define the consequences of Pelo deletion on male germ cells, we temporally induced deletion of the gene at both embryonic and postnatal stages. Deletion of Pelo in adult mice resulted in a complete loss of whole-germ cell lineages after 45 days of deletion. The absence of newly emerging spermatogenic cycles in mutants confirmed that spermatogonial stem cells (SSCs) were unable to maintain spermatogenesis in the absence of PELO protein. However, germ cells beyond the undifferentiated SSC stage were capable of completing spermatogenesis and producing spermatozoa, even in the absence of PELO. Following the deletion of Pelo during embryonic development, we found that although PELO is dispensable for maintaining gonocytes, it is necessary for the transition of gonocytes to SSCs. Immunohistological and protein analyses revealed the attenuation of FOXO1 transcriptional activity, which induces the expression of many SSC self-renewal genes. The decreased transcriptional activity of FOXO1 in mutant testes was due to enhanced activity of the PI3K/AKT signaling pathway, which led to phosphorylation and cytoplasmic sequestration of FOXO1. These results suggest that PELO negatively regulates the PI3K/AKT pathway and that the enhanced activity of PI3K/AKT and subsequent FOXO1 inhibition are responsible for the impaired development of SSCs in mutant testes.

Mouse Dazl and its novel splice variant functions in translational repression of target mRNAs in embryonic stem cells.

Dazl (deleted in azoospermia-like) is an RNA binding protein that is important for germ cell differentiation in vertebrates. In the present study, we report the identification of a novel Dazl isoform (Dazl_Δ8) that results from alternative splicing of exon8 of mouse Dazl. We observed the expression of Dazl_Δ8 in various pluripotent cell types, but not in somatic cells. Furthermore, the Dazl_Δ8 splice variant was expressed along with the full-length isoform of Dazl (Dazl_FL) throughout male germ-cell development and in the ovary. Sub-cellular localization studies of Dazl_Δ8 revealed a diffused cytoplasmic and large granular pattern, which is similar to the localization patterns of Dazl_FL protein. In contrast to the well documented translation stimulation function in germ cells, overexpression and downregulation studies of Dazl isoforms (Dazl_FL and Dazl_Δ8) revealed a role for Dazl in the negative translational regulation of Mvh, a known target of Dazl, as well as Oct3/4 and Sox2 in embryonic stem cells (ESCs). In line with these observations, a luciferase reporter assay with the 3'UTRs of Oct3/4 and Mvh confirmed the translational repressive role of Dazl isoforms in ESCs but not in germ cells derived cell line GC-1. Further, we identified several putative target mRNAs of Dazl_FL and Dazl_Δ8 in ESCs through RNA-binding immunoprecipitation followed by whole genome transcriptome analysis. Collectively, our results show a translation repression function of Dazl in pluripotent stem cells.

Embryo implantation failure and other reproductive defects in Ube2q1-deficient female mice.

The ubiquitination process is indispensable for proteome regulation. Three classes of ubiquitin (Ub)-related proteins can be distinguished: E1, E2 and E3. Proteins from the E2 class are responsible for the transfer of Ubls from E1 to the target protein. For this activity, interaction with class E3 ligases is usually required. Ub-conjugating enzyme E2Q 1 (UBE2Q1) belongs to the E2 class of Ub-related enzymes and is demonstrated to be involved in the regulation of membrane B4GALT1 protein. Here, we demonstrate that human UBE2Q1 and mouse Ube2q1 are widely expressed and highly conserved genes. To elucidate the function of UBE2Q1 protein, we generated knockout mouse model. No overt phenotype was detected in UBE2Q1-deficient males, but in mutant females, pleiotropic reproductive defects were observed including altered oestrus cycle, abnormal sexual behaviour and reduced offspring care. Moreover, in the uterus of mutant females, significantly increased embryonic lethality and decreased implantation capacity of homozygous mutant embryos were noticed. We found that Ube2q1 is not expressed in the uterus of non-pregnant females but its expression is up-regulated during pregnancy. Taken together, Ube2q1 is involved in different aspects of female fertility.

MicroRNA signature in various cell types of mouse spermatogenesis: evidence for stage-specifically expressed miRNA-221, -203 and -34b-5p mediated spermatogenesis regulation.

BACKGROUND INFORMATION: Recently, it became apparent that microRNAs (miRNAs) can regulate gene expression post-transcriptionally. Despite the advances in identifying the testis-expressed miRNAs and their role in spermatogenesis, only few data are available showing the spatiotemporal expression of miRNAs during this process. RESULTS: To understand how different miRNAs can regulate germ cells differentiation, we generated a transgenic mouse model and purified pure populations of premeiotic (PrM) cells and primary spermatocytes (meiotic cells). We also established spermatogonial stem cell (SSC) culture using relatively simple and robust culture conditions. Comparison of global miRNA expression in these germ cell populations revealed 17 SSC-, 11 PrM- and 13 meiotic-specific miRNAs. We identified nine miRNAs as specific for both SSC and PrM cells and another nine miRNAs as specific for PrM and meiotic cells. Additionally, 45 miRNAs were identified as commonly expressed in all three cell types. Several of PrM- and meiotic-specific miRNAs were identified as exclusively/preferentially expressed in testis. We were able to identify the relevant target genes for many of these miRNAs. The luciferase reporter assays with SSC (miR-221)-, PrM (miR-203)- and meiotic (miR-34b-5p)-specific miRNAs and 3'-untranslated region constructs of their targets, c-Kit, Rbm44 and Cdk6, respectively, showed an approximately 30%-40% decrease in reporter activity. Moreover, we observed a reduced expression of endogenous proteins, c-Kit and Cdk6, when the testis-derived cell lines, GC-1 and GC-4, were transfected with miRNA mimics for miR-221 and miR-34b-5p, respectively. CONCLUSIONS: Taken together, we established the miRNA signature of SSC, PrM and meiotic cells and show evidence for their functional relevance during the process of spermatogenesis by target prediction and validation. Through our observations, we propose a working model in which the stage-specific miRNAs such as miR-221, -203 and -34b-5p coordinate the regulation of spermatogenesis.

Targeted disruption of Hspa4 gene leads to cardiac hypertrophy and fibrosis.

Failure of molecular chaperones to direct the correct folding of newly synthesized proteins leads to the accumulation of misfolded proteins in cells. HSPA4 is a member of the heat shock protein 110 family (HSP110) that acts as a nucleotide exchange factor of HSP70 chaperones. We found that the expression of HSPA4 is upregulated in murine hearts subjected to pressure overload and in failing human hearts. To investigate the cardiac function of HSPA4, Hspa4 knockout (KO) mice were generated and exhibited cardiac hypertrophy and fibrosis. Hspa4 KO hearts were characterized by a significant increase in heart weight/body weight ratio, elevated expression of hypertrophic and fibrotic gene markers, and concentric hypertrophy with preserved contractile function. In response to pressure overload, cardiac hypertrophy and remodeling were further aggravated in the Hspa4 KO compared to wild type (WT) mice. Cardiac hypertrophy in Hspa4 KO hearts was associated with enhanced activation of gp130-STAT3, CaMKII, and calcineurin-NFAT signaling. Protein blot and immunofluorescent analyses showed a significant accumulation of polyubiquitinated proteins in cardiac cells of Hspa4 KO mice. These results suggest that the myocardial remodeling of Hspa4 KO mice is due to accumulation of misfolded proteins resulting from impaired chaperone activity. Further analyses revealed a significant increase in cross sectional area of cardiomyocytes, and in expression levels of hypertrophic markers in cultured neonatal Hspa4 KO cardiomyocytes suggesting that the hypertrophy of mutant mice was a result of primary defects in cardiomyocytes. Gene expression profile in hearts of 3.5-week-old mice revealed a differentially expressed gene sets related to ion channels, muscle-specific contractile proteins and stress response. Taken together, our in vivo data demonstrate that Hspa4 gene ablation results in cardiac hypertrophy and fibrosis, possibly, through its role in protein quality control mechanism.

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.

Multipotent adult germline stem cells and embryonic stem cells functional proteomics revealed an important role of eukaryotic initiation factor 5A (Eif5a) in stem cell differentiation.

Multipotent adult germline stem cells (maGSCs) are pluripotent cells that can be differentiated into somatic cells of the three primary germ layers. To highlight the protein profile changes associated with stem cell differentiation, retinoic acid (RA) treated mouse stem cells (maGSCs and ESCs) were compared to nontreated stem cells. 2-DE and DIGE reference maps were created, and differentially expressed proteins were further processed for identification. In both stem cell types, the RA induced differentiation resulted in an alteration of 36 proteins of which 18 were down-regulated and might be potential pluripotency associated proteins, whereas the other 18 proteins were up-regulated. These might be correlated to stem cell differentiation. Surprisingly, eukaryotic initiation factor 5A (Eif5a), a protein which is essential for cell proliferation and differentiation, was significantly down-regulated under RA treatment. A time-dependent investigation of Eif5a showed that the RA treatment of stem cells resulted in a significant up-regulation of the Eif5a in the first 48 h followed by a progressive down-regulation thereafter. This effect could be blocked by the hypusination inhibitor ciclopirox olamine (CPX). The alteration of Eif5a hypusination, as confirmed by mass spectrometry, exerts an antiproliferative effect on ESCs and maGSCs in vitro, but does not affect the cell pluripotency. Our data highlights the important role of Eif5a and its hypusination for stem cell differentiation and proliferation.

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.

Global and gene-specific histone modification profiles of mouse multipotent adult germline stem cells.

We previously reported the generation of multipotent adult germline stem cells (maGSCs) from spermatogonial stem cells (SSCs) isolated from adult mouse testis. In a later study, we substantiated the pluripotency of maGSCs by demonstrating their close similarity to pluripotent male embryonic stem cells (ESCs) at the epigenetic level of global and gene-specific DNA methylation. Here, we extended the comparative epigenetic analysis of maGSCs and male ESCs by investigating the second main epigenetic modification in mammals, i.e. global and gene-specific modifications of histones (H3K4 trimethylation, H3K9 acetylation, H3K9 trimethylation and H3K27 trimethylation). Using immunofluorescence staining, flow cytometry and western blot analysis, we show that maGSCs are very similar to male ESCs with regard to global levels and nuclear distribution patterns of these modifications. Chromatin immunoprecipitation real-time PCR analysis of these modifications at the gene-specific level further revealed modification patterns of the pluripotency marker genes Oct4, Sox2 and Nanog in maGSCs that are nearly identical to those of male ESCs. These genes were enriched for activating histone modifications including H3K4me3 and H3K9ac and depleted of repressive histone modifications including H3K27me3 and H3K9me3. In addition, Hoxa11, a key regulator of early embryonic development showed the ESC-typical bivalent chromatin conformation with enrichment of both the activating H3K4me3 and the repressive H3K27me3 modification also in maGSCs. Collectively, our results demonstrate that maGSCs also closely resemble ESCs with regard to their chromatin state and further evidence their pluripotent nature.

Heat-shock protein HSPA4 is required for progression of spermatogenesis.

Heat-shock protein 110 (HSP110) family members act as nucleotide exchange factors (NEF) of mammalian and yeast HSP70 chaperones during the ATP hydrolysis cycle. In this study, we describe the expression pattern of murine HSPA4, a member of the HSP110 family, during testis development and the consequence of HSPA4 deficiency on male fertility. HSPA4 is ubiquitously expressed in all the examined tissues. During prenatal and postnatal development of gonad, HSPA4 is expressed in both somatic and germ cells; however, expression was much higher in germ cells of prenatal gonads. Analyses of Hspa4-deficient mice revealed that all homozygous mice on the hybrid C57BL/6J×129/Sv genetic background were apparently healthy. Although HSPA4 is expressed as early as E13.5 in male gonad, a lack of histological differences between Hspa4(-/-) and control littermates suggests that Hspa4 deficiency does not impair the gonocytes or their development to spermatogonia. Remarkably, an increased number of the Hspa4-deficient males displayed impaired fertility, whereas females were fertile. The total number of spermatozoa and their motility were drastically reduced in infertile Hspa4-deficient mice compared with wild-type littermates. The majority of pachytene spermatocytes in the juvenile Hspa4(-/-) mice failed to complete the first meiotic prophase and became apoptotic. Furthermore, down-regulation of transcription levels of genes known to be expressed in spermatocytes at late stages of prophase I and post-meiotic spermatids leads to suggest that the development of most spermatogenic cells is arrested at late stages of meiotic prophase I. These results provide evidence that HSPA4 is required for normal spermatogenesis.

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells.

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

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.

Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton.

BACKGROUND: Pelota (PELO) is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation. RESULTS: We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC), we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments. CONCLUSION: We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.

Embryonic stem cell-related miRNAs are involved in differentiation of pluripotent cells originating from the germ line.

Cells originating from the germ cell lineage retain the remarkable property under special culture conditions to give rise to cells with embryonic stem cell (ESC) properties, such as the multipotent adult germline stem cells (maGSCs) derived from adult mouse testis. To get an insight into the mechanisms that control pluripotency and differentiation in these cells, we studied how differences observed during in vitro differentiation between ESCs and maGSCs are associated with differences at the level of microRNAs (miRNAs). In this work, we provide for a first time a connection between germ cell origin of maGSCs and their specific miRNA expression profile. We found that maGSCs express higher levels of germ cell markers characteristic for primordial germ cells (PGCs) and spermatogonia compared with ESCs. Retained expression of miR-290 cluster has been previously reported in maGSCs during differentiation and it was associated with higher Oct-4 levels. Here, we show that this property is also shared by another pluripotent cell line originating from the germ line, the embryonic germ cells. In addition, we provide proof that the specific miRNA expression profile of maGSCs has an impact on their differentiation potential. Low levels of miR-302 in maGSCs during the first 10 days of leukaemia inhibitory factor deprivation are shown to be necessary for the maintenance of high levels of early germ cell markers.

Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia.

To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3(-/-) animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3(-/-) males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3(-/-) males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

Members of the miR-290 cluster modulate in vitro differentiation of mouse embryonic stem cells.

We report the biological effects of miR-290 cluster via gain-of-function or loss-of-function experiments in mouse embryonic stem cells (ESCs) cultured under differentiation conditions. Under these conditions we found that overexpression of miR-290 cluster in ESCs cannot prevent downregulation of Oct-4, but inhibition results in earlier downregulation of Oct-4 compared with the negative control. In consistence with previous findings that report ectopic expression of Brachyury during gastrulation in Argonaute-2 KO mice due to impaired miRNA function, we show that miR-290 cluster regulates negatively differentiation of ESCs towards mesodermal and germ cell lineage. These results suggest that although incapable to maintain pluripotent state alone, miR-290 cluster inhibits ESC differentiation and it is involved in the pathways controlling mesoderm and primordial germ cell differentiation. Finally, we provide proofs that members of this cluster target Dkk-1 gene, a Wnt pathway inhibitor, and affect this pathway, which can partially explain why miR-290 cluster favours pluripotency against differentiation.

Multipotent adult germline stem cells and embryonic stem cells: comparative proteomic approach.

Spermatogonial stem cells isolated from the adult mouse testis acquire under certain culture conditions pluripotency and become so-called multipotent adult germline stem cells (maGSCs). They can be differentiated into somatic cells of the three germ layers. We investigated a subset of the maGSCs and ESCs proteomes using cell lines derived from two different mouse strains, narrow range immobilized pH gradients to favor the detection of less abundant proteins, and DIGE to ensure confident comparison between the two cell types. 2-D reference maps of maGSCs and ESCs in the pI ranges 3-6 and 5-8 were created, and protein entities were further processed for protein identification. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 409 proteins was identified, corresponding to a library of 166 nonredundant stem cell-associated proteins. The identified proteins were classified according to their main known/postulated functions using bioinformatics. Furthermore, we used DIGE to highlight the ESC-like nature of maGSCs on the proteome scale. We concluded that the proteome of maGSCs is highly similar to that of ESCs as we could identify only a small subset of 18 proteins to be differentially expressed between the two cell types. Moreover, comparative analysis of the cell line proteomes from two different mouse strains showed that the interindividual differences in maGSCs proteomes are minimal. With our study, we created for the first time a proteomic map for maGSCs and compared it to the ESCs proteome from the same mouse. We confirmed on the proteome level the ESC-like nature of maGSCs.

Comparative methylation profiles and telomerase biology of mouse multipotent adult germline stem cells and embryonic stem cells.

Recently, several groups described the isolation of mouse spermatogonial stem cells (SSCs) and their potential to develop to embryonic stem cell (ESC)-like cells, so-called multipotent germline stem cells (mGSCs). We were the first to derive such mGSCs from SSCs isolated from adult mouse testis and, therefore, called these mGSCs multipotent adult germline stem cells (maGSCs). Here, we comparatively analyzed gene-specific and global DNA methylation profiles as well as the telomerase biology of several maGSC and male ESC lines. We show that undifferentiated maGSCs are very similar to undifferentiated male ESCs with regard to global DNA methylation, methylation of pluripotency marker gene loci, telomerase activity and telomere length. Imprinted gene methylation levels were generally lower in undifferentiated maGSCs than in undifferentiated male ESCs, but, compared with undifferentiated mGSCs derived by other groups, more similar to those of male ESCs. Differentiation of maGSCs increased the methylation of three of the four analyzed imprinted genes to almost somatic methylation patterns, but dramatically decreased global DNA methylation. Our findings further substantiate the pluripotency of maGSCs and their potential for regenerative medicine.

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.