Multipotent Adult Germline Stem Cells Research Articles

Prediction of novel pluripotent proteins involved in reprogramming of male Germline stem cells (GSCs) into multipotent adult Germline stem cells (maGSCs) by network analysis

Germline stem cells (GSCs) are known to transmit genetic information from parents to offspring. These GSCs can undergo reprogramming to transform themselves into pluripotent stem cells, called as Multipotent adult Germline stem cells (maGSCs). The mechanism of the reprogramming of GSCs to maGSCs is elusive. To investigate novel factors that may govern the process of reprogramming, the RNA-seq data of both GSCs and maGSCs were retrieved and subjected to Tuxedo protocol using Galaxy server. Total 1558 differentially expressed genes were identified from the analysis. Protein sequence in the FASTA format of all 1558 differentially expressed genes was retrieved and submitted to Pluripred web server to predict whether the proteins were pluripotent or not. A total of 232 proteins were predicted as pluripotent, and to identify the novel proteins, these were submitted to STRING database to obtain an interaction map. The obtained interaction map was submitted to Cytoscape, and various apps such as MCODE and Centiscape were used to identify the clusters and centrality measures between the nodes of the generated network. Five clusters were identified and ranked according to their score. Novel pluripotent proteins like cadherin related cdh5, cdh10 were predicted. Phox2b, Nrp2, Dll1, Shh, Gbx2, Nodal, Lefty1, Wnt7b, Pitx2, fgf4, Pou5f1, Nanog, Tet1, trim8, alx2, Dppa2, Prdm14,Sox11, Esrrb were predicted to be involved in the stem cell development. Dppa2, Sox11, Sox2, Bmp4, Shh, and Otp were predicted to be involved in positive regulation of the stem cell proliferation. Pathway analysis further revealed that signaling pathways such as Wnt, Jak-Stat and PI3K may play important role in the pluripotency of the maGSCs. Novel proteins involved in pluripotency, which were predicted by our findings, can be experimentally researched in future.

The Tumorigenicity of Multipotent Adult Germline Stem Cells Transplanted into the Heart Is Affected by Natural Killer Cells and by Cyclosporine A Independent of Its Immunosuppressive Effects

Transplantation of stem cells represents an upcoming therapy for many degenerative diseases. For clinical use, transplantation of pluripotent stem cell-derived cells should lead to integration of functional grafts without immune rejection or teratoma formation. Our previous studies showed that the risk of teratoma formation is highly influenced by the immune system of the recipients. In this study, we have observed a higher teratoma formation rate when undifferentiated so-called multipotent adult germline stem cells (maGSCs) were transplanted into the heart of T, B, and natural killer (NK) cell-deficient RAG2−/−γc−/− mice than in RAG2−/− mice, which still have NK cells. Notably, in both strains, the teratoma formation rate was significantly reduced by the immunosuppressive drug cyclosporine A (CsA). Thus, CsA had a profound effect on teratoma formation independent of its immunosuppressive effects. The transplantation into RAG2−/− mice led to an activation of NK cells, which reached the maximum 14 days after transplantation and was not affected by CsA. The in vivo-activated NK cells efficiently killed YAC-1 and also maGSC target cells. This NK cell activation was confirmed in C57BL/6 wild-type mice whether treated with CsA or not. Sham operations in wild-type mice indicated that the inflammatory response to open heart surgery rather than the transplantation of maGSCs activated the NK cell system. An activation of NK cells during the transplantation of stem cell-derived in vitro differentiated grafts might be clinically beneficial by reducing the risk of teratoma formation by residual pluripotent cells.

Impact of the antiproliferative agent ciclopirox olamine treatment on stem cells proteome

To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells. Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA), which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics. Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We could highlight, that CPX treatment of stem cells, with subsequent proliferation inhibition, resulted in an alteration of the expression of 56 proteins compared to non-treated cells, and 54 proteins compared to RA treated cells. Bioinformatics analysis of the regulated proteins demonstrated their involvement in various biological processes. To our interest, a number of proteins have potential roles in the regulation of cell proliferation either directly or indirectly. Furthermore the classification of the altered polypeptides according to their main known/postulated functions revealed that the majority of these proteins are involved in molecular functions like nucleotide binding and metal ion binding, and biological processes like nucleotide biosynthetic processes, gene expression, embryonic development, regulation of transcription, cell cycle processes, RNA and mRNA processing. Proteins, which are involved in nucleotide biosynthetic process and proteolysis, were downregulated in CPX treated cells compared to control, as well as in RA treated cells, which may explain the cell cycle arrest. Moreover, proteins which were involved in cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis, and phosphorylation were downregulated in RA treated cells compared to control and CPX treated cells. The CPX treatment of SCs results in downregulation of nucleotide binding proteins and leads to cell cycle stop without impairment of pluripotency.

Apoptosis-Related Gene Expression Profiles of Mouse ESCs and maGSCs: Role of Fgf4 and Mnda in Pluripotent Cell Responses to Genotoxicity

Stem cells in the developing embryo proliferate and differentiate while maintaining genomic integrity, failure of which may lead to accumulation of mutations and subsequent damage to the embryo. Embryonic stem cells (ESCs), the in vitro counterpart of embryo stem cells are highly sensitive to genotoxic stress. Defective ESCs undergo either efficient DNA damage repair or apoptosis, thus maintaining genomic integrity. However, the genotoxicity- and apoptosis-related processes in germ-line derived pluripotent cells, multipotent adult germ-line stem cells (maGSCs), are currently unknown. Here, we analyzed the expression of apoptosis-related genes using OligoGEArray in undifferentiated maGSCs and ESCs and identified a similar set of genes expressed in both cell types. We detected the expression of intrinsic, but not extrinsic, apoptotic pathway genes in both cell types. Further, we found that apoptosis-related gene expression patterns of differentiated ESCs and maGSCs are identical to each other. Comparative analysis revealed that several pro- and anti-apoptotic genes are expressed specifically in pluripotent cells, but markedly downregulated in the differentiated counterparts of these cells. Activation of the intrinsic apoptotic pathway cause approximately ∼35% of both ESCs and maGSCs to adopt an early-apoptotic phenotype. Moreover, we performed transcriptome studies using early-apoptotic cells to identify novel pluripotency- and apoptosis-related genes. From these transcriptome studies, we selected Fgf4 (Fibroblast growth factor 4) and Mnda (Myeloid cell nuclear differentiating antigen), which are highly downregulated in early-apoptotic cells, as novel candidates and analyzed their roles in apoptosis and genotoxicity responses in ESCs. Collectively, our results show the existence of common molecular mechanisms for maintaining the pristine stem cell pool of both ESCs and maGSCs.

Generation and Characterization of Yeast Two-Hybrid cDNA Libraries Derived From Two Distinct Mouse Pluripotent Cell Types

Pluripotent stem cells have the therapeutic potential in future regenerative medicine applications. Therefore, it is highly important to understand the molecular mechanisms governing the pluripotency and differentiation potential of these cells. Our current knowledge of pluripotent cells is largely limited owing to the candidate gene/protein approach rather than studying the complex interactions of the proteins. Experimentally, yeast two-hybrid system (Y2H) is by far the most useful and widely used method to detect the protein–protein interactions in high-throughput screenings. Unfortunately, currently there is no GAL4-based pluripotent stem cell-specific cDNA library available for screening the interaction proteins impeding the large-scale studies. In this study, we report the construction of Y2H cDNA libraries derived from mouse pluripotent embryonic stem cells (ESCs) and multipotent adult germ-line stem cells (maGSCs) in GAL4-based Y2H vector system with very high transformation efficiency. Furthermore, we have constructed two different baits and screened for interaction partners in an effort to characterize the libraries and also as a part of our ongoing studies. Consequently, many putative interaction proteins were identified in both cases and their interaction was further validated by direct-Y2H. The observed interactions between bait proteins and their respective analyzed putative interaction proteins were further confirmed using two independent approaches in mammalian cells, thus highlighting the biological significance of the identified interactor (s). Finally, we would like to make these cDNA libraries as a resource that can be distributed to the research community.

Differentiation of Multipotent Adult Germline Stem Cells Derived from Mouse Testis into Functional Endothelial Cells

Pluripotent stem cells hold great promise for the treatment of cardiovascular disease. We previously described multipotent adult germline stem cells (maGSCs) from mouse testis with differentiation potential similar to embryonic stem cells. The aim of this work was to differentiate maGSCs into functional endothelial cells and to study their potential for vasculogenesis. MaGSCs were cocultivated with OP9 stromal cells to induce differentiation into cardiovascular progenitors, i.e. fetal liver kinase 1-positive (Flk-1⁺) cells. Five days later, Flk-1⁺ cells were separated using fluorescence-activated cell sorting, followed by cultivation on collagen type IV under endothelial differentiation conditions. At different time points, maGSC-derived endothelial-like cells were characterized using RT-PCR, flow cytometry, immunofluorescence and functional assays. Cultivation of Flk-1⁺ cells resulted in the progressive upregulation of endothelial cell markers, including VE-cadherin, von Willebrand factor and endothelial nitric oxide synthase. Moreover, Flk-1⁺ maGSC-derived endothelial-like cells were able to branch and form networks in vitro and promoted functional blood vessel formation in vivo. Importantly, Flk-1⁺ cells retained their potential to proliferate and could be continuously expanded, while the ability of contact inhibition was preserved. Thus, maGSCs may provide a useful source of endothelial-like cells to study the basic mechanisms of vasculogenesis or endothelial differentiation.

Differential Genomic Imprinting and Expression of Imprinted microRNAs in Testes-Derived Male Germ-Line Stem Cells in Mouse

BackgroundTestis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings.Methods and FindingsUsing mouse as a model system, here we show that, similar to sperm, expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher (P<0.001), while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower (P<0.001) in GS cells than in control embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA.ConclusionsOur data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.

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.

302 IMPRINTED microRNA ARE DIFFERENTIALLY EXPRESSED IN ADULT MOUSE TESTES-DERIVED MALE GERM-LINE STEM CELLS

Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells, can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which, upon testicular transplantation, produce teratomas instead of initiating spermatogenesis. This study evaluated the DNA methylation and expression of imprinted microRNA (miRNA) in mouse GS and maGS cells. The GS and maGS cell lines were established essentially as described earlier (Jung et al. 2010 Mol. Hum. Reprod. PMID: 20610616) and were quantified for maternally (miR-296-3p, miR-296-5p, miR-483) and paternally (miR-127, miR-127-5p) imprinted miRNA by real-time TaqMan® MicroRNA assay and for DNA methylation at imprinting control regions of respective miRNA (Gnas-Nespas DMR, Igf2-H19 ICR, and Dlk1-Dio3 IG-DMR) by bisulfite genomic sequencing. Sperm and embryonic stem (ES) cells were used as controls for comparison. Results showed that, similar to sperm, expression of maternally imprinted miRNA was consistently higher (P &lt; 0.001), whereas that of paternally imprinted miRNA was consistently lower (P &lt; 0.001) in GS cells than in control ES cells. The DNA methylation analyses further confirmed that imprinted miRNA were androgenetic in GS cells. On the other hand, DNA methylation of maGS cells resembled that of ES cells, but the expression pattern of imprinted miRNA was intermediate between that of GS cells and ES cells. The expression of imprinted miRNA in GS and maGS cells was also altered during their in vitro differentiation but varied with both the differentiation stage and the miRNA. In conclusion, our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNA which changes to an ES cell-like pattern upon their conversion to maGS cells and, therefore, may serve as an epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells. This work was supported by a grant (Code #200901FHT010305191) from BioGreen 21 Program, RDA, Republic of Korea.

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.

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.

Pluripotent embryonic stem cells and multipotent adult germline stem cells reveal similar transcriptomes including pluripotency-related genes

DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs).

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.

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.

Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W v mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

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.

H19 gene is epigenetically stable in mouse multipotent germline stem cells.

Testis-derived germline stem (GS) cells can undergo re-programming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.

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.

Isolation and cultivation of stem cells from adult mouse testes

The successful isolation and cultivation of spermatogonial stem cells (SSCs) as well as induction of SSCs into pluripotent stem cells will allow us to study their biological characteristics and their applications in therapeutic approaches. Here we provide step-by-step procedures on the basis of previous work in our laboratory for: the isolation of testicular cells from adolescent mice by a modified enzymatic procedure; the enrichment of undifferentiated spermatogonia by laminin selection or genetic selection using Stra8-EGFP (enhanced green fluorescent protein) transgenic mice; the cultivation and conversion of undifferentiated spermatogonia into embryonic stem-like cells, so-called multipotent adult germline stem cells (maGSCs); and characterization of these cells. Normally, it will take about 16 weeks to obtain stable maGSC lines starting from the isolation of testicular cells.

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

BackgroundMultipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs).ResultsWe have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide.ConclusionPluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells.ReviewersThis article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter.