The Experts below are selected from a list of 8052 Experts worldwide ranked by ideXlab platform
Ralph L Brinster - One of the best experts on this subject based on the ideXlab platform.
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Isolation of the Spermatogonial Stem Cell-Containing Fraction from Testes.
Cold Spring Harbor protocols, 2017Co-Authors: Shaun M. Goodyear, Ralph L BrinsterAbstract:Abstract A testis Cell fraction enriched for Spermatogonial Stem Cells (SSCs) compared with the unfractionated total testis Cell population can be isolated from testes after birth and adult stages. This protocol involves dissociating the seminiferous tubules into a single-Cell suspension, using enzymes and mechanical disruption. This is followed by the selection of the Thy1(+) Cell fraction that contains nearly the entire SSC population and is enriched for these Cells compared with the total testis Cell population. This isolated fraction can be used for molecular studies, transfer into culture to establish primary cultures, or transplantation into the testes of recipients for spermatogenesis. Typically, if the SSCs are destined for transplantation, a marker is included to visualize the clones of spermatogenesis (e.g., lacZ or green fluorescent protein [GFP] transgenes). It is important to note that the isolated Thy1(+) Cell fraction does not consist of pure SSCs and that most germ Cells are non-Stem-Cell progenitors, and a smaller portion of the population is somatic Cells.
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Spermatogonial Stem Cell Transplantation to the Testis.
Cold Spring Harbor protocols, 2017Co-Authors: Shaun M. Goodyear, Ralph L BrinsterAbstract:Spermatogonial Stem Cells (SSCs) are located in the basal region of the seminiferous tubules and provide a self-renewing reservoir from which progenitor spermatogonia arise and transiently amplify in number before transition to a differentiating state. SSC transplantation involves grafting mouse testicular Cells into the seminiferous tubules of an aspermic recipient testis that is generated either by chemical (e.g., busulfan) treatment or genetic mutations.
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Spermatogonial Stem Cell self renewal requires etv5 mediated downstream activation of brachyury in mice
Biology of Reproduction, 2011Co-Authors: Shaun M. Goodyear, Mary R Avarbock, John W Tobias, Ralph L BrinsterAbstract:Insight regarding mechanisms controlling gene expression in the Spermatogonial Stem Cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In the present study, we explored the global gene expression profiles of the glial Cell line-derived neurotrophic factor (GDNF)-regulated transcription factors Ets (E-twenty-six) variant gene 5 (Etv5); B-Cell chronic lymphocytic leukemia (CLL)/lymphoma 6, member B (Bcl6b); and POU domain, class-3 transcription factor 1 (Pou3f1). We reasoned that these three factors may function as a core set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient siRNA oligonucleotides to individually target Etv5, Bcl6b, and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. Only modest overlaps in the target genes regulated by the three factors were noted, but ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes, including Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury (T) and CXC chemokine receptor, type 4 (Cxcr4), and we showed that ETV5 binding to the Brachyury (T) gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury (T) significantly reduced the number of donor Cell-derived colonies formed within recipient mouse testes. These results suggest Brachyury is of biological importance and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro.
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glial Cell line derived neurotrophic factor and endothelial Cells promote self renewal of rabbit germ Cells with Spermatogonial Stem Cell properties
The FASEB Journal, 2011Co-Authors: Hiroshi Kubota, Mary R Avarbock, Shaun M. Goodyear, Ralph L BrinsterAbstract:Previous studies suggest that exogenous factors crucial for Spermatogonial Stem Cell (SSC) self-renewal are conserved among several mammalian species. Since glial Cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ Cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as Cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ Cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial Cell (C166) feeder layers. Proliferation of rabbit germ Cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ Cells colonized seminiferous tubules of immunodeficient mice, proliferated for at le...
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in vivo and in vitro aging is detrimental to mouse Spermatogonial Stem Cell function
Biology of Reproduction, 2011Co-Authors: Jonathan A. Schmidt, Mary R Avarbock, Hiroshi Kubota, Lara K Abramowitz, Zhiyv Niu, John W Tobias, Marisa S Bartolomei, Ralph L BrinsterAbstract:The development of techniques to maintain the Spermatogonial Stem Cell (SSC) in vivo and in vitro for extended periods essentially allows for the indefinite continuation of an individual germline. Recent evidence indicates that the aging of male reproductive function is due to failure of the SSC niche. SSCs are routinely cultured for 6 mo, and no apparent effect of culture over this period has been observed. To determine the effects of SSC aging, we utilized an in vitro culture syStem, followed by quantitative transplantation experiments. After culture for 6 mo, SSCs that had been aged in vivo for 1500 days had a slower proliferation rate than SSCs that were aged in vivo to 8 or 300 days. Examination of methylation patterns revealed no apparent difference in DNA methylation between SSCs that were aged 8, 300, or 1500 days before culture. Long-term culture periods resulted in a loss of Stem Cell potential without an obvious change in the visual appearance of the culture. DNA microarray analysis of in vivo- and in vitro-aged SSCs identified the differential expression of several genes important for SSC function, including B-Cell CLL/lymphoma 6, member B (Bcl6b), Lim homeobox protein 1 (Lhx1), and thymus Cell antigen 1, theta (Thy1). Collectively, these data indicate that, although both in vitro and in vivo aging are detrimental to SSC function, in vitro aging results in greater loss of function, potentially due to a decrease in core SSC self-renewal gene expression and an increase in germ Cell differentiation gene expression.
Karim Nayernia - One of the best experts on this subject based on the ideXlab platform.
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Stem Cell protein Piwil2 modulates expression of murine Spermatogonial Stem Cell expressed genes
Molecular Reproduction and Development, 2006Co-Authors: Wolfgang Engel, Karim NayerniaAbstract:The piwi family genes are highly conserved during evolution and play essential roles in Stem Cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ Cells of adult testis, suggesting that this gene functions in Spermatogonial Stem Cell self-renewal. In order to find molecular mechanisms underlying Stem Cell activity mediated by Piwil2 gene, an in vitro gain of function Cell culture model was established. Messenger RNAs isolated from Cells expressing Piwil2 and mRNAs isolated from Cells without Piwil2 expression were compared using a Stem Cell array technique. It was shown that Piwil2 modulates expression of Stem Cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and Spermatogonial Cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in Stem Cells proliferation (Pdgfrb), energy metabolism (Slc2a1), Cell adhesion, Cell–Cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ Cell differentiation (Stra8). The expression of these markers in Spermatogonial Stem Cells and other nongerminal Stem Cells suggests that these Cells share elements of common molecular machinery with Stem Cells in other tissues which are modulated by Stem Cell protein Piwil2. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.
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Stem Cell protein piwil2 modulates expression of murine Spermatogonial Stem Cell expressed genes
Molecular Reproduction and Development, 2006Co-Authors: Jae Ho Lee, Wolfgang Engel, Karim NayerniaAbstract:The piwi family genes are highly conserved during evolution and play essential roles in Stem Cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ Cells of adult testis, suggesting that this gene functions in Spermatogonial Stem Cell self-renewal. In order to find molecular mechanisms underlying Stem Cell activity mediated by Piwil2 gene, an in vitro gain of function Cell culture model was established. Messenger RNAs isolated from Cells expressing Piwil2 and mRNAs isolated from Cells without Piwil2 expression were compared using a Stem Cell array technique. It was shown that Piwil2 modulates expression of Stem Cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and Spermatogonial Cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in Stem Cells proliferation (Pdgfrb), energy metabolism (Slc2a1), Cell adhesion, Cell-Cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ Cell differentiation (Stra8). The expression of these markers in Spermatogonial Stem Cells and other nongerminal Stem Cells suggests that these Cells share elements of common molecular machinery with Stem Cells in other tissues which are modulated by Stem Cell protein Piwil2.
Jon M Oatley - One of the best experts on this subject based on the ideXlab platform.
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Spermatogonial Stem Cell transplantation insights and outlook for domestic animals
Annual Review of Animal Biosciences, 2019Co-Authors: Mariana Ianello Giassetti, Michela Ciccarelli, Jon M OatleyAbstract:The demand for food will increase to an unprecedented level over the next 30 years owing to human population expansion, thus necessitating an evolution that improves the efficiency of livestock production. Genetic gain to improve production traits of domestic animal populations is most effectively achieved via selective use of gametes from animals deemed to be elite, and this principle has been the basis of selective breeding strategies employed by humans for thousands of years. In modern-day animal agriculture, artificial insemination (AI) has been the staple of selective breeding programs, but it has inherent limitations for applications in beef cattle and pig production syStems. In this review, we discuss the potential and current state of development for a concept termed Surrogate Sires as a next-generation breeding tool in livestock production. The scheme capitalizes on the capacity of Spermatogonial Stem Cells to regenerate sperm production after isolation from donor testicular tissue and transfer into the testes of a recipient male that lacks endogenous germline, thereby allowing the surrogate male to produce offspring with the donor haplotype via natural mating. This concept provides an effective selective breeding tool to achieve genetic gain that is conducive for livestock production syStems in which AI is difficult to implement.
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Functional assessment of Spermatogonial Stem Cell purity in experimental Cell populations
Elsevier, 2018Co-Authors: Tessa Lord, Jon M OatleyAbstract:Historically, research in Spermatogonial biology has been hindered by a lack of validated approaches to identify and isolate pure populations of the various Spermatogonial subsets for in-depth analysis. In particular, although a number of markers of the undifferentiated Spermatogonial population have now been characterized, standardized methodology for assessing their specificity to the Spermatogonial Stem Cell (SSC) and transit amplifying progenitor pools has been lacking. To date, SSC content within an undefined population of spermatogonia has been inferred using either lineage tracing or Spermatogonial transplantation analyses which generate qualitative and quantitative data, respectively. Therefore, these techniques are not directly comparable, and are subject to variable interpretations as to a readout that is representative of a ‘pure’ SSC population. We propose standardization across the field for determining the SSC purity of a population via use of a limiting dilution transplantation assay that would eliminate subjectivity and help to minimize the generation of inconsistent data on ‘SSC’ populations. In the limiting dilution transplantation assay, a population of LacZ-expressing spermatogonia are selected based on a putative SSC marker, and a small, defined number of Cells (i.e. 10 Cells) are microinjected into the testis of a germ Cell-deficient recipient mouse. Using colony counts and an estimated colonization efficiency of 5%; a quantitative value can be calculated that represents SSC purity in the starting population. The utilization of this technique would not only be useful to link functional relevance to novel markers that will be identified in the future, but also for providing validation of purity for marker-selected populations of spermatogonia that are commonly considered to be SSCs by many researchers in the field of spermatogenesis and Stem Cell biology. Keywords: Spermatogonial Stem Cell, Limiting dilution, Transplantatio
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Spermatogonial Stem Cell and Niche
Encyclopedia of Reproduction, 2018Co-Authors: Nathan C. Law, Jon M OatleyAbstract:Spermatogonial Stem Cells (SSCs) are the foundation of spermatogenesis. The SSC microenvironment, known as the niche , controls fate decisions between self-renewal to maintain Stem Cell numbers and progenitor formation to produce differentiating spermatogonia that will ultimately form spermatozoa. Somatic Cells, other germ Cells, and the syStemic circulation contribute to dynamic changes in niche composition that regulate SSC function and maintain continuous spermatogenesis during a male's reproductive lifespan.
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dmrt1 is required for mouse Spermatogonial Stem Cell maintenance and replenishment
PLOS Genetics, 2016Co-Authors: Teng Zhang, Jon M Oatley, Vivian J Bardwell, David ZarkowerAbstract:Male mammals produce sperm for most of postnatal life and therefore require a robust germ line Stem Cell syStem, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse Spermatogonial Stem Cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ Cell depletion. Committed progenitor Cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ Cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the Stem Cell pool under normal conditions; and enabling progenitor Cells to help restore the Stem Cell pool after germ Cell depletion.
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Spermatogonial Stem Cell functions in physiological and pathological conditions
Current Topics in Developmental Biology, 2014Co-Authors: Qien Yang, Jon M OatleyAbstract:Sperm have a vital role in the continuity of a species by contributing genetic information to the next generation. Production of these specialized gametes in numbers sufficient to confer normal fertility occurs via cycling of the spermatogenic lineage, a process referred to as spermatogenesis. Continuity relies on the activities of a self-renewing reservoir of Spermatogonial Stem Cells (SSCs) from which progenitors will arise that transiently amplify in number before committing to a pathway of terminal differentiation. A primary population of SSCs is established during neonatal development from a pool of quiescent gonocyte precursors that forms in embryogenesis. Disruption of this process has dire consequences on maintenance of a cycling spermatogenic lineage in adulthood. At present, the molecular mechanisms underlying initial formation of the SSC pool are largely undefined. However, several transcription factors and posttranscriptional regulators have been identified as important regulators of SSC self-renewal from studies with mutant mouse models and experimental manipulation within primary cultures of mouse SSCs. Importantly, loss of function of these self-renewal factors may be underlying causes of infertility. Furthermore, disruption in the establishment of the SSC state within gonocytes or misregulation of self-renewal may manifest as testicular germ Cell tumors in postnatal life.
Takashi Shinohara - One of the best experts on this subject based on the ideXlab platform.
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Spermatogonial Stem Cell self renewal and development
Annual Review of Cell and Developmental Biology, 2013Co-Authors: Mito Kanatsushinohara, Takashi ShinoharaAbstract:Spermatogenesis originates from Spermatogonial Stem Cells (SSCs). Development of the Spermatogonial transplantation technique in 1994 provided the first functional assay to characterize SSCs. In 2000, glial Cell line–derived neurotrophic factor was identified as a SSC self-renewal factor. This discovery not only provided a clue to understand SSC self-renewing mechanisms but also made it possible to derive germline Stem (GS) Cell cultures in 2003. In vitro culture of GS Cells demonstrated their potential pluripotency and their utility in germline modification. However, in vivo SSC analyses have challenged the traditional concept of SSC self-renewal and have revealed their relationship with the microenvironment. An improved understanding of SSC self-renewal through functional assays promises to uncover fundamental principles of Stem Cell biology and will enable us to use these Cells for applications in animal transgenesis and medicine.
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ros are required for mouse Spermatogonial Stem Cell self renewal
Cell Stem Cell, 2013Co-Authors: Hiroko Morimoto, Takeshi Morimoto, Narumi Ogonuki, Kazumi Iwata, Kimiko Inoue, Ogura Atsuo, Mito Kanatsushinohara, Chihiro Yabenishimura, Takashi ShinoharaAbstract:Summary Reactive oxygen species (ROS) generation is implicated in Stem Cell self-renewal in several tissues but is thought to be detrimental for spermatogenesis as well as Spermatogonial Stem Cells (SSCs). Using cultured SSCs, we show that ROS are generated via the AKT and MEK signaling pathways under conditions where the growth factors glial Cell line-derived neurotrophic factor and fibroblast growth factor 2 drive SSC self-renewal and, instead, stimulate self-renewal at physiological levels. SSCs depleted of ROS stopped proliferating, but they showed enhanced self-renewal when ROS levels were increased by the addition of hydrogen peroxide, which induced the phosphorylation of stress kinases p38 mitogen-activated protein kinase (MAPK) and c- jun N-terminal kinase (JNK). Moreover, ROS depletion in vivo decreased SSC number in the testis, and NADPH oxidase 1 (Nox1)-deficient SSCs exhibited reduced self-renewal division upon serial transplantation. These results suggest that ROS generated by Nox1 play critical roles in SSC self-renewal via the activation of the p38 MAPK and JNK pathways.
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reconstitution of mouse Spermatogonial Stem Cell niches in culture
Cell Stem Cell, 2012Co-Authors: Mito Kanatsushinohara, Hiroko Morimoto, Narumi Ogonuki, Atsuo Ogura, Kimiko Inoue, Seiji Takashima, Masanori Takehashi, Takashi Nagasawa, Takashi ShinoharaAbstract:Spermatogonial Stem Cells (SSCs) reside in specific niches within seminiferous tubules. These niches are thought to secrete chemotactic factors for SSCs, because SSCs migrate to them upon transplantation. However, the identity of these chemotactic molecules remains unknown. Here, we established a testis feeder Cell culture syStem and used it to identify SSC chemotactic factors. When seeded on testis Cells from infertile mice, SSCs migrated beneath the Sertoli Cells and formed colonies with a cobblestone appearance that were very similar to those produced by hematopoietic Stem Cells. Cultured Cells maintained SSC activity and fertility for at least 5 months. Cobblestone colony formation depended on GDNF and CXCL12, and dominant-negative GDNF receptor transfection or CXCL12 receptor deficiency reduced SSC colonization. Moreover, GDNF upregulated CXCL12 receptor expression, and CXCL12 transfection in Sertoli Cells increased homing efficiency. Overall, our findings identify GDNF and CXCL12 as SSC chemotactic factors in vitro and in vivo.
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fgf2 mediates mouse Spermatogonial Stem Cell self renewal via upregulation of etv5 and bcl6b through map2k1 activation
Development, 2012Co-Authors: Kei Ishii, Mito Kanatsushinohara, Shinya Toyokuni, Takashi ShinoharaAbstract:Fibroblast growth factor 2 (FGF2) and glial Cell line-derived neurotrophic factor (GDNF) are required to recapitulate Spermatogonial Stem Cell (SSC) self-renewal in vitro. Although studies have revealed the role of the GDNF signaling pathway in SSCs, little is known about how FGF2 is involved. In the present study, we assessed the role of the FGF2 signaling pathway using a mouse germline Stem (GS) Cell culture syStem that allows in vitro expansion of SSCs. Adding GDNF or FGF2 induced phosphorylation of MAPK1/3, and adding the MAP2K1 inhibitor PD0325091 reduced GS Cell proliferation and MAPK1/3 phosphorylation. Moreover, GS Cells transfected with an activated form of Map2k1 not only upregulated Etv5 and Bcl6b gene expression, but also proliferated in an FGF2-independent manner, suggesting that they act downstream of MAP2K1 signaling to drive SSC self-renewal. Although GS Cells transfected with Map2k1, Etv5 or Bcl6b showed normal Spermatogonial markers, transplanting GS Cells expressing Bcl6b into infertile mouse testes resulted in the formation of a germ Cell tumor, suggesting that excessive self-renewal signals causes tumorigenic conversion. These results show that FGF2 depends on MAP2K1 signaling to drive SSC self-renewal via upregulation of the Etv5 and Bcl6b genes.
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Pluripotency of a Single Spermatogonial Stem Cell in Mice
Biology of reproduction, 2008Co-Authors: Mito Kanatsu-shinohara, Narumi Ogonuki, Atsuo Ogura, Kimiko Inoue, Jiyoung Lee, Hiromi Miki, Shinya Toyokuni, Masahito Ikawa, Tomoyuki Nakamura, Takashi ShinoharaAbstract:Although pluripotent Stem Cells were recently discovered in postnatal testis, attempts to analyze their developmental potential have led to conflicting claims that Spermatogonial Stem Cells are pluripotent or that they lose spermatogenic potential after conversion into pluripotent Stem Cells. To examine this issue, we analyzed the developmental fate of a single Spermatogonial Stem Cell that appeared during transfection experiments. After transfection of a neomycin-resistance gene into germline Stem Cells, we obtained an embryonic Stem-like, multipotent germline Stem Cell line. Southern blot analysis revealed that the germline Stem and multipotent germline Stem clones have the same transgene integration pattern, demonstrating their identical origin. The two lines, however, have different DNA methylation patterns. The multipotent germline Stem Cells formed chimeras after blastocyst injection but did not produce sperm after germ Cell transplantation, whereas the germline Stem Cells could produce only spermatozoa and did not differentiate into somatic Cells. Interestingly, the germline Stem Cells expressed several transcription factors (Pou5f1, Sox2, Myc, and Klf4) required for reprogramming fibroblasts into a pluripotent state, suggesting that they are potentially pluripotent. Thus, our study provides evidence that a single Spermatogonial Stem Cell can acquire pluripotentiality but that conversion into a pluripotent Cell type is accompanied by loss of spermatogenic potential.
Ellen Goossens - One of the best experts on this subject based on the ideXlab platform.
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human Spermatogonial Stem Cells display limited proliferation in vitro under mouse Spermatogonial Stem Cell culture conditions
Fertility and Sterility, 2016Co-Authors: Jose V Medrano, Charlotte Rombaut, Carlos Simon, Antonio Pellicer, Ellen GoossensAbstract:Objective To study the ability of human Spermatogonial Stem Cells (hSSCs) to proliferate in vitro under mouse Spermatogonial Stem Cell (mSSC) culture conditions. Design Experimental basic science study. Setting Reproductive biology laboratory. Patient(s) Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Intervention(s) Testicular Cells used to create in vitro Cell cultures corresponding to the following groups: [1] unsorted human testicular Cells, [2] differentially plated human testicular Cells, and [3] Cells enriched with major histocompatibility complex class 1 (HLA − )/epithelial Cell surface antigen (EPCAM + ) in coculture with inactivated testicular feeders from the same patient. Main Outcome Measure(s) Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ Cell markers, testosterone and inhibin B quantification, and TUNEL assay. Result(s) Putative hSSCs appeared in singlets, doublets, or small groups of up to four Cells in vitro only when testicular Cells were cultured in StemPro-34 medium supplemented with glial Cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated Cell sorting with HLA − /EPCAM + resulted in an enrichment of 27% VASA + /UTF1 + hSSCs, compared to 13% in unsorted controls. Coculture of sorted Cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm 2 after 2 weeks in vitro compared with unsorted Cells (61 hSSCs/cm 2 ) and differentially plated Cells (49 hSSCS/cm 2 ). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. Conclusion(s) We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA − /EPCAM + sorted Cells with testicular feeders improved the germ Cell/somatic Cell ratio.
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Spermatogonial Stem Cell preservation and transplantation from research to clinic
Human Reproduction, 2013Co-Authors: Ellen Goossens, Dorien Van Saen, Herman TournayeAbstract:Study question What issues remain to be solved before fertility preservation and transplantation can be offered to prepubertal boys? Summary answer The main issues that need further investigation are malignant Cell decontamination, improvement of in vivo fertility restoration and in vitro maturation. What is known already Prepubertal boys who need gonadotoxic treatment might render sterile for the rest of their life. As these boys do not yet produce sperm Cells, they cannot benefit from sperm banking. Spermatogonial Stem Cell (SSC) banking followed by autologous transplantation has been proposed as a fertility preservation strategy. But before this technique can be applied in the clinic, some important issues have to be resolved. Study design, size duration Original articles as well as review articles published in English were included in a search of the literature. Participants/materials, setting, methods Relevant studies were selected by an extensive Medline search. Search terms were fertility preservation, cryopreservation, prepubertal, SSC, testis tissue, transplantation, grafting and in vitro spermatogenesis. The final number of studies selected for this review was 102. Main results and the role of chance Cryopreservation protocols for testicular tissue have been developed and are already being used in the clinic. Since the efficiency and safety of SSC transplantation have been reported in mice, transplantation methods are now being adapted to the human testes. Very recently, a few publications reported on in vitro spermatogenesis in mice, but this technique is still far from being applied in a clinical setting. Limitations, reasons for caution Using tissue from cancer patients holds a potential risk for contamination of the collected testicular tissue. Therefore, it is of immense importance to separate malignant Cells from the Cell suspension before transplantation. Because biopsies obtained from young boys are small and contain only few SSCs, propagation of these Cells in vitro will be necessary. Wider implications of the findings The ultimate use of the banked tissue will depend on the patient's disease. If the patient was suffering from a non-malignant disease, tissue grafting might be offered. In cancer patients, decontaminated Cell suspensions will be injected in the testis. For patients with Klinefelter syndrome, the only option would be in vitro spermatogenesis. However, at present, restoring fertility in cancer and Klinefelter patients is not yet possible. Study funding/competing interest(s) Research Foundation, Flanders (G.0385.08 to H.T.), the Institute for the Agency for Innovation, Belgium (IWT/SB/111245 to E.G.), the Flemish League against Cancer (to E.G.), Kom op tegen kanker (G.0547.11 to H.T.) and the Fund Willy Gepts (to HT). E.G. is a Postdoctoral Fellow of the FWO, Research Foundation, Flanders. There are no conflicts of interest.
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Functional sperm produced after Spermatogonial Stem Cell transplantation into rhesus
Asian journal of andrology, 2013Co-Authors: Ellen Goossens, Herman TournayeAbstract:Spermatogonial Stem Cell (SSC) transplantation is a promising technique to circumvent sterility in prepubertal boys undergoing gonadotoxic treatments. While the cryopreservation of Spermatogonial Stem Cells is being introduced in clinical practices worldwide, a lot of unanswered questions remain regarding their eventual transplantation. In this paper autologous and allogeneic SSC transplantations in the testes of sterilized macaques were performed and spermatogenesis could be restored from donor SSCs. The spermatozoa obtained were competent to fertilize oocytes. This report proves the feasability of SSC transplantation in a primate model, hence reinforcing the hope that this strategy will eventually find its way into clinical practice. Spermatogonial Stem Cells (SSCs) are
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Spermatogonial Stem Cell preservation in boys with klinefelter syndrome to bank or not to bank that s the question
Fertility and Sterility, 2012Co-Authors: Inge Gies, Ellen Goossens, Jean De Schepper, Dorien Van Saen, Guido Pennings, Herman TournayeAbstract:Although early development of testis appears normal in boys with Klinefelter syndrome (KS), Spermatogonial Stem Cell (SSC) depletion occurs in midpuberty, leading to infertility. Therefore, freezing of semen samples or testicular tissue sampling could be offered to boys with KS at onset of puberty. However, only in about half of patients with KS, adult or prepubertal, spermatozoa or SSCs can be observed, and to date, no clinical parameters are available to detect patients who might benefit from these techniques. Furthermore, strategies for the further use of the cryopreserved material are still under investigation. Retrieval of Spermatogonial Cells in prepubertal boys with KS should therefore still be viewed as experimental and patients and their parents must be counseled accordingly.
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can pubertal boys with klinefelter syndrome benefit from Spermatogonial Stem Cell banking
Human Reproduction, 2012Co-Authors: Dorien Van Saen, Inge Gies, Herman Tournaye, Jean De Schepper, Ellen GoossensAbstract:BACKGROUND Although early development of testes appears normal in boys with Klinefelter syndrome (KS), Spermatogonial Stem Cell (SSC) depletion occurs in mid puberty, leading to infertility. Cryopreservation of SSCs prior to Stem Cell loss is an option that is currently offered to boys who have to undergo gonadotoxic treatments. This study aimed to explore the possibility of preserving SSCs in pubertal KS adolescents by testicular tissue banking. METHODS A retrospective study was conducted in seven non-mosaic 47,XXY adolescents, aged 13-16 years, who were invited for an experimental testicular tissue banking programme during their follow-up at the Paediatric Endocrinology Department of the UZ Brussel between 2009 and 2011. Paraffin-embedded testicular tissue was sectioned and stained with haematoxylin-eosin, and immunostainings were performed for Mage-A4, anti-Mullerian hormone, Inhibin α and steroidogenic acute regulatory protein. The presence of spermatogenesis and/or spermatogonia was evaluated. RESULTS Massive fibrosis and hyalinization was observed in all but one KS patients. Although spermatogonia were seen in five patients, spermatogonia were only present in tubules showing normal architecture in the youngest patient who also had normal follicle-stimulating hormone and inhibin B concentrations. CONCLUSIONS Testicular tissue cryopreservation in KS adolescents should be recommended as soon as possible, probably before hormonal changes of failing Sertoli Cell function are detected.