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germline stem cell

Sarita Panula, Ahmed Reda, Jan-Bernd Stukenborg, Cyril Ramathal, Meena Sukhwani, Halima Albalushi, Daniel Edsgärd, Michiko Nakamura, Olle Söder, Kyle E Orwig, Shinya Yamanaka, Renee A Reijo Pera, Outi Hovatta
The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation...
2016: PloS One
Orie Hikabe, Nobuhiko Hamazaki, Go Nagamatsu, Yayoi Obata, Yuji Hirao, Norio Hamada, So Shimamoto, Takuya Imamura, Kinichi Nakashima, Mitinori Saitou, Katsuhiko Hayashi
The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts...
October 17, 2016: Nature
Aileen R Helsel, Jon M Oatley
In mammals, the activities of spermatogonial stem cells (SSCs) provide the foundation for continual spermatogenesis throughout a male's reproductive lifetime. At present, the defining characteristics of SSCs and mechanisms controlling their fate decisions are not well understood. Transplantation is a definitive functional measure of stem cell capacity for male germ cells that can be used as an assay to provide an unequivocal quantification of the SSC content in an experimental cell population. Here, we discuss the procedure for mice and provide protocols for preparing donor germ cell suspensions from testes directly or primary cultures of spermatogonia for transplantation, enriching for SSCs, preparing recipient males, microinjection into recipient testes, and considerations for experimental design...
2017: Methods in Molecular Biology
Gina M Aloisio, Ileana Cuevas, Yuji Nakada, Christopher G Peña, Diego H Castrillon
The precise identity of spermatogonial stem cells-the germline stem cell of the adult testis-remains a controversial topic. Technical limitations have included the lack of specific markers and methods for lineage tracing of Asingle spermatogonia and their subsets. Immunolocalization of proteins in tissue sections has been a standard tool for the in situ identification and visualization of rare cellular subsets. However, these studies are limited by the need for faithful and reliable protein markers to define these cell types, as well as the availability of specific antibodies to these markers...
2017: Methods in Molecular Biology
Nevine A Shalaby, Michael Buszczak
The Drosophila ovary represents a key in vivo model used to study germline stem cell (GSC) maintenance and stem cell daughter differentiation because these cells and their somatic cell neighbors can be identified at single-cell resolution within their native environment. Here we describe a fluorescent-based technique for the acquisition of 4D datasets of the Drosophila ovariole for periods that can exceed 12 consecutive hours. Live-cell imaging facilitates the investigation of molecular and cellular dynamics that were not previously possible using still images...
2017: Methods in Molecular Biology
Dana Gancz, Lilach Gilboa
In the fruit fly Drosophila melanogaster, ovarian germline stem cells (GSCs) and their niches form during larval development. This process is poorly studied partly due to technical difficulties in isolating early larval ovaries. In addition, purifying RNA from larval ovaries proves to be more challenging than purifying it from other organs. Here we describe a technique for dissecting ovaries from early larvae and advise on how to extract RNA with maximum yield and purity. RNA isolation allows assaying gene expression in a direct and quantitative manner, which is invaluable for understanding molecular events underlying ovarian niche formation and GSC establishment...
2017: Methods in Molecular Biology
Mayu Inaba, Yukiko M Yamashita
Asymmetric cell division (ACD) is utilized in many stem cell systems to produce two daughter cells with different cell fates. Despite the fundamental importance of ACD during development and tissue homeostasis, the nature of ACD is far from being fully understood. Step-by-step observation of events during ACD allows us to understand processes that lead to ACD. Here we describe examples of how we evaluate ACD in vivo using the Drosophila male germline stem cell system.
2017: Methods in Molecular Biology
Sarah L Crittenden, Hannah S Seidel, Judith Kimble
The Caenorhabditis elegans germline is an excellent model for studying the regulation of a pool of stem cells and progression of cells from a stem cell state to a differentiated state. At the tissue level, the germline is organized in an assembly line with the germline stem cell (GSC) pool at one end and differentiated cells at the other. A simple mesenchymal niche caps the GSC region of the germline and maintains GSCs in an undifferentiated state by signaling through the conserved Notch pathway. Downstream of Notch signaling, key regulators include novel LST-1 and SYGL-1 proteins and a network of RNA regulatory proteins...
2017: Methods in Molecular Biology
Dan Wang, Lei Hou, Shuhei Nakamura, Ming Su, Fang Li, Weiyang Chen, Yizhen Yan, Christopher D Green, Di Chen, Hong Zhang, Adam Antebi, Jing-Dong J Han
The RNA-binding protein LIN-28 was first found to control developmental timing in Caenorhabditis elegans. Later, it was found to play important roles in pluripotency, metabolism, and cancer in mammals. Here we report that a low dosage of lin-28 enhanced stress tolerance and longevity, and reduced germline stem/progenitor cell number in C. elegans. The germline LIN-28-regulated microRNA let-7 was required for these effects by targeting akt-1/2 and decreasing their protein levels. AKT-1/2 and the downstream DAF-16 transcription factor were both required for the lifespan and germline stem cell effects of lin-28...
October 11, 2016: Aging Cell
J Matthew Watson, Alexander Platzer, Anita Kazda, Svetlana Akimcheva, Sona Valuchova, Viktoria Nizhynska, Magnus Nordborg, Karel Riha
In plants, gametogenesis occurs late in development, and somatic mutations can therefore be transmitted to the next generation. Longer periods of growth are believed to result in an increase in the number of cell divisions before gametogenesis, with a concomitant increase in mutations arising due to replication errors. However, there is little experimental evidence addressing how many cell divisions occur before gametogenesis. Here, we measured loss of telomeric DNA and accumulation of replication errors in Arabidopsis with short and long life spans to determine the number of replications in lineages leading to gametes...
October 11, 2016: Proceedings of the National Academy of Sciences of the United States of America
Xing Ma, Yingying Han, Xiaoqing Song, Trieu Do, Zhihao Yang, Jianquan Ni, Ting Xie
Stem cells in adult tissues are constantly exposed to genotoxic stress and also accumulate DNA damage with age. DNA damage has been proposed to cause stem cell loss and cancer formation. However, it remains a mystery how DNA damage leads to both stem cell loss and cancer formation. In this study, we use germline stem cells (GSCs) in the Drosophila ovary to show that DNA damage retards stem cell self-renewal and lineage differentiation in a CHK2 kinase-dependent manner. Both heatshock-inducible endonuclease I-CreI expression and X-ray irradiation can efficiently introduce double-strand breaks in GSCs and their progeny, resulting in a rapid GSC loss and an accumulation of ill-differentiated GSC progeny...
October 11, 2016: Development
Kaitlin M Laws, Daniela Drummond-Barbosa
Multiple aspects of organismal physiology influence the number and activity of stem cells and their progeny, including nutritional status. Previous studies demonstrated that Drosophila germline stem cells (GSCs), follicle stem cells (FSCs), and their progeny sense and respond to diet via complex mechanisms involving many systemic and local signals. AMP-activated protein kinase, or AMPK, is a highly conserved regulator of energy homeostasis known to be activated under low cellular energy conditions; however, its role in the ovarian response to diet has not been investigated...
October 8, 2016: Developmental Biology
Ferdinand von Meyenn, Rebecca V Berrens, Simon Andrews, Fátima Santos, Amanda J Collier, Felix Krueger, Rodrigo Osorno, Wendy Dean, Peter J Rugg-Gunn, Wolf Reik
Primordial germ cell (PGC) development is characterized by global epigenetic remodeling, which resets genomic potential and establishes an epigenetic ground state. Here we recapitulate PGC specification in vitro from naive embryonic stem cells and characterize the early events of epigenetic reprogramming during the formation of the human and mouse germline. Following rapid de novo DNA methylation during priming to epiblast-like cells, methylation is globally erased in PGC-like cells. Repressive chromatin marks (H3K9me2/3) and transposable elements are enriched at demethylation-resistant regions, while active chromatin marks (H3K4me3 or H3K27ac) are more prominent at regions that demethylate faster...
October 10, 2016: Developmental Cell
Yoshiko Murakami
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired GPI deficiency caused by somatic mutation of the PIGA gene in one or several hematopoietic stem cells. Recently, PNH caused by somatic mutation of one allele of the PIGT gene in combination with a germline mutation of the other allele was reported, showing that PIGA is not the only gene responsible for PNH, though other causes are rare. These mutant cells become GPI deficient, expand clonally and differentiate into all of the hematopoietic lineages. When GPI deficient erythrocytes increase in proportion, massive hemolysis occurs due to activated complement attack during infection...
2016: [Rinshō Ketsueki] the Japanese Journal of Clinical Hematology
Michelle A Carmell, Gregoriy A Dokshin, Helen Skaletsky, Yueh-Chiang Hu, Josien C van Wolfswinkel, Kyomi J Igarashi, Daniel W Bellott, Michael Nefedov, Peter W Reddien, George C Enders, Vladimir N Uversky, Craig C Mello, David C Page
The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. Gcna gene expression is enriched in reproductive cells across eukarya - either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells of diverse multicellular animals...
October 8, 2016: ELife
ChangHwan Lee, Erika B Sorensen, Tina R Lynch, Judith Kimble
C. elegans Notch signaling maintains a pool of germline stem cells within their single-celled mesenchymal niche. Here we investigate the Notch transcriptional response in germline stem cells using single-molecule fluorescence in situ hybridization coupled with automated, high-throughput quantitation. This approach allows us to distinguish Notch-dependent nascent transcripts in the nucleus from mature mRNAs in the cytoplasm. We find that Notch-dependent active transcription sites occur in a probabilistic fashion and, unexpectedly, do so in a steep gradient across the stem cell pool...
October 5, 2016: ELife
R Kumar Pramod, Bo Ram Lee, Young Min Kim, Hong Jo Lee, Young Hyun Park, Tamao Ono, Jeong Mook Lim, Jae Yong Han
We reported previously testis-mediated germline chimera production and characterization of germline stem cell-like cells from chicken testes. The present study aimed to establish an in vitro system for culture of quail spermatogonia stem cells (SSCs) for practical applications in germline preservation and transgenesis. Testicular cells from juvenile (4 weeks old) or adult (8 weeks old) quail testis were isolated using sequential enzymatic digestion. The percentages of viability of isolated testicular cells were 91...
October 5, 2016: Stem Cells and Development
Minoru Tanaka
Egg or sperm? The mechanism of sexual fate decision in germ cells has been a long-standing issue in biology. A recent analysis identified foxl3 as a gene that determines the sexual fate decision of germ cells in the teleost fish, medaka. foxl3/Foxl3 acts in female germline stem cells to repress commitment into male fate (spermatogenesis), indicating that the presence of mitotic germ cells in the female is critical for continuous sexual fate decision of germ cells in medaka gonads. Interestingly, foxl3 is found in most vertebrate genomes except for mammals...
October 4, 2016: BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology
Dhananjay Chaturvedi, Mayu Inaba, Shane Scoggin, Michael Buszczak
Conserved from yeast to humans, the Paf1 complex participates in a number of diverse processes including transcriptional initiation and polyadenylation. This complex typically includes 5 proteins: Paf1, Rtf1, Cdc73, Leo1 and Ctr9. Previous efforts identified clear Drosophila homologs of Paf1, Rtf1 and Cdc73 based on sequence similarity. Further work showed that these proteins help to regulate gene expression and are required for viability. To date, a Drosophila homolog of Ctr9 has remained uncharacterized. Here, we show that the gene CG2469 encodes a functional Drosophila Ctr9 homolog...
September 27, 2016: G3: Genes—Genomes—Genetics
Kazuhito Sakamoto, Barbara L Wehde, Patrick D Rädler, Aleata A Triplett, Kay-Uwe Wagner
The biological functions of the Janus kinase 1 (JAK1) are suggested to be pleiotropic since this signal transducer is ubiquitously expressed and coupled to a variety of cytokine receptors. Consequently, mice that are deficient in this tyrosine kinase were reported to die shortly after birth. To facilitate studies that address the biological and molecular functions of JAK1 during postnatal development, we performed gene targeting in embryonic stem cells and generated a Cre/lox-based conditional knockout mouse model...
September 27, 2016: Genesis: the Journal of Genetics and Development
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