scholarly journals HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Dinar Yunusov ◽  
Leticia Anderson ◽  
Lucas Ferreira DaSilva ◽  
Joanna Wysocka ◽  
Toshihiko Ezashi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Germ Cells ◽  
Expression Patterns ◽  
Primordial Germ Cells ◽  
Population Level ◽  
Human Embryos ◽  
Cell Stage ◽  
Lncrna Gene ◽  
Stable Cell Lines ◽  
The Individual

Abstract Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines.

scholarly journals Erratum: Corrigendum: HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Dinar Yunusov ◽  
Leticia Anderson ◽  
Lucas Ferreira DaSilva ◽  
Joanna Wysocka ◽  
Toshihiko Ezashi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Human Embryos ◽  
Stable Cell Lines

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells

Zygote ◽  
2019 ◽  
Vol 27 (02) ◽  
pp. 82-88 ◽  
Author(s):  
Vivek Pandey ◽  
Anima Tripathi ◽  
Pawan K. Dubey
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Germ Cells ◽  
Expression Patterns ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Single Type ◽  
Rt Pcr

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

Immunomagnetic Isolation of Primordial Germ Cells and the Establishment of Embryonic Germ Cell Lines in the Mouse

Cloning ◽  
1999 ◽  
Vol 1 (4) ◽  
pp. 217-224 ◽  
Author(s):  
Gabriela Durcova-Hills ◽  
Tomoyuki Tokunaga ◽  
Satoshi Kurosaka ◽  
Manabu Yamaguchi ◽  
Seiya Takahashi ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Immunomagnetic Isolation

scholarly journals Bayesian inference of transcriptional branching identifies regulators of early germ cell development in humans

2017 ◽  
Author(s):  
Christopher A. Penfold ◽  
Anastasiya Sybirna ◽  
John Reid ◽  
Aracely Castillo Venzor ◽  
Elena Drousioti ◽  
...  
Keyword(s):  
Cell Fate ◽  
Germ Cells ◽  
Temporal Dynamics ◽  
Primordial Germ Cells ◽  
Series Data ◽  
Human Embryos ◽  
Marker Genes ◽  
Cell Fate Decisions ◽  
Stage Of Development

AbstractDuring embryonic development, cells undertake a series of fate decisions to form a complete organism comprised of various cell types, epitomising a branching process. A striking example of branching occurs in humans around the time of implantation, when primordial germ cells (PGCs), precursors of sperm and eggs, and somatic lineages are specified. Due to inaccessibility of human embryos at this stage of development, understanding the mechanisms of PGC specification remains difficult. The integrative modelling of single cell transcriptomics data from embryos and appropriate in vitro models should prove to be a useful resource for investigating this system, provided that the cells can be suitably ordered over a developmental axis. Unfortunately, most methods for inferring cell ordering were not designed with structured (time series) data in mind. Although some probabilistic approaches address these limitations by incorporating prior information about the developmental stage (capture time) of the cell, they do not allow the ordering of cells over processes with more than one terminal cell fate. To investigate the mechanisms of PGC specification, we develop a probabilistic pseudotime approach, branch-recombinant Gaussian process latent variable models (B-RGPLVMs), that use an explicit model of transcriptional branching in individual marker genes, allowing the ordering of cells over developmental trajectories with arbitrary numbers of branches. We use first demonstrate the advantage of our approach over existing pseudotime algorithms and subsequently use it to investigate early human development, as primordial germ cells (PGCs) and somatic cells diverge. We identify known master regulators of human PGCs, and predict roles for a variety of signalling pathways, transcription factors, and epigenetic modifiers. By concentrating on the earliest branched signalling events, we identified an antagonistic role for FGF receptor (FGFR) signalling pathway in the acquisition of competence for human PGC fate, and identify putative roles for PRC1 and PRC2 in PGC specification. We experimentally validate our predictions using pharmacological blocking of FGFR or its downstream effectors (MEK, PI3K and JAK), and demonstrate enhanced competency for PGC fate in vitro, whilst small molecule inhibition of the enzymatic component of PRC1/PRC2 reveals reduced capacity of cells to form PGCs in vitro. Thus, B-RGPLVMs represent a powerful and flexible data-driven approach for dissecting the temporal dynamics of cell fate decisions, providing unique insights into the mechanisms of early embryogenesis. Scripts relating to this analysis are available from: https://github.com/cap76/PGCPseudotime

scholarly journals 178 ESTABLISHMENT OF MOUSE PLURIPOTENT STEM CELLS GENERATED FROM PRIMORDIAL GERM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 239
Author(s):  
S.W. Shim ◽  
S.J. Song ◽  
H.S. Shim ◽  
S.J. Uhm ◽  
B.Y. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Cultured Cells ◽  
Cell Layer ◽  
Es Cells ◽  
Primordial Germ Cells ◽  
Pcr Analysis ◽  
Feeder Cell

Pluripotent stem cells have been generated from two embryonic sources: ES cells generated from ICM of blastocyst stage embryos, and embryonic germ (EG) cells generated from primordial germ cells (PGCs). Both ES and EG cells are pluripotent and exhibit important characteristics such as high alkaline phosphatase (AP) activity, multicellular colony formation, normal and stable karyotype, continuous passaging ability, and capacity to differentiate into three embryonic germ layers. This study was performed to establish the culture system for mouse EG cells derived from mouse PGCs. PGCs collected from the genital ridge of Day 11.5, 12.5, and 13.5 mouse embryos (C57BL/6 × DBA/2) were cultured and subsequently passaged on mitotically inactivated STO feeder cell layer. Cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, 1,000 units/mL of leukemia inhibiting factor (LIF), 6 ng/mL of SCF, and 10 ng/mL of bFGF at culture conditions of 5% CO2 in air, 95% relative humidity, 37°C temperature. Cells were routinely passaged every 3–4 days. Over a period of 7–10 days in primary culture, PGCs proliferated to form small, densely packed, multicellular colonies consisting of AP-positive cells that morphologically resembled undifferentiated ES cells. RT-PCR analysis confirmed mRNA expression of transcription factors Oct-4 and Nanog in these cells. Cultured cells could be maintained on the feeder cell layer for at least 10 passages and still retain normal karyotype. These results suggest that cell lines derived from mouse primordial germ cells are presumably EG cell lines and could be useful for transgenic animal production and ES cell study.

Events in the germ cell lineage after entry of the primordial germ cells into the genital ridges in normal and u.v.-irradiated Xenopus laevis

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 33-46
Author(s):  
Brigitta Züst ◽  
K. E. Dixon
Keyword(s):  
Xenopus Laevis ◽  
Germ Cell ◽  
Germ Cells ◽  
Dorsal Root ◽  
Primordial Germ Cells ◽  
Cell Lineage ◽  
Cell Stage ◽  
Comparison Of The Results ◽  
New Generation

Approximately 20–25 primordial germ cells leave the endoderm between stages 38–41 and localize in the dorsal root of the mesentery by stage 43/44. At this time all the cells contain large quantities of yolk which is gradually resorbed. The cells begin dividing between stages 48–52. The number and size of the germ cells were measured in tadpoles between stages 48–54 of development. The results indicate that in females the germ cells divide more often than in males. In both sexes the mitoses are grossly unequal, leading to the formation of a new generation of germ cells which are considerably smaller (one-tenth to one-fifth) than the size of the primordial germ cells at stage 48. The germ cells in male tadpoles at stage 54 are larger than in female tadpoles at the same stage. In tadpoles which developed from eggs irradiated in the vegetal hemisphere with u.v. light at the 2- to 4-cell-stage, primordial germ cells migrate into the genital ridges much later (stage 46–48) than in unirradiated embryos. They also differ morphologically from germ cells in control animals at this stage in that they are approximately one-tenth the size, lacking yolk in the cytoplasm and have a more highly lobed nucleus. Comparison of the results in unirradiated and irradiated animals suggests that the germ cell lineage is composed of a series of ordered, predictable events, and serious disruption of one of the events deranges later events.

Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3197-3204 ◽  
Author(s):  
P.A. Labosky ◽  
D.P. Barlow ◽  
B.L. Hogan
Keyword(s):  
Growth Factor ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Coat Color ◽  
Primordial Germ Cells ◽  
Methylation Status ◽  
Embryonic Stem ◽  
Insulin Like Growth Factor ◽  
Significant Difference

Primordial germ cells of the mouse cultured on feeder layers with leukemia inhibitory factor, Steel factor and basic fibroblast growth factor give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells can be induced to differentiate extensively in culture, form teratocarcinomas when injected into nude mice and contribute to chimeras when injected into host blastocysts. Here, we report the derivation of multiple embryonic germ cell lines from 8.5 days post coitum embryos of C57BL/6 inbred mice. Four independent embryonic germ cell lines with normal male karyotypes have formed chimeras when injected into BALB/c host blastocysts and two of these lines have transmitted coat color markers through the germline. We also show that pluripotent cell lines capable of forming teratocarcinomas and coat color chimeras can be established from primordial germ cells of 8.0 days p.c. embryos and 12.5 days p.c. genital ridges. We have examined the methylation status of the putative imprinting box of the insulin-like growth factor type 2 receptor gene (Igf2r) in these embryonic germ cell lines. No correlation was found between methylation pattern and germline competence. A significant difference was observed between embryonic stem cell and embryonic germ cell lines in their ability to maintain the methylation imprint of the Igf2r gene in culture. This may illustrate a fundamental difference between these two cell types.

Germ cells and pluripotent stem cells in the mouse

2001 ◽  
Vol 13 (8) ◽  
pp. 661 ◽  
Author(s):  
Anne McLaren ◽  
Gabriela Durcova-Hills
Keyword(s):  
Growth Factors ◽  
Cell Lines ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Individual Growth ◽  
Developmental Potential

For many years, attempts to achieve long-term culture of mouse primordial germ cells (PGCs) proved unsuccessful, even when feeder layers were used and individual growth factors were added to the medium. However, when three growth factors were added simultaneously to the medium, some of the cells continued to proliferate indefinitely. Similar to embryonic stem cell lines, these embryonic germ (EG) cell lines were capable of giving rise to embryoid bodies in vitro, and colonizing all cell lineages in chimeras, including the germline. Initially, EG cells were made from PGCs before migration, 8.5 days post coitum (dpc), and after entry into the genital ridge, 11.5 and 12.5 dpc. New EG cell lines from 9.5 dpc (migrating) and 11.5 dpc PGCs, carrying either a LacZ or GFP transgene, are described here. The developmental potential of the new EG cell lines in vitro, in vivoin chimeras, and in tissue aggregates in organ culture was studied. The EG cells were compared with PGCs at the stage from which the EG cells were derived. The two cell types show several similarities, but also some differences in gene expression and cell behaviour, which require further exploration.

scholarly journals Primordial germ cells do not migrate along nerve fibres in marmoset monkey and mouse embryos

Reproduction ◽  
2019 ◽  
pp. 101-109 ◽  
Author(s):  
E Wolff ◽  
M M Suplicki ◽  
R Behr
Keyword(s):  
Germ Cells ◽  
Peripheral Nerves ◽  
Developmental Stages ◽  
Primordial Germ Cells ◽  
Spatial Association ◽  
General Mechanism ◽  
Human Embryos ◽  
General Strategy ◽  
Marmoset Monkey ◽  
Specific Finding

Primordial germ cells (PGCs) are the embryonic precursors of spermatozoa and eggs. In mammals, PGCs arise early in embryonic development and migrate from their tissue of specification over a significant distance to reach their destinations, the genital ridges. However, the exact mechanism of translocation is still debated. A study on human embryos demonstrated a very close spatial association between migrating PGCs and developing peripheral nerves. Thus, it was proposed that peripheral nerves act as guiding structures for migrating PGCs. The goal of the present study is to test whether the association between nerves and PGCs may be a human-specific finding or whether this represents a general strategy to guide PGCs in mammals. Therefore, we investigated embryos of different developmental stages from the mouse and a non-human primate, the marmoset monkey (Callithrix jacchus), covering the phase from PGC emergence to their arrival in the gonadal ridge. Embryo sections were immunohistochemically co-stained for tubulin beta-3 chain (TUBB3) to visualise neurons and Octamer-binding protein 4 (OCT4 (POU5F1)) as marker for PGCs. The distance between PGCs and the nearest detectable neuron was measured. We discovered that in all embryos analysed of both species, the majority of PGCs (>94%) was found at a minimum distance of 50 µm to the closest neuron and, more importantly, that the PGCs had reached the gonads before any TUBB3 signal could be detected in the vicinity of the gonads. In conclusion, our data indicate that PGC migration along peripheral nerves is not a general mechanism in mammals.

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