Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid

An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0–5 μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh), α6 integrin, β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3 μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs.

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Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽

10.1038/s41422-021-00477-x ◽

2021 ◽

Author(s):

Xiaoxiao Wang ◽

Yunlong Xiang ◽

Yang Yu ◽

Ran Wang ◽

Yu Zhang ◽

...

Keyword(s):

Stem Cells ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Large Set ◽

Mouse Escs ◽

Epiblast Stem Cells ◽

Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

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Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells

Development ◽

10.1242/dev.122.4.1235 ◽

1996 ◽

Vol 122 (4) ◽

pp. 1235-1242 ◽

Cited By ~ 3

Author(s):

U. Koshimizu ◽

T. Taga ◽

M. Watanabe ◽

M. Saito ◽

Y. Shirayoshi ◽

...

Keyword(s):

Germ Cells ◽

Intracellular Signaling ◽

Primordial Germ Cells ◽

Cell Formation ◽

Embryonic Stem ◽

Oncostatin M ◽

Receptor Complexes ◽

Binding Subunit

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

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154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab154 ◽

2016 ◽

Vol 28 (2) ◽

pp. 207

Author(s):

J. Galiguis ◽

C. E. Pope ◽

C. Dumas ◽

G. Wang ◽

R. A. MacLean ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Germ Cells ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Embryonic Stem ◽

Transcript Abundance ◽

Domestic Cat ◽

Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

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In Vitro Culture of Chicken Circulating and Gonadal Primordial Germ Cells on a Somatic Feeder Layer of Avian Origin

Animals ◽

10.3390/ani10101769 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1769

Author(s):

Agata Szczerba ◽

Takashi Kuwana ◽

Michelle Paradowska ◽

Marek Bednarczyk

Keyword(s):

Germ Cells ◽

Mammalian Species ◽

Primordial Germ Cells ◽

Feeder Layer ◽

Feeder Cells ◽

Conventional Culture ◽

Routine Identification ◽

Avian Origin ◽

The Right

The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo’s feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture.

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Human Second Trimester Amniotic Fluid Cells are Able to Create Embryoid Body-Like Structures in Vitro and to Show Typical Expression Profiles of Embryonic and Primordial Germ Cells

Cell Transplantation ◽

10.3727/096368914x678553 ◽

2014 ◽

Vol 23 (12) ◽

pp. 1501-1515 ◽

Cited By ~ 24

Author(s):

Ivana Antonucci ◽

Roberta Di Pietro ◽

Melissa Alfonsi ◽

Maria Antonietta Centurione ◽

Lucia Centurione ◽

...

Keyword(s):

Amniotic Fluid ◽

Germ Cells ◽

Embryoid Body ◽

Expression Profiles ◽

Primordial Germ Cells ◽

Second Trimester ◽

Amniotic Fluid Cells

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Retinoic Acid Is a Potent Growth Activator of Mouse Primordial Germ Cells in Vitro

Developmental Biology ◽

10.1006/dbio.1995.1113 ◽

1995 ◽

Vol 168 (2) ◽

pp. 683-685 ◽

Cited By ~ 93

Author(s):

Uichi Koshimizu ◽

Miho Watanabe ◽

Norio Nakatsuji

Keyword(s):

Retinoic Acid ◽

Germ Cells ◽

Primordial Germ Cells ◽

Potent Growth

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Primary culture of porcine PGCs requires LIF and porcine membrane-bound stem cell factor

Zygote ◽

10.1017/s0967199498000215 ◽

1998 ◽

Vol 6 (3) ◽

pp. 271-275 ◽

Cited By ~ 17

Author(s):

Gabriela Durcova-Hills ◽

Katja Prelle ◽

Sigrid Müller ◽

Miodrag Stojkovic ◽

Jan Motlik ◽

...

Keyword(s):

Stem Cell ◽

Primary Culture ◽

Germ Cells ◽

Stem Cell Factor ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Leukaemia Inhibitory Factor ◽

Feeder Cells ◽

Membrane Bound

We studied the effect of murine leukaemia inhibitory factor (LIF), human basic fibroblast growth factor (bFGF) and porcine stem cell factor (SCF) on the survival and/or proliferation of porcine primordial germ cells (PGCs) obtained from 27-day-old embryos in vitro. PGCs were cultured in embryonic stem cell (ESC) medium supplemented with or without either LIF (1000 IU/ml) alone or LIF together with bFGF (10 ng/ml). They were seeded on mitotically inactivated feeder cells, either STO or transfected STO cells (STO#8), expressing the membrane-bound form of porcine SCF. PGCs were identified by their alkaline phosphatase (AP) activity and counted after 1, 3 and 5 days in culture. After 1 day of culture, PGCs cultured on STO#8 cells showed significantly higher survival than PGCs cultured on STO cells (p < 0.05). The combined effect of SCF and LIF caused a significant increase in PGC number by day 3 of culture when PGCs were cultured on either STO cells (p < 0.01) or STO#8 (p < 0.001). When SCF and LIF were used together with bFGF no increase in the PGC number was observed. Our results suggest that the membrane-bound form of porcine SCF plays a pivotal role in the primary culture of porcine PGCs and that bFGF is not required in vitro.

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When Regenerative Medicine Faces the Challenges of Reproductive Medicine: A Review Study on Recent Advances in the Strategies for Derivation of Gametes from Stem Cells

EMJ Reproductive Health ◽

10.33590/emjreprohealth/20-00096 ◽

2020 ◽

pp. 42-52

Author(s):

María Gil Juliá ◽

José V. Medrano

Keyword(s):

Stem Cells ◽

Germ Cells ◽

Developmental Stages ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Differentiation Strategy ◽

Stem Cell Source ◽

Potential Applications ◽

Key Events

The murine model has allowed for the replication of all developmental stages of the mammalian germline in vitro, from embryonic stem cells to epiblast cells, primordial germ cells, and finally into functional haploid gametes. However, because of interspecies differences between mice and humans, these results are yet to be replicated in our species. Reports on the use of stem cells as a source of gametes, retrieved from public scientific databases, were analysed and classified according to the animal model used, the stem cell source and type, the differentiation strategy, and its potential application. This review offers a comprehensive compilation of recent publications of key events in the derivation of germ cells and gametogenesis in vitro, in both mice and human models. Additionally, studies intending to replicate the different stages in human cells in vitro, in order to obtain cells with a phenotype akin to functional human gametes, are also depicted. The authors present options for deriving gametes from stem cells in vitro and different reproductive options for specific groups of patients. Lastly, the potential applications of in vitro human gametogenesis are evaluated as well as the main limitations of the techniques employed. Even though it appears that we are far from being able to obtain gametes from pluripotent stem cells in vitro as a viable reproductive option, its current academic and clinical implications are extremely promising.

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