scholarly journals The reversible developmental unipotency of germ cells in chicken

Reproduction ◽  
2010 ◽  
Vol 139 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Jin Gyoung Jung ◽  
Young Mok Lee ◽  
Jin Nam Kim ◽  
Tae Min Kim ◽  
Ji Hye Shin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Developmental Stages ◽  
Production Systems ◽  
Primordial Germ Cells ◽  
Transmission Efficiency ◽  
Germline Transmission ◽  
Adult Testis

We recently developed bimodal germline chimera production approaches by transfer of primordial germ cells (PGCs) or embryonic germ cells (EGCs) into embryos and by transplantation of spermatogonial stem cells (SSCs) or germline stem cells (GSCs) into adult testes. This study was undertaken to investigate the reversible developmental unipotency of chicken germ cells using our established germline chimera production systems. First, we transferred freshly isolated SSCs from adult testis or in vitro cultured GSCs into stage X and stage 14–16 embryos, and we found that these transferred SSCs/GSCs could migrate to the recipient embryonic gonads. Of the 527 embryos that received SSCs or GSCs, 135 yielded hatchlings. Of 17 sexually mature males (35.3%), six were confirmed as germline chimeras through testcross analysis resulting in an average germline transmission efficiency of 1.3%. Second, PGCs/EGCs, germ cells isolated from embryonic gonads were transplanted into adult testes. The EGC transplantation induced germline transmission, whereas the PGC transplantation did not. The germline transmission efficiency was 12.5 fold higher (16.3 vs 1.3%) in EGC transplantation into testis (EGCs to adult testis) than that in SSC/GSC transfer into embryos (testicular germ cells to embryo stage). In conclusion, chicken germ cells from different developmental stages can (de)differentiate into gametes even after the germ cell developmental clock is set back or ahead. Use of germ cell reversible unipotency might improve the efficiency of germ cell-mediated germline transmission.

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

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.

scholarly journals 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

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.

scholarly journals In vivo and in vitro differentiation of male germ cells in the mouse

Reproduction ◽  
2004 ◽  
Vol 128 (2) ◽  
pp. 147-152 ◽  
Author(s):  
Orly Lacham-Kaplan
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cellular Interactions ◽  
Germ Cell Differentiation

Primordial germ cells appear in the embryo at about day 7 after coitum. They proliferate and migrate towards the genital ridge. Once there, they undergo differentiation into germ stem cells, known as ‘A spermatogonia’. These cells are the foundation of spermatogenesis. A spermatogonia commit to spermatogenesis, stay undifferentiated or degenerate. The differentiation of primordial germ cells to migratory, postmigratory and germ stem cells is dependent on gene expression and cellular interactions. Some of the genes that play a crucial role in germ cell differentiation are Steel, c-Kit, VASA, DAZL, fragilis, miwi, mili, mil1 and mil2. Their expression is stage specific, therefore allowing solid identification of germ cells at different developmental phases. In addition to the expression of these genes, other markers associated with germ cell development are nonspecific alkaline phosphatase activity, the stage specific embryonic antigen, the transcription factor Oct3/4 and β1- and α6-integrins. Commitment of cells to primordial germ cells and to A spermatogonia is also dependent on induction by the bone morphogenetic protein (BMP)-4. With this knowledge, researchers were able to isolate germ stem cells from embryonic stem cell-derived embryoid bodies, and drive these into gametes either in vivo or in vitro. Although no viable embryos were obtained from these gametes, the prospects are that this goal is not too far from being accomplished.

183 In Vitro Generation and Characterization of Putative Primordial Germ Cells Derived from Induced Pluripotent Stem Cells in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 231
Author(s):  
F. F. Bressan ◽  
M. A. Lima ◽  
L. S. Machado ◽  
N. C. G. Pieiri ◽  
P. Fantinato-Neto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Growth Factor ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Farm Animals ◽  
Induced Pluripotent

Embryonic pluripotent stem cells (ESC) and induced pluripotent stem cells (iPSC) were reported capable of differentiating into primordial germ cell-like (PGCL) and functional gametes in vitro in the murine model (Hikabe et al. 2016 Nature 539, 299-303). The in vitro generation of primordial germ cells (PGC) and gametes from farm animals would greatly contribute to enhance animal production technologies and to the creation of adequate models for several disorders. The present study aimed at the generation of PGC in vitro (iPGC) from iPSC in cattle and their characterisation through pluripotency and germ cell markers. For that, bovine iPSC previously generated and characterised (Bressan et al. 2015 Reprod. Fertil. Dev. 27, 254) were submitted to in vitro differentiation into epiblast-like cells (EpiLC) and iPGC by the protocol adapted from mice (Hayashi et al. 2011 Cell 146, 519-532). The biPS cells were induced into EpiLC by culture in fibronectin-coated (16.7 µg mL−1) 6-well plates in N2B27 culture medium supplemented with 20 ng mL−1 activin A, 12 ng mL−1 basic fibroblast growth factor (bFGF), and 1% knockout serum replacement (KSR) for 48 h and further differentiated into iPGC by non-adherent culture (Agreewell plates, StemCell Technologies, Vancouver, BC, Canada) with GK15 medium (GMEM supplemented with 15% KSR, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 2 mm l-glutamine, and 1% antibiotics) in the presence of 500 ng mL−1 BMP4, 100 ng mL−1 SCF, 500 ng mL−1 BMP8b, and 50 ng mL−1 epidermal growth factor for 4 days. The cells were then characterised regarding morphology, detection of alkaline phosphatase, immunofluorescence for OCT4, DDX4, VASA, and c-Kit proteins, and transcripts of pluripotency-related genes OCT4 and SOX2, as well as of imprinted genes (H19, SNRPN) and imprinted-related (DNMT1, DNMT3B) genes were analysed through RT-qPCR and compared with constitutive genes GAPDH, NAT1, and ACTB. Alkaline phosphatase and immunofluorescence analysis were positive for all specific markers. Interestingly, although OCT4 and SOX2 expression was present in iPS, EpiLC, and iPGC, this last group presented greater OCT4 and lesser SOX2 transcript amounts compared with other groups, suggesting, as expected, that PGC are still pluripotent but may already be differentiating into germ-cell lineages. The expression of H19 was increased in iPGC, whereas the expression of SNRPN was decreased only in the fibroblast group, potentially indicating epigenetic reprogramming process in these cells. Expression of DNMT1 and DNMT3B was not different between pluripotent groups but subtly increased when compared with that in fibroblasts. The results obtained herein represent an important first step in the in vitro generation of PGC and gametes from domestic farm animals, an unprecedented and desirable tool for enhancing new reproductive technologies and providing new understanding of cellular reprogramming and pluripotent germ cell biology. Financially supported by FAPESP grants 2013/08135-2, 2013/13686-8, 2015/26818-5; CNPq 482163/2013-5.

scholarly journals Germ Cell Derivation from Pluripotent Stem Cells for Understanding In Vitro Gametogenesis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1889
Author(s):  
Tae-Kyung Hong ◽  
Jae-Hoon Song ◽  
So-Been Lee ◽  
Jeong-Tae Do
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Assisted Reproductive Technologies ◽  
Primordial Germ Cells ◽  
Reproductive Technologies ◽  
Obstructive Azoospermia ◽  
Female Germ Cell

Assisted reproductive technologies (ARTs) have developed considerably in recent years; however, they cannot rectify germ cell aplasia, such as non-obstructive azoospermia (NOA) and oocyte maturation failure syndrome. In vitro gametogenesis is a promising technology to overcome infertility, particularly germ cell aplasia. Early germ cells, such as primordial germ cells, can be relatively easily derived from pluripotent stem cells (PSCs); however, further progression to post-meiotic germ cells usually requires a gonadal niche and signals from gonadal somatic cells. Here, we review the recent advances in in vitro male and female germ cell derivation from PSCs and discuss how this technique is used to understand the biological mechanism of gamete development and gain insight into its application in infertility.

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

Cell Research ◽  
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.

scholarly journals Ligand–Receptor Interactions Elucidate Sex-Specific Pathways in the Trajectory From Primordial Germ Cells to Gonia During Human Development

2021 ◽  
Vol 9 ◽  
Author(s):  
Arend W. Overeem ◽  
Yolanda W. Chang ◽  
Jeroen Spruit ◽  
Celine M. Roelse ◽  
Susana M. Chuva De Sousa Lopes
Keyword(s):  
Germ Cell ◽  
Signaling Pathways ◽  
Germ Cells ◽  
Tyrosine Kinases ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Cell Lineage ◽  
Systematic Analysis ◽  
Receptor Interactions

The human germ cell lineage originates from primordial germ cells (PGCs), which are specified at approximately the third week of development. Our understanding of the signaling pathways that control this event has significantly increased in recent years and that has enabled the generation of PGC-like cells (PGCLCs) from pluripotent stem cells in vitro. However, the signaling pathways that drive the transition of PGCs into gonia (prospermatogonia in males or premeiotic oogonia in females) remain unclear, and we are presently unable to mimic this step in vitro in the absence of gonadal tissue. Therefore, we have analyzed single-cell transcriptomics data of human fetal gonads to map the molecular interactions during the sex-specific transition from PGCs to gonia. The CellPhoneDB algorithm was used to identify significant ligand–receptor interactions between germ cells and their sex-specific neighboring gonadal somatic cells, focusing on four major signaling pathways WNT, NOTCH, TGFβ/BMP, and receptor tyrosine kinases (RTK). Subsequently, the expression and intracellular localization of key effectors for these pathways were validated in human fetal gonads by immunostaining. This approach provided a systematic analysis of the signaling environment in developing human gonads and revealed sex-specific signaling pathways during human premeiotic germ cell development. This work serves as a foundation to understand the transition from PGCs to premeiotic oogonia or prospermatogonia and identifies sex-specific signaling pathways that are of interest in the step-by-step reconstitution of human gametogenesis in vitro.

229 LONG-TERM PROPAGATION AND CRYOPRESERVATION OF CAT SPERMATOGONIAL STEM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 246
Author(s):  
L. M. Vansandt ◽  
M. Dickson ◽  
R. Zhou ◽  
L. Li ◽  
B. S. Pukazhenthi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Domestic Cat ◽  
Feeder Cell ◽  
Cell Clusters ◽  
Fetal Fibroblasts

Spermatogonial stem cells (SSC) are unique adult stem cells that reside within the seminiferous tubules of the testis. As stem cells, SSC maintain the ability to self-replicate, providing a potentially unlimited supply of cells and an alternate source for preservation of the male genome. While self-renewing, long-term SSC culture has been achieved in mice, there is virtually no information regarding culture requirements of felid SSC. Therefore, the objectives of this study were to (1) evaluate the ability of 3 feeder cell lines to support germ cell colony establishment in domestic cats (Felis catus), and (2) assess long-term culture using the best feeder(s). Cells isolated enzymatically from peripubertal cat testes (n = 4) and enriched by differential plating were cultured on mouse embryonic fibroblasts (STO line), mouse-derived C166 endothelial cells, and primary cat fetal fibroblasts (cFF). Colony morphology was assessed every other day and immunocytochemistry (ICC) was performed to investigate expression of SSC markers. At 5 days in vitro (DIV), a cluster forming activity assay was used to estimate the number of SSC supported by each feeder cell line. Differences among treatments were compared using Tukey-Kramer adjustment for pair-wise mean comparisons. Data were expressed as mean cluster number ± SE per 105 cells input. When cultured on STO feeders, cat germ cells were distributed as individual cells. On both C166 cells and cFF feeders, germ cell clumps (morphologically consistent with SSC colonies in other species) were observed. Immunocytochemistry revealed that the single germ cells present on STO feeders were positive for UCHL1 and weakly expressed PLZF and OCT4. Cells within the germ cell clumps on C166 cells and cFF co-expressed all 3 SSC markers. The C166 cells supported a higher number of germ cell clusters (77.4 ± 13.8) compared with STO (3.5 ± 1.1, P = 0.0003) or cFF (22.7 ± 1.0, P = 0.0024). Therefore, subsequent subculture experiments were performed exclusively with C166 feeder layers. Cultures from 2 donors were passaged at 12 DIV and periodically as needed thereafter. Germ cell clumps consistently reestablished following each subculture and immunocytochemistry analysis confirmed maintenance of all 3 SSC markers. Cells were also positive for alkaline phosphatase activity. Cells that had been cryopreserved in culture medium with 5% (vol/vol) dimethyl sulphoxide after144 DIV (7 passages) were thawed and cultured for an additional 18 days. These cells continued to express SSC markers and form germ cell clusters. Taken together, these data demonstrate that C166 feeder cells can facilitate colony establishment and in vitro propagation of germ cell clumps in the domestic cat. This represents an important first step towards attainment and optimization of a long-term SSC culture system in the cat. This system would provide a mechanism to explore regulation of spermatogenesis, test species-specific drugs, and produce transgenic biomedical models.

An Evidence That Murine Marrow-Derived CXCR4+ SSEA-1+ Oct-4+ Very Small Embryonic-Like (VSEL) Stem Cells Are Pluripotent and Express Several Primordial Germ Cell (PGC) Markers - Hypotesis for Developmental Deposition of PGC in Various Organs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1676-1676 ◽  
Author(s):  
Magda Kucia ◽  
Ewa Zuba-Surma ◽  
Ryan Reca ◽  
Janina Ratajczak ◽  
Mariusz Ratajczak
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Fetal Liver ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
C2c12 Cells

Abstract Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia2006;20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for embryonic stem cells such as i) small size (2–4 um in diameter), ii) large nuclei surrounded by a narrow rim of cytoplasm, and iii) open-type chromatin (euchromatin). We also found that VSELs may be released from BM and circulate in peripheral blood during tissue/organ injuries (e.g., heart infarct, stroke). Recently we noticed that ~5–10% of purified VSELs if plated over a C2C12 murine sarcoma cell feeder layer are able to form spheres that resemble embryoid bodies. Cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as purified VSELs are CXCR4+SSEA-1+Oct-4+. Furthermore, VSEL-DS after replating over C2C12 cells may again (up to 5–7 passages) grow new spheres or if plated into cultures promoting tissue differentiation expand into cells from all three germ-cell layers. The formation of VSEL-DS was observed in a presence of C2C12 cells obtained from different sources. Furthermore, VSELs isolated from GFP+ mice grew GFP+ VSEL-DS which show a diploid content of DNA. This suggests that VSEL-DS are in fact derived from VSELs and not from the supportive C2C12 cell line as well as excludes the possibility of cell fusion to the observed phenomenon. Similar spheres were also formed by VSELs isolated from murine fetal liver, spleen and thymus. Interestingly formation of VSEL-DS was associated with a young age, and no VSEL-DS were observed by cells isolated from old mice (> 2 years). We also found that cells isolated from VSEL-DS similarly as embryonic stem cells grow tumors after injection into immunodeficient NOD/SCID mice (51/52 inoculated mice). Since VSELs isolated by us express several markers of primordial germ cells (fetal-type alkaline phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella, Fragilis, Nobox, Hdac6) we hypothesize that VSELs are closely related to a population of primordial germ cells. These cells are specified during early gastrulation in the proximal epiblast and subsequently migrate in a CXCR4-SDF-1 dependent manner through the embryo proper to their final destination in genital ridges. It is possible that some of these cells or a population of cells closely related to them migrate astray being chemoattracted by SDF-1 to fetal liver and subsequently, during the third trimester of gestation seed together with hematopoietic stem cells in bone marrow and perhaps other organs as well. In conclusion, we postulate that VSELs identified by us and purified at the single cell level could become an important source of pluripotent stem cells for regeneration.

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