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

Szczerba, Agata; Kuwana, Takashi; Paradowska, Michelle; Bednarczyk, Marek

doi:10.3390/ani10101769

Cited by 7 publications

(8 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been confirmed that defined culture conditions help to minimize extrinsic molecules needed to establish and maintain PGCs lines that have self‐renewal and the ability to proliferate (Macdonald et al, 2010). Research has been performed under different culture conditions, including the use of a combination of growth factors, conditioned medium, and feeder cell layers, which led to improved PGC culture procedures (Naito et al, 2015d; Szczerba et al, 2020; Whyte et al, 2015). Raucci et al (2015) used an embryonic stem avian complete medium with differing FGF concentrations and an irradiated STO feeder layer for chicken PGCs (Raucci et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

Zare

Mirhoseini

Ghovvati

et al. 2023

Molecular Reproduction Devel

View full text Add to dashboard Cite

In many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF-β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26−28 Hamburger−Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder-free substrates. The results showed that TGF-β signaling agonists (IDE1 and Activin-A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF-β, disrupted PGCs' proliferation. However, the transfection of PGCs with constitutively active SMAD2/3 (SMAD2/3CA) resulted in improved PGC proliferation for more than 5 weeks. The results confirmed the interactions between overexpressed SMAD2/3CA and pluripotency-associated genes NANOG, OCT4, and SOX2. According to the results, the application of SMAD2/3CA could represent a step toward achieving an efficient expansion of avian PGCs.

show abstract

Section: Discussionmentioning

confidence: 99%

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

Zare

Mirhoseini

Ghovvati

et al. 2023

Molecular Reproduction Devel

View full text Add to dashboard Cite

show abstract

“…Beyond developmental biology, our study has important implications for the long-term maintenance of zebra finch PGCs in vitro . In chicken, HH28 gonads are used as a source for PGCs for stable cultures (Choi et al, 2010; Han et al, 2002; Shiue et al, 2009; Szczerba et al, 2020), and in previous work we successfully cultured zebra finch gonadal PGCs for several days, injected them in host embryonic gonads, and identified some host gonad colonization (Jung et al, 2019). This highlights the value of embryonic songbird gonads for gene manipulation and biobanking applications.…”

Section: Discussionmentioning

confidence: 99%

Pronounced early differentiation underlies zebra finch gonadal germ cell development

Biegler,

Belay,

Wang

et al. 2023

Preprint

View full text Add to dashboard Cite

The diversity of germ cell developmental strategies has been well documented across many vertebrate clades. However, much of our understanding of avian primordial germ cell (PGC) specification and differentiation has derived from only one species, the chicken (Gallus gallus). Of the three major classes of birds, chickens belong to Galloanserae, representing less than 4% of species, while nearly 95% of extant bird species belong to Neoaves. This represents a significant gap in our knowledge of germ cell development across avian species, hampering efforts to adapt genome editing and reproductive technologies developed in chicken to other birds. We therefore applied single-cell RNA sequencing to investigate inter-species differences in germ cell development between chicken and zebra finch (Taeniopygia castanotis), a Neoaves songbird species and a common model of vocal learning. Analysis of early embryonic male and female gonads revealed the presence of two distinct early germ cell types in zebra finch and only one in chicken. Both germ cell types expressed zebra finch Germline Restricted Chromosome (GRC) genes, present only in songbirds among birds. One of the zebra finch germ cell types expressed the canonical PGC markers, as did chicken, but with expression differences in several signaling pathways and biological processes. The second zebra finch germ cell cluster was marked by proliferation and fate determination markers, indicating beginning of differentiation. Notably, these two zebra finch germ cell populations were present in both male and female zebra finch gonads as early as HH25. Using additional chicken developmental stages, similar germ cell heterogeneity was identified in the more developed gonads of females, but not males. Overall, our study demonstrates a substantial heterochrony in zebra finch germ cell development compared to chicken, indicating a richer diversity of avian germ cell developmental strategies than previously known.

show abstract

“…PGCs play a critical role as the exclusive progenitors of sperm and eggs in developing embryos. They possess unique genetic programs designed to ensure the safe transfer of genetic material to the next generation, thereby facilitating the preservation of both male and female genes through sexual reproduction [16,17].…”

Section: Introductionmentioning

confidence: 99%

PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens

Wei,

Kinoshita,

Tanabe

et al. 2024

Preprint

View full text Add to dashboard Cite

Primordial germ cells (PGCs) are essential precursors for sperm and eggs. PGCs are highly reliable for conserving chicken germplasm and producing genetically modified chickens. Currently, efforts to conserve native chicken germplasm through PGCs and research on genetically modified chickens using CRISPR/Cas9 are limited. In this study, we successfully established 289 PGC lines from eight Chinese chicken populations with an 81.6% derivation rate. Furthermore, we successfully regenerated Piao chickens derived from these PGCs by repropagating cryopreserved PGCs and transplanting them into recipient chickens, resulting in a 12.7% efficiency rate. The regenerated chickens carried mitochondrial DNA from the female PGC donor and the rumplessness (Rp) mutation from male and female PGC donors in a homozygous condition. As a further application, we utilized CRISPR/Cas9 genome editing to disrupt the TYRP1 gene and created KO PGC lines. Transplanting KO cells into male recipients and mating them with wild-type hens enabled the successful generation of four TYRP1 KO chickens. Molecular analysis confirmed a 73 bp deletion in these four chicks, resulting in a frameshift mutation in the TYRP1 target region. These KO chickens had brown plumage due to reduced eumelanin production, observed in both homozygous and hemizygous mutations, compared to wild-type chickens. In conclusion, we have efficiently cultured and cryopreserved chicken PGCs, used them to regenerate chickens, and generated gene-edited chickens with TYRP1 dysfunction. These achievements will greatly impact the conservation of chicken genetic diversity, create new mutation models, and have potential applications in biomedical and avian research and breeding advancements.

show abstract

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

Cited by 7 publications

References 31 publications

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

Pronounced early differentiation underlies zebra finch gonadal germ cell development

PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens

Contact Info

Product

Resources

About