Three-dimensional culture of chicken primordial germ cells in chemically defined media containing the functional polymer FP003

Mapping Intimacies ◽

10.1101/358952 ◽

2018 ◽

Author(s):

Yi-Chen Chen ◽

Wei-Che Chang ◽

Shau-Ping Lin ◽

Masataka Minami ◽

Christian Jean ◽

...

Keyword(s):

Recombinant Protein ◽

Germ Cells ◽

Large Scale ◽

Fluorescent Proteins ◽

Primordial Germ Cells ◽

Three Dimensional ◽

3D Culture ◽

Defined Medium ◽

Virus Growth ◽

Functional Polymer

AbstractScalable production of avian suspension cell exhibits a valuable potential on therapeutic application by producing recombinant protein and as the substrate for virus growth. This study sought to establish a system with chemically defined components for three-dimensional (3D) culture of chicken primordial germ cells (cPGCs), a pluripotent avian cell type. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium, and cPGCs did not sediment,and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1week of culture in 3D-FAot medium, an aseric chemically defined medium containing FP003, indicating that this medium enhances the expansion of cPGCs. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. cPGCs harboring both PGK:EGFP and VASA:tdTOMATO robustly expressed both fluorescent proteins upon culture in 3D-FAot, suggesting that this approach is perspective for recombinant protein production. In summary,this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring or loss of cellular properties. This system provides a platform for large-scale culture ofcPGCs in industry.

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Three-dimensional culture of chicken primordial germ cells (cPGCs) in defined media containing the functional polymer FP003

PLoS ONE ◽

10.1371/journal.pone.0200515 ◽

2018 ◽

Vol 13 (9) ◽

pp. e0200515 ◽

Cited By ~ 1

Author(s):

Yi-Chen Chen ◽

Wei-Che Chang ◽

Shau-Ping Lin ◽

Masataka Minami ◽

Christian Jean ◽

...

Keyword(s):

Germ Cells ◽

Primordial Germ Cells ◽

Three Dimensional ◽

Functional Polymer ◽

Defined Media ◽

Three Dimensional Culture

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Collagen-alginate microspheres as a 3D culture system for mouse embryonic stem cells differentiation to primordial germ cells

Biologicals ◽

10.1016/j.biologicals.2017.04.003 ◽

2017 ◽

Vol 48 ◽

pp. 114-120 ◽

Cited By ~ 4

Author(s):

Vahid Mansouri ◽

Mohammad Salehi ◽

Mir davood Omrani ◽

Zahra Niknam ◽

Abdolreza Ardeshirylajimi

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Germ Cells ◽

Culture System ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

3D Culture ◽

Mouse Embryonic Stem Cells ◽

3D Culture System

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Knockout of MMP3 Weakened Solid Tumor Organoids and Cancer Extracellular Vesicles

10.20944/preprints202003.0371.v1 ◽

2020 ◽

Author(s):

Eman A. Taha ◽

Chiharu Sogawa ◽

Yuka Okusha ◽

Hotaka Kawai ◽

May Wathone Oo ◽

...

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Extracellular Vesicles ◽

Fluorescent Proteins ◽

Three Dimensional ◽

3D Culture ◽

Proliferating Cells ◽

Ki 67 ◽

Additional Release

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (300-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 leads to a significant reduction in tumoroid size and to the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. These weakened phenotypes by MMP3 KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated into recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3 resulting in increasing the proliferation and CD9+ tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

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Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Cancers ◽

10.3390/cancers12051260 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1260 ◽

Cited By ~ 4

Author(s):

Eman Taha ◽

Chiharu Sogawa ◽

Yuka Okusha ◽

Hotaka Kawai ◽

May Oo ◽

...

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Extracellular Vesicles ◽

Fluorescent Proteins ◽

Three Dimensional ◽

3D Culture ◽

Proliferating Cells ◽

Ki 67 ◽

Additional Release

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

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Microfluidic-Based Droplets for Advanced Regenerative Medicine: Current Challenges and Future Trends

Biosensors ◽

10.3390/bios12010020 ◽

2021 ◽

Vol 12 (1) ◽

pp. 20

Author(s):

Hojjatollah Nazari ◽

Asieh Heirani-Tabasi ◽

Sadegh Ghorbani ◽

Hossein Eyni ◽

Sajad Razavi Bazaz ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Regenerative Medicine ◽

Cell Therapy ◽

Large Scale ◽

Three Dimensional ◽

3D Culture ◽

Cell Encapsulation ◽

Cost Effective ◽

Culture Methods

Microfluidics is a promising approach for the facile and large-scale fabrication of monodispersed droplets for various applications in biomedicine. This technology has demonstrated great potential to address the limitations of regenerative medicine. Microfluidics provides safe, accurate, reliable, and cost-effective methods for encapsulating different stem cells, gametes, biomaterials, biomolecules, reagents, genes, and nanoparticles inside picoliter-sized droplets or droplet-derived microgels for different applications. Moreover, microenvironments made using such droplets can mimic niches of stem cells for cell therapy purposes, simulate native extracellular matrix (ECM) for tissue engineering applications, and remove challenges in cell encapsulation and three-dimensional (3D) culture methods. The fabrication of droplets using microfluidics also provides controllable microenvironments for manipulating gametes, fertilization, and embryo cultures for reproductive medicine. This review focuses on the relevant studies, and the latest progress in applying droplets in stem cell therapy, tissue engineering, reproductive biology, and gene therapy are separately evaluated. In the end, we discuss the challenges ahead in the field of microfluidics-based droplets for advanced regenerative medicine.

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Bright multicolor labeling of neuronal circuits with fluorescent proteins and chemical tags

eLife ◽

10.7554/elife.40350 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 13

Author(s):

Richi Sakaguchi ◽

Marcus N Leiwe ◽

Takeshi Imai

Keyword(s):

Olfactory System ◽

Large Scale ◽

Fluorescent Proteins ◽

Three Dimensional ◽

Neuronal Tracing ◽

Operator System ◽

Enhanced Expression ◽

Chemical Tags ◽

Labeling Method ◽

Powerful Strategy

The stochastic multicolor labeling method ‘Brainbow’ is a powerful strategy to label multiple neurons differentially with fluorescent proteins; however, the fluorescence levels provided by the original attempts to use this strategy were inadequate. In the present study, we developed a stochastic multicolor labeling method with enhanced expression levels that uses a tetracycline-operator system (Tetbow). We optimized Tetbow for either plasmid or virus vector-mediated multicolor labeling. When combined with tissue clearing, Tetbow was powerful enough to visualize the three-dimensional architecture of individual neurons. Using Tetbow, we were able to visualize the axonal projection patterns of individual mitral/tufted cells along several millimeters in the mouse olfactory system. We also developed a Tetbow system with chemical tags, in which genetically encoded chemical tags were labeled with synthetic fluorophores. This was useful in expanding the repertoire of the fluorescence labels and the applications of the Tetbow system. Together, these new tools facilitate light-microscopy-based neuronal tracing at both a large scale and a high resolution.

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Intercellular bridges coordinate the transition from pluripotency to meiosis in mouse fetal oocytes

Science Advances ◽

10.1126/sciadv.abc6747 ◽

2021 ◽

Vol 7 (15) ◽

pp. eabc6747

Author(s):

B. Soygur ◽

R. G. Jaszczak ◽

A. Fries ◽

D. H. Nguyen ◽

S. Malki ◽

...

Keyword(s):

Germ Cells ◽

State Transition ◽

Primordial Germ Cells ◽

Three Dimensional ◽

Female Fertility ◽

Intercellular Bridges ◽

Regulatory Factors ◽

Fetal Ovary ◽

Meiotic Initiation ◽

Anterior Posterior

Meiosis is critical to generating oocytes and ensuring female fertility; however, the mechanisms regulating the switch from mitotic primordial germ cells to meiotic germ cells are poorly understood. Here, we implicate intercellular bridges (ICBs) in this state transition. We used three-dimensional in toto imaging to map meiotic initiation in the mouse fetal ovary and revealed a radial geometry of this transition that precedes the established anterior-posterior wave. Our studies reveal that appropriate timing of meiotic entry across the ovary and coordination of mitotic-meiotic transition within a cyst depend on the ICB component Tex14, which we show is required for functional cytoplasmic sharing. We find that Tex14 mutants more rapidly attenuate the pluripotency transcript Dppa3 upon meiotic initiation, and Dppa3 mutants undergo premature meiosis similar to Tex14. Together, these results lead to a model that ICBs coordinate and buffer the transition from pluripotency to meiosis through dilution of regulatory factors.

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Hexachlorobenzene toxicity in the rat ovary: II. Ultrastructure induced by medium (10 mg/kg) dose exposure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012374x ◽

1992 ◽

Vol 50 (1) ◽

pp. 668-669

Author(s):

Amreek Singh ◽

Warren G. Foster ◽

Anna Dykeman ◽

David C. Villeneuve

Keyword(s):

Germ Cells ◽

Primordial Germ Cells ◽

Surface Epithelium ◽

Morphological Parameters ◽

Comprehensive Investigation ◽

Ovary Surface Epithelium ◽

Rat Ovary ◽

High Doses

Hexachlorobenzene (HCB) is a known toxicant that is found in the environment as a by-product during manufacture of certain pesticides. This chlorinated chemical has been isolated from many tissues including ovary. When administered in high doses, HCB causes degeneration of primordial germ cells and ovary surface epithelium in sub-human primates. A purpose of this experiment was to determine a no-effect dose of the chemical on the rat ovary. The study is part of a comprehensive investigation on the effects of the compound on the biochemical, hematological, and morphological parameters in the monkey and rat.

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