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1.
Anim Biosci ; 36(12): 1905-1917, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37641830

ABSTRACT

OBJECTIVE: Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. METHODS: A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. RESULTS: In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. CONCLUSION: Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

2.
Data Brief ; 48: 109212, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37213550

ABSTRACT

Fertilized embryos develop and move freely in the reproductive tract until implantation. Subsequently, the embryos continue to develop after attachment to the uterus. Because of the absence of the uterus, in vitro culturing of embryos is limited to a period of approximately a week. Hatched blastocysts were seeded on feeder cells to extend the culture period. We cultured the colonies formed from the blastocysts for an additional 14 days. From the colonies, four types of cells were established, and each type was isolated to extract RNA. RNA sequencing was conducted using NovaSeq6000. Sequencing reads were aligned to genes and transcripts. Raw data from our previous study were used to compare these samples with the cultured cell lines. We analyzed differentially expressed genes and Gene Ontology terms between new samples and cultured cell lines. Our data can provide essential information for extending the period of embryo culture in vitro.

3.
Anim Biosci ; 36(8): 1180-1189, 2023 Aug.
Article in English | MEDLINE | ID: mdl-36915922

ABSTRACT

OBJECTIVE: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. METHODS: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. RESULTS: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. CONCLUSION: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.

4.
Biomed Pharmacother ; 156: 113864, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36252351

ABSTRACT

Skin aging is a major risk factor for the dermal diseases, and interventions to attenuate cellular senescence are expected to reduce the risk for age-related diseases involving skin atrophy. However, blocking cell death or extending proliferation causally results in side effects and an increased cancer risk. For identification of a safer approach, we focused on PDK1 inhibition, which could revert cellular senescence and reduce senescence factors in skin in vitro, in a human skin equivalent model and in an exploratory, placebo-controlled, interventional trial. Natural phytochemical kaempferol tetrasaccharides resulted in a significant reduction in cellular senescence, and an increase in collagen fiber was observed in the skin cell and human skin equivalent. Clinical enhancement in skin appearance was noted in multiple participants, and an immunohistochemical study revealed improvement in the histological appearance of skin tissue and extracellular matrix. This change was associated with relative improvement in histological markers of senescence and clinical appearance of the aged skin and an increase in collagen fiber, an essential factor for preventing skin atrophy and consistency of the basement membrane. These results indicate that PDK1 inhibition is a potentially effective antiaging intervention, suggesting a diagnostic role and preventive actions of PDK1 in senescence-associated skin atrophy.


Subject(s)
Fibroblasts , Kaempferols , Humans , Aged , Kaempferols/pharmacology , Kaempferols/therapeutic use , Skin , Cellular Senescence , Collagen/metabolism , Atrophy/drug therapy , Atrophy/metabolism
5.
Front Cell Dev Biol ; 10: 918222, 2022.
Article in English | MEDLINE | ID: mdl-36172290

ABSTRACT

Many types of embryonic stem cells have been induced from pre-implantation blastocysts to study the specification of early lineages. Various cell lines have been established using chemicals, including excessive inhibitory molecules. Previous studies have also aimed to purify cell populations representing a single embryonic lineage from a protocol. In this study, we used a novel culture condition to induce cells from blastocyst seeding and analyzed their characteristics. Next, signaling inhibitors were introduced during the cell culture period. Furthermore, we investigated the cell types using RNA sequencing. Each type of cell population showed a distinct morphology and reactivity with alkaline phosphatase. Marker proteins enabled each cell type to be distinguished by immunocytochemistry, and genes such as Sox17, Gata4, Gata6, T, and Cdx2 showed applicability for the discrimination of cell types. Signaling inhibitors suppressed the production of some cell types, and gene expression and marker protein patterns were collapsed. RNA-sequencing suggested cell-type-specific marker genes and the correlation among samples. In conclusion, four types of cells could be induced from porcine embryos using a single protocol, and they could be isolated manually. Our data will help promote the study of lineage segregation based on embryonic cells.

6.
Cell Prolif ; 55(11): e13313, 2022 Nov.
Article in English | MEDLINE | ID: mdl-35883229

ABSTRACT

OBJECTIVES: Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic development, has long been an issue. OCT4 has a species-specific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4-related pluripotency networks and second-cell fate decisions. MATERIALS AND METHODS: To determine the functions of OCT4 in lineage specification and embryo proliferation, loss- and gain-of-function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real-time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes. RESULTS: In OCT4-targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real-time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4-targeted blastocysts, but trophoblast-related genes were increased. In OCT4-overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real-time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased. CONCLUSION: Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation.


Subject(s)
Gene Expression Regulation, Developmental , Octamer Transcription Factor-3 , Pregnancy , Female , Swine , Animals , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Blastocyst/metabolism , Cell Differentiation/genetics , Cell Proliferation , Mammals/genetics , Mammals/metabolism
7.
Stem Cells Int ; 2022: 6337532, 2022.
Article in English | MEDLINE | ID: mdl-35846983

ABSTRACT

The present study examined the activity and function of the pig OCT4 enhancer in the porcine early embryonic development stage and porcine authentic embryonic stem cells. OCT4 is known as a pluripotent regulator, and its upstream regulatory region-based dual-fluorescence protein reporter system controlled by distal and proximal enhancers is broadly used in studies examining the states and mechanism of pluripotency. We analyzed how this reporter system functions during early embryo development and in stem cells using a previously established porcine-specific reporter system. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated with different expression patterns simultaneously as the expression of pluripotent marker genes changed during the development of in vitro pathenotes and the establishment of porcine embryonic stem cells (ESCs). This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual-fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.

8.
Theriogenology ; 187: 173-181, 2022 Jul 15.
Article in English | MEDLINE | ID: mdl-35596974

ABSTRACT

Fatty acid has a various role in preimplantation embryo development. Especially, Linoleic acid, polyunsaturated fatty acid, has been reported to affect the apoptosis pathway via nuclear transcription factor-kappa B. But to date, the function of NF-κB has not been demonstrated in porcine preimplantation embryos. We demonstrated that linoleic acid had a positive effect on embryo development at a certain concentration(25 µM), but developmental failure was observed at higher concentration. Furthermore, the expression level of NF-κB increased, unlike that of IL-6, as the concentration of linoleic acid increased. Interestingly, the concentration of NF-κB was found to increase even at the concentration of linoleic acid at which embryo development decreased. We found that pro-apoptotic gene expression was downregulated in the linoleic acid-treated group. It was also found that MCL-1, an anti-apoptotic gene known to be unaffected by IL-6, was found to be increased at the mRNA level in the linoleic acid-treated group. As the concentration of NF-kB increased, the nuclear translocation of C-JUN gradually increased dependent on the linoleic acid concentration. It was confirmed that NF-κB is an important factor in porcine embryos by treated ammonium pyrrolidinedithiocarbamate (APDC 0.1 µM, an inhibitor of NF-κB) affected NF-κB protein expression, IL-6 expression, and blastocyst production. These data supported porcine embryos can use exogenous linoleic acid as a metabolic energy source via NF-κB.


Subject(s)
Linoleic Acid , NF-kappa B , Animals , Apoptosis , Female , Interleukin-6 , Linoleic Acid/pharmacology , NF-kappa B/metabolism , Pregnancy , RNA, Messenger/metabolism , Swine
9.
Stem Cell Res ; 57: 102609, 2021 Nov 27.
Article in English | MEDLINE | ID: mdl-34861590

ABSTRACT

Here, we report pig embryonic stem cells (ESCs) originating from parthenogenetic blastocysts which were developed from 2-cell embryos micro-injected with porcine OCT4 reporter system cultured in previous reported chemically defined culture media. The ESCs with reporter system expressed pluripotency markers and fluorescent signals produced by OCT4 reporter system. Also, they were capable of forming teratomas following subcutaneous injection into nude mice. Since reporter system enables the non-destructive classification of the condition of live pluripotent stem cells, this reporter cell line could be a useful resource for research on species-specific pluripotency.

10.
Nutrients ; 13(12)2021 Dec 06.
Article in English | MEDLINE | ID: mdl-34959923

ABSTRACT

The stratum corneum (SC) is the outermost layer of the epidermis and plays an important role in maintaining skin moisture and protecting the skin from the external environment. Ceramide and natural moisturizing factor (NMF) are the major SC components that maintain skin moisture. In this study, we investigated whether the oral intake of enzymatically decomposed AP collagen peptides (APCPs) can improve skin moisture and barrier function by assessing changes in the ceramide and NMF contents in the SC after APCP ingestion with the aim to develop a skin functional food. Fifty participants orally ingested APCP (1000 mg) or placebo for 12 weeks, and then, skin hydration and skin texture were evaluated. SC samples were collected to analyze skin scaling, ceramide, and NMF contents. Participants in the APCP group exhibited improved skin moisture content by 7.33% (p = 0.031) and roughness by -4.09% (p = 0.036) when compared with those in the placebo group. NMF content; the amounts of amino acids (AA), including glycine and proline; and AA derivatives were significantly increased in the APCP group (31.98 µg/mg protein) compared to those in the placebo group (-16.01 µg/mg protein) (p = 0.006). The amounts of total ceramides and ceramide subclasses were significantly higher in the APCP group than in the placebo group (p = 0.014). In conclusion, our results demonstrate that APCP intake improves skin moisture and increase the ceramide and NMF contents in the SC, thereby enhancing the skin barrier function.


Subject(s)
Body Water/metabolism , Ceramides/metabolism , Collagen/administration & dosage , Collagen/pharmacology , Dietary Supplements , Eating/physiology , Epidermis/metabolism , Adult , Female , Humans , Male , Middle Aged , Water Loss, Insensible/drug effects
11.
Cell Prolif ; 54(8): e13097, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34250657

ABSTRACT

OBJECTIVES: Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre-implantation embryo development. The extraordinarily longer pre-implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre-implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre-implantation embryos between species. MATERIALS AND METHODS: To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss- and gain-of-function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real-time PCR. RESULTS: Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2-disrupted blastocysts, the expression of the ICM-related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes, excluding OCT4, and proliferation-related genes were decreased in SOX2-targeted blastocysts. In SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. CONCLUSIONS: Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early-stage embryogenesis.


Subject(s)
Embryonic Development , SOXB1 Transcription Factors/metabolism , Animals , Blastocyst/cytology , Blastocyst/metabolism , CRISPR-Cas Systems/genetics , Cell Lineage , Cell Proliferation , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , RNA, Guide, Kinetoplastida/metabolism , SOXB1 Transcription Factors/genetics , Swine , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome
12.
Cell Reprogram ; 23(3): 168-179, 2021 06.
Article in English | MEDLINE | ID: mdl-34037424

ABSTRACT

This study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4, which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in fibroblast growth factor (FGF)- or leukemia inhibitory factor (LIF)-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. Porcine embryonic fibroblasts were coinfected with the pOCT4-ΔPE-eGFP (distal enhancer [DE]-green fluorescent protein [GFP]) and pOCT4-ΔDE-DsRed2 (proximal enhancer [PE]-red fluorescent protein [RFP]) vectors, and GFP and RFP expression were verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 DE and PE were activated in different expression patterns simultaneously as changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems confirmed that porcine iPSCs are in primed state after reprogramming in FGF2- or LIF-containing media. This work demonstrates the applicability of porcine OCT4 upstream region-derived dual fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.


Subject(s)
Cell Differentiation , Cellular Reprogramming , Embryonic Stem Cells/cytology , Fibroblasts/cytology , Green Fluorescent Proteins/metabolism , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/cytology , Animals , Cells, Cultured , Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Green Fluorescent Proteins/genetics , Pluripotent Stem Cells/metabolism , Swine
13.
J Anim Sci Biotechnol ; 12(1): 64, 2021 May 13.
Article in English | MEDLINE | ID: mdl-33980301

ABSTRACT

BACKGROUND: Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression, which is facilitated by various signaling pathways associated with myogenesis. In this study, we attempted to transdifferentiate pig embryonic fibroblasts (PEFs) myogenically into skeletal muscle through overexpression of the pig MYOD1 gene and modulation of the FGF, TGF-ß, WNT, and cAMP signaling pathways. RESULTS: The MYOD1 overexpression vector was constructed based on comparative sequence analysis, demonstrating that pig MYOD1 has evolutionarily conserved domains across various species. Although forced MYOD1 expression through these vectors triggered the expression of endogenous muscle markers, transdifferentiated muscle cells from fibroblasts were not observed. Therefore, various signaling molecules, including FGF2, SB431542, CHIR99021, and forskolin, along with MYOD1 overexpression were applied to enhance the myogenic reprogramming. The modified conditions led to the derivation of myotubes and activation of muscle markers in PEFs, as determined by qPCR and immunostaining. Notably, a sarcomere-like structure was observed, indicating that terminally differentiated skeletal muscle could be obtained from transdifferentiated cells. CONCLUSIONS: In summary, we established a protocol for reprogramming MYOD1-overexpressing PEFs into the mature skeletal muscle using signaling molecules. Our myogenic reprogramming can be used as a cell source for muscle disease models in regenerative medicine and the production of cultured meat in cellular agriculture.

14.
J Cosmet Dermatol ; 20(9): 2851-2859, 2021 Sep.
Article in English | MEDLINE | ID: mdl-33533074

ABSTRACT

BACKGROUND: Skin darkening because of increased and irregular synthesis of melanin causes melasma, solar lentigo, and freckles. Melasolv™, produced in the early 2000s, shows potent depigmenting effect and has low cytotoxicity. It has been used as a brightening agent in cosmetics for decades. AIMS: This study was conducted to investigate whether Melasolv™ is effective for the skin of ASEAN (Southeast Asia) women. METHODS: We recruited ASEAN women in Singapore and divided them into two groups (active group vs. placebo group). Melasolv™ and placebo formulations were applied twice a day for 12 weeks. The changes in the pigmented spots were visually evaluated by an expert and assessed using a spectrophotometer and Mexameter at 0, 4, 8, and 12 weeks. RESULTS: The visual evaluation revealed significant improvements, in both size and color intensity, in the active group compared with those in the placebo group at 12 weeks. In the spectrophotometric evaluation, the L* value of the pigmented spots in the active group was significantly higher than that in the placebo group at 12 weeks. Similar results were obtained in the evaluation using the Mexameter. After 12 weeks, the melanin index of the pigmented spots significantly decreased, and it was significantly higher than that in the placebo group. There was no significant change in the erythema index. In the image analysis, there were no significant differences in skin color brightness and evenness in the active group compared with those in the placebo group. CONCLUSION: Melasolv™ can be effective used for skin brightening.


Subject(s)
Skin Lightening Preparations , Thymol , Cinnamates , Esters , Female , Humans , Skin Pigmentation
15.
Cancer Drug Resist ; 4(4): 866-880, 2021.
Article in English | MEDLINE | ID: mdl-35582384

ABSTRACT

Aim: Therapy to overcome drug resistance by modulating epidermal growth factor receptor (EGFR) is a viable approach to suppress the proliferation of human non-small cell lung cancer (NSCLC) cells. A previous study demonstrated that the seeds of an aqueous Brucea javanica (BJ) (L.) Merr (Simaroubaceae) extract containing quassinoid mixtures effectively inhibited the growth and alleviated tumorigenesis in H1975 cells of NSCLC by targeting T790M/L858R EGFR. This study aimed to further determine whether the aqueous BJ extract affects the enriched H1975 spheroids in suspension culture and mouse xenograft tumor models. Methods: The spheroids of NSCLC adenocarcinoma H1975 cells were enriched in a serum-free media. The growth rate of sphere propagation by aqueous BJ extract was determined in suspended culture and in colony-formation assay. BJ extract was fed orally to nude mice bearing xenograft tumors. The resected tumors were analyzed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and proliferating cell nuclear antigen assessment. Various markers were used to determine the pluripotency of tumors from mice treated with different concentrations of BJ extract. Results: BJ extract was demonstrated to be effective against the propagation of the enriched spheroids. In animal models, oral administration of the aqueous BJ extract reduced spheroid tumorigenicity. The alleviated growth of the established xenograft tumors can be attributed to the reduced drug resistance and induced apoptosis without distinct adverse effects. More evidence supports activated apoptotic death attenuated spheroid stemness of tumors. Conclusion: As an effective treatment regime to assuage lung cancer, the indigenous BJ extract promises to obliterate drug resistance and the growth of cancer stem cell tumors from NSCLC cells harboring T790M/L858R EGFR.

16.
Data Brief ; 33: 106563, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33294536

ABSTRACT

Typical models of pluripotency, humans and mice, have been used to analyse the characteristics of pluripotent stem cells. However, these species exhibit molecular differences in many aspects. With similar physiology and genomics as humans, pigs are promising model for the research of pluripotency. The data of porcine pluripotent cells would be helpful in understanding the molecular network of human pluripotency. Pluripotent cells of humans and mice exhibit specific MicroRNA (miRNA) expression patterns to maintain the pluripotent state. Information about miRNA expression in pig pluripotent cells is not sufficient, so we analysed miRNAs in pluripotent (blastocysts and ES-like) and somatic cell samples (PEB and PFF). We screened cell-type specific miRNAs and identified their target genes. Functional annotation of the target genes was also conducted. Our data may facilitate miRNA-based induction and maintenance of the pluripotent state of porcine cells and provide support to fill the gap between the pluripotency networks of humans and mice.

17.
Stem Cell Res ; 49: 102093, 2020 12.
Article in English | MEDLINE | ID: mdl-33232901

ABSTRACT

Here, we report in vivo developmental competent pig embryonic stem cells (ESCs) originating from in vitro-fertilized and parthenogenetic embryos cultured in chemically defined culture media. The pig ESC lines expressed pluripotency markers and were capable of forming teratomas following subcutaneous injection into nude mice. These cell lines would be a useful resource for comparative developmental biology and agricultural biotechnology.


Subject(s)
Embryonic Stem Cells , Parthenogenesis , Animals , Cell Differentiation , Cell Line , Mice , Mice, Nude , Swine
18.
Int J Mol Sci ; 21(19)2020 Oct 01.
Article in English | MEDLINE | ID: mdl-33019677

ABSTRACT

Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.


Subject(s)
Adaptor Proteins, Vesicular Transport/genetics , Blastocyst/metabolism , Cell Lineage/genetics , Ectoderm/metabolism , Gene Expression Regulation, Developmental , Adaptor Proteins, Vesicular Transport/antagonists & inhibitors , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Biomarkers/metabolism , Blastocyst/cytology , Ectoderm/cytology , Ectoderm/growth & development , Embryonic Development , Female , Fertilization in Vitro , Gene Expression Profiling , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Male , Oocytes/cytology , Oocytes/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Spermatozoa/cytology , Spermatozoa/metabolism , Swine , Transcription, Genetic
19.
Diagnostics (Basel) ; 10(6)2020 Jun 05.
Article in English | MEDLINE | ID: mdl-32517113

ABSTRACT

Magnetic resonance cholangiopancreatography (MRCP) has been widely used in clinical practice, and recently developed compressed-sensing accelerated MRCP (CS-MRCP) has shown great potential in shortening the acquisition time. The purpose of this prospective study was to evaluate the clinical feasibility and image quality of optimized breath-hold CS-MRCP (BH-CS-MRCP) and conventional navigator-triggered MRCP. Data from 124 consecutive patients with suspected pancreaticobiliary diseases were analyzed by two radiologists using a five-point Likert-type scale. Communication between a cyst and the pancreatic duct (PD) was analyzed. Signal-to-noise ratio (SNR) of the common bile duct (CBD), contrast ratio between the CBD and periductal tissue, and contrast-to-noise ratio (CNR) of the CBD and liver were measured. Optimized BH-CS-MRCP showed significantly fewer artifacts with better background suppression and overall image quality. Optimized BH-CS-MRCP demonstrated communication between a cyst and the PD better than conventional MRCP (96.7% vs. 76.7%, p = 0.048). SNR, contrast ratio, and CNR were significantly higher with optimized BH-CS-MRCP (p < 0.001). Optimized BH-CS-MRCP showed comparable or even better image quality than conventional MRCP, with improved visualization of communication between a cyst and the PD.

20.
BMC Res Notes ; 13(1): 144, 2020 Mar 11.
Article in English | MEDLINE | ID: mdl-32160914

ABSTRACT

OBJECTIVES: Pig pluripotent stem cells have tremendous potential because the pig is a valuable animal as both an agricultural resource and as a preclinical model of human therapy. To date, a lack of understanding of pig pluripotency has inhibited the derivation of embryonic stem cells (ESCs) and transgene-free induced pluripotent stem cells. Therefore, there has been no accessible or reliable transcriptome data for researching the genuine pig pluripotency network. Our previous study isolated authentic pig ESCs, which had teratoma-forming and direct differentiation ability, that were derived by activating the FGF2, ACTIVIN A, and WNT pathways. Here, we aimed to provide detailed information on transcriptome data of the newly derived pig ESCs and perform a comparative analysis with pig preimplantation embryo transcriptomes in a public database. DATA DESCRIPTION: The transcriptome data of ESCs derived from in vitro fertilized and parthenogenetic embryos were generated by HiSeq 2500. Then, differentially expressed genes (DEGs) from each sample were compared with fibroblasts, and gene expression profiling was carried out for comparative analysis. Our data, as the first transcriptome dataset for genuine pig pluripotent cells, could be a general reference for explaining the molecular mechanism of species-specific pluripotency and improving understanding of the embryo development of domestic animals.


Subject(s)
Embryo, Mammalian/cytology , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Pluripotent Stem Cells/metabolism , Swine/genetics , Animals , Quality Control
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