Expression Study of Pluripotency Marker Genes  in Gold Fish, Carassius auratrus

Platelet-rich plasma (PRP) and its byproduct platelet-poor plasma (PPP) are rich sources of cytokines in tissue damage repair. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have received more and more attention for their ability to treat multiple diseases. The purpose of our study was to investigate the biologic action of PPP and PRP on BM-MSCs. The adipogenic potential of BM-MSCs revealed no obvious change, but the osteogenic ability of BM-MSCs was enhanced after treated with PRP. CCK8 assays and cell colony formation assays showed that PRP promoted cell proliferation, while this effect of PPP was not obvious. No obvious difference was found in cell cycle and apoptosis of BM-MSCs between PRP and PPP treatment. Expression of β-galactosidase, a biological marker of senescence, was decreased upon PRP treatment which indicated that PRP provided significant protection against cellular senescence. The migratory capacity of BM-MSCs was detected by scratch and transwell assays. The results indicated that PRP could affect the migration ability of BM-MSCs. From immunofluorescence detection and western blot, we demonstrated that the level of epithelial-mesenchymal transition-related proteins was changed and several pluripotency marker genes, including Sox2, Sall4, Oct4, and Nanog, were increased. Finally, the expression of the key signal pathway such as PI3K/AKT was examined. Our findings suggested that PRP promoted cell migration of BM-MSCs via stimulating the signaling pathway of PI3K/AKT.

Download Full-text

82 Stage-specific expression of lineage marker genes and pluripotency marker distribution in porcine pre-implantation embryos

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab82 ◽

2020 ◽

Vol 32 (2) ◽

pp. 167

Author(s):

J. Oh ◽

M. Lee ◽

D. Lee ◽

K. Choi ◽

S. Kim ◽

...

Keyword(s):

Internal Control ◽

Inner Cell Mass ◽

Electric Pulse ◽

Cell Mass ◽

Expression Level ◽

Marker Genes ◽

Lineage Specification ◽

Cell Stage ◽

Specific Expression ◽

Pluripotency Marker

Lineage specification in pre-implantation embryos has been revealed, and it was expedited recently by single cell studies. However, data on expression marker genes and proteins in porcine embryos were still missing. We aimed to investigate the expression and distribution of marker genes and proteins, respectively, in IVF and parthenogenetically activated (PA) embryos. For this, cumulus-free oocytes were co-incubated with sperm in modified Tris-buffered medium (mTBM) for 5h and PA was performed using an electric pulse in activation medium. Following this, the embryos were incubated in porcine zygote medium 3 (PZM3). We first tested gene expression level of lineage candidates (internal control; β-actin). In IVF embryos (30, 25, 20, 15, 10, and 5 embryos pooled on Day 2, 3, 4, 5, 6, and 7; replicated 3 times), trophectoderm (TE)-specific genes (Dab2, Gata3) showed peaks on Day 4-5. Within the 2 genes, Dab2 had an earlier peak than Gata3. Inner cell mass (ICM) marker candidates (Nanog, Sox2, and Hnf4a) had diverse patterns. The Nanog and Sox2 genes had peak expression on Day 3. The Nanog expression dropped gradually, but Sox2 dropped suddenly on Day 4. Otherwise, Hnf4a expressed little in Day 3 and expression was sustained from Day 4 to 7. Primitive endoderm markers showed the highest expression on Day 4. We also checked expression level of ICM markers (Sox2, Oct4, and Nanog) in PA embryos (20, 20, 20, 10, and 5 embryos were pooled in 2, 4, 6-8 cells, morula, early, and late blastocyst stages; replicated 3 times). Expression of markers was similar (the highest in the 6-8-cell stage; at least 7.3-, 4.5-, and 3.7-fold compared with the other stages in Sox2, Oct4, and Nanog). We used analysis of variance and Tukey's test for statistical analysis. Following this, we conducted immunocytochemistry with both IVF and PA embryos (20 in each condition). Primary antibodies were treated overnight at 4°C and appropriate secondary antibodies were treated 1h at room temperature. In the case of IVF, well-known ICM markers (SOX2, OCT4, NANOG, and SOX17) showed restricted distribution in nuclei of ICM cells. However, DAB2 was distributed in the cytoplasm of TE cells. In PA embryos, SOX2 and NANOG distributions were similar to IVF. The OCT4 in ICM cells from morula to early blastocyst was restricted, but not in Day 7 embryos. In conclusion, marker genes showed diverse expression pattern in IVF, but all ICM-specific genes had a similar pattern in PA. Also, ICM marker proteins were restricted in nuclei of ICM cells only except Day 7 PA. Our results provide eye-opening information on marker contribution to lineage specification of porcine embryos. This work was supported by the National Research Foundation in Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03032256, NRF-2019R1C1C1004514).

Download Full-text

Spheroid Culture of Mesenchymal Stem Cells

Stem Cells International ◽

10.1155/2016/9176357 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 138

Author(s):

Zoe Cesarz ◽

Kenichi Tamama

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Biology ◽

Molecular Mechanisms ◽

Replicative Senescence ◽

Marker Genes ◽

Adherent Cell ◽

Pluripotency Marker ◽

Adherent Cell Culture ◽

Microarray Analyses

Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.

Download Full-text

656: Comparative Expression Study of Selected Marker Genes on Paired Prostate Tissue Samples

The Journal of Urology ◽

10.1016/s0022-5347(18)30896-6 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 220-220

Author(s):

Susanne Fuessel ◽

Susanne Unversucht ◽

Rainer Koch ◽

Gustavo B. Baretton ◽

Michael Froehner ◽

...

Keyword(s):

Prostate Tissue ◽

Marker Genes ◽

Expression Study ◽

Tissue Samples

Download Full-text

266 IDENTIFICATION OF Oct-4 AND Nanog, THE TWO PLURIPOTENCY MARKER GENES IN RABBIT PRE-IMPLANTATION-STAGE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab266 ◽

2006 ◽

Vol 18 (2) ◽

pp. 240

Author(s):

Z. Tancos ◽

J. Kobolak ◽

A. Baji Gal ◽

A. Dinnyes

Keyword(s):

Detection System ◽

Direct Sequencing ◽

Free Water ◽

Marker Genes ◽

Fluorescence Measurement ◽

Coding Region ◽

Sequence Detection ◽

Pluripotency Marker ◽

Mouse Sequence ◽

Stage Embryo

Several genes, including Oct-4 and Nanog, coordinate the embryogenesis of mammalian embryos. Whereas Oct-4 has an activator effect, the Nanog protein blocks the transcription of several genes in early stages; however, the product of these two genes appears parallel and directs early embryogenesis. The goal of this work was to isolate the Oct-4 and Nanog genes from rabbit, based on the sequences of other species published so far. The sequence of known genes has been analyzed, and primers have been designed based on similarity of sequences. Oocyte-to-blastocyt-stage embryos were collected from superovulated rabbits in RNase-free water. Embryonic mRNA was isolated by using the Dynal mRNA isolation KIT (Dynal, Biotech, Oslo, Norway). Real-time PCR was performed in a ABI PRISM� 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The reaction mixture consisted SYBR� Green PCR Master Mix (Applied Biosystems), 300 nM of each primer, and an 1/8 aliquot of the embryo cDNA in a 25-�L final volume. The cDNA template was denatured by heating to 95�C for 10 min and amplified by 45 cycles of 95�C for 15 s and 60�C for 1 min, with a single fluorescence measurement at each cycle. When the reaction was finished, melting curves were plotted to confirm product purity. The reaction mix was electrophoretically separated to facilitate fragment isolation. In the case of Nanog, a 131-bp fragment was cloned. Sequence analysis revealed significant homology with the mouse sequence. Based on this finding, new primers were designed in order to isolate a larger fragment as well as the genomic copy of the gene. In the case of Oct-4, several combinations of primers were tested, because of the rather conservative sequence of the Oct-4 transcription factor family. As a result, an optimal primer pair was found that yielded a 450 bp fragment, expected according to known sequences. After direct sequencing, there was a high similarity to Oct-4 genes, indicating that the isolated cDNA is probably part of the rabbit Oct-4 cDNA. For further analysis, the cDNA fragments of both genes were isolated (MinElute Gel Extraction Kit; Qiagen, Valencia, CA, USA) and cloned into bacterial plasmids (TOP 10 Cloning KIT; Invitrogen, Carlsbad, CA, USA). At this point, it can be stated that: (1) both genes have been successfully identified in rabbit genome, (2) a method has been developed to detect expression of genes, and (3) gene-specific primers have been produced. Our further goal is to clone the whole coding region of the genes and to identify the sequence. The cloned fragments will be used for in situ hybridization in implanted stage embryo sections. This research was supported by EU-FP6-MEXT-CT-2003-59582, Wellcome Trust (Grant No. 070246), OTKA T046171, and NKTH BIO-00017/2002.

Download Full-text

In Vitro Derivation of Functional Sertoli-Like Cells from Mouse Embryonic Stem Cells

Cell Transplantation ◽

10.1177/0963689718797053 ◽

2018 ◽

Vol 27 (10) ◽

pp. 1523-1534 ◽

Cited By ~ 2

Author(s):

Dong-Won Seol ◽

Seah Park ◽

Eun Young Shin ◽

Jae Ho Chang ◽

Dong Ryul Lee

Keyword(s):

Immune Modulation ◽

Marker Gene ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Marker Genes ◽

Basal Region ◽

Supporting Cells ◽

Pluripotency Marker ◽

Immune Related Genes

Sertoli cells (SCs) in the mammalian testes are well known as supporting cells of spermatogenesis, but have recently become an attractive source of cell therapy because of their capacity for immune modulation and trophic effects. In order to increase their applicable efficacy, we demonstrate a novel differentiation method for mouse embryonic stem cell (ESC)-derived Sertoli-like cells (SLCs) via the intermediate mesoderm (IM). We show that IM derived from an induction of 6 days expressed markers such as Wt1, Lhx1, Pax2 and Osr1, and that a sequential induction of 6 days resulted in ESC-SLCs. The SLCs expressed their marker genes ( Sf1, Sox9, Gata4, Wt1, Fshr and Scf), but the pluripotency-marker gene Oct4 was decreased. After sorting by FSHR expression, high-purity (> 90%) SLCs were collected that showed distinct characteristics of SCs such as high phagocytic and immune modulation activities as well as the expression of immune-related genes. In addition, when transplanted into the seminiferous tubule of busulfan-treated mice, SLCs re-located and were maintained in the basal region of the tubule. These results demonstrated that our robust sequential differentiation system produced functional SLCs from mouse ESCs in vitro.

Download Full-text