Establishment of Bovine Trophoblast Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos Using Two Inhibitors
Xianghua HuangXuejie HanUyunbilig BorjiginManling ZhangShuguang DuoYongchun ZuoYuhang ZhaoTing YunDapeng TaiChen WangJinhua LiXueling LiRongfeng Li
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The trophoblast (TR) is the first to differentiate during mammalian embryogenesis and play a pivotal role in the development of the placenta. We used a dual inhibitor system (PD0325901 and CHIR99021) with mixed feeders to successfully obtain bovine trophoblast stem-like (bTS) cells, which were similar in phenotype to mouse trophoblast stem cells (TSCs). The bTS cells that were generated using this system continually proliferated, displayed a normal diploid karyotype, and had no signs of altered morphology or differentiation even after 150 passages. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81, and TR lineage markers such as CDX2, as determined by both immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta TR cells in vitro. The microarray analysis of bTS cells showed high expression levels of many TR markers, such as TEAD4, EOMES, GATA3, ETS2, TFAP2A, ELF5, SMARCA4 (BRG1), CDH3, MASH2, HSD17B1, CYP11A1, PPARG, ID2, GCM1, HAND1, TDK, PAG, IFN-τ, and THAP11. The expression of many pluripotency markers, such as OCT4, SOX2, NANOG, and GDF3, was lower in bTS cells compared with in vitro-produced blastocysts; however, compared with bovine fetal fibroblasts, the expression of these pluripotency markers was elevated in bTS cells. The DNA methylation status of the promoter regions of OCT4, NANOG, and SOX2 was investigated, which were significantly higher in bTS cells (OCT4 23.90%, NANOG 74.40%, and SOX2 8.50%) compared with blastocysts (OCT4 8.90%, NANOG 34.4%, and SOX2 3.80%). In contrast, two promoter regions of CDX2 were hypomethylated in bTS cells (13.80% and 3.90%) compared with blastocysts (18.80% and 9.10%). The TSC lines that were established in this study may be used either for basic research that is focused on peri-implantation and placenta development or as donor cells for transgenic animal production.Keywords:
Homeobox protein NANOG
Trophoblast
Inner cell mass
Stem cell marker
The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.
Homeobox protein NANOG
Stem cell marker
Nanog Homeobox Protein
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Abstract Background: Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detail morphological and molecular characteristics. Methods: CRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment condition and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes ( KLF4 , OCT4 , SOX2 , NANOG , C-MYC ) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related ( Vimentin , SNAIL1 , TWIST1 , N-Cadherin , E-Cadherin , ZEB1 ) and multi-drug resistant ( ABCB1 , ABCC1 , ABCG2 ) genes was evaluated. Results: Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2 , C-MYC, NANOG and OCT4 in HT-29 and SOX2 , C-MYC and KLF4 in Caco-2 spheroids ( p-value<0.05 ) and increased expression of CRC-CSC markers compared to parental cells ( p-value<0.05 ). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 ( p-value=0.02 ). The significant up-regulation of promoting EMT genes, ZEB1 , Twist1 , E-cadherin and SNAIL1 in HT-29 spheroids ( p-value=0.03 ), SNAIL1 and Vimentin in Caco-2 spheroids ( p-value<0.05 ) and N-cadherin down-regulation in both spheroids were observed. Conclusion: Based on enrichment of CSC-related features in HT-29 and Caco-2 spheroids, our findings suggest CRC spheroids culture as simple, cost-effective and efficient model to imitate the complexity of in vivo CRC tumors including self-renewal, drug resistance and invasion potential for CSC research.
Homeobox protein NANOG
KLF4
Stem cell marker
Spheroid
BMI1
LIN28
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Abstract Background Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics. Methods CRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes ( KLF4 , OCT4 , SOX2 , NANOG , C-MYC ) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related ( Vimentin , SNAIL1 , TWIST1 , N-cadherin , E-cadherin , ZEB1 ) and multi-drug resistant ( ABCB1 , ABCC1 , ABCG2 ) genes was evaluated. Results Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2 , C-MYC, NANOG and OCT4 in HT-29 and SOX2 , C-MYC and KLF4 in Caco-2 spheroids ( p-value < 0.05 ) and increased expression of CRC-CSC markers compared to parental cells ( p-value < 0.05 ). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 ( p-value = 0.02 ). The significant up-regulation of promoting EMT genes, ZEB1 , TWIST1 , E-cadherin and SNAIL1 in HT-29 spheroids ( p-value = 0.03 ), SNAIL1 and Vimentin in Caco-2 spheroids ( p-value < 0.05 ) and N-cadherin down-regulation in both spheroids were observed. Conclusion Enrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.
Homeobox protein NANOG
KLF4
Spheroid
LIN28
Stem cell marker
BMI1
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Homeobox protein NANOG
Nanog Homeobox Protein
Tumor progression
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The transcription factors, including OCT4, NANOG, and SOX2, played crucial roles in the maintenance of self-renewal and pluripotency in embryonic stem cells (ESCs). They expressed in preimplantation mammalian development with spatio-temporal pattern and took part in regulation of development. However, their expression and roles in goat had not been reported. In the present study, the expression of OCT4, NANOG, and SOX2 in goat preimplantation embryos both in vivo and in vitro were detected by real-time RCR and immunofluorescence. For in vivo fertilized embryos, the transcripts of OCT4, NANOG, and SOX2 could be detected from oocytes to blastocyst stage, their expression in morula and blastocyst stages was much higher than other stage. OCT4 protein was detected from oocyte to blastocyst, but the fluorescence was more located-intensive with nuclei from 8-cell stage, its expression present in both inner cell mass (ICM) and trophoblast cells (TE) at blastocyse stage. NANOG protein was similar to OCT4, the signaling of fluorescence completely focused on cell nuclei, while the SOX2 firstly showed nuclei location in morula. Comparing to in vivo fertilized embryo, the mRNA of these three transcription factors could be detected at 8-cell stage in parthenogenetic embryos (in vitro). Thereafter, the expressional level rose gradually along with embryo development. The locations of OCT4 and NANOG proteins were similar to in vivo fertilized embryos, and they located in cell nuclei from morula to blastocyst stage, while SOX2 protein firstly could be detected in cell nuclei at 8-cell stage. These differences suggested that OCT4, NANOG, and SOX2 played different function in regulating development of goat preimplantation embryos. These results may provide a novel insight to goat embryo development and be useful for goat ESCs isolation.
Homeobox protein NANOG
Inner cell mass
Rex1
Nanog Homeobox Protein
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Two critical points of early development are the first and second lineage segregations, which are regulated by a wide spectrum of molecular and cellular factors. Gene regulatory networks, are one of the important components which handle inner cell mass (ICM) and trophectoderm (TE) fates and the pluripotency status across different mammalian species. Considering the importance of goats in agriculture and biotechnology, this study set out to investigate the dynamics of expression of the core pluripotency markers at the mRNA and protein levels.In this experimental study, the expression pattern of three pluripotency markers (Oct4, Nanog and Sox2) and the linage specific markers (Rex1, Gata4 and Cdx2) were quantitatively assessed in in vitro matured (MII) oocytes and embryos at three distinctive stages: 8-16 cell stage, day-7 (D7) blastocysts and D14 blastocysts. Moreover, expression of Nanog, Oct4, Sox2 proteins, and their localization in the goat blastocyst was observed through immunocytochemistry.Relative levels of mRNA transcripts for Nanog and Sox2 in D3 (8-16 cell) embryos were significantly higher than D7 blastocysts and mature oocytes, while Oct4 was only significantly higher than D7 blastocysts. However, the expression pattern of Rex1, as an epiblast linage marker, decreased from the oocyte to the D14 stage. The expression pattern of Gata4 and Cdx2, as extra embryonic linage markers, also showed a similar trend from oocyte to D3 while their expressions were up-regulated in D14 blastocysts.Reduction in Nanog, Oct4, Sox2 mRNA transcription and a late increase in extra embryonic linage markers suggests that the developmental program of linage differentiation is retarded in goat embryos compared to previously reported data on mice and humans. This is likely related to late the implantation in goats.
Homeobox protein NANOG
Rex1
Inner cell mass
Nanog Homeobox Protein
Epiblast
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NANOG, as a key regulator of pluripotency and acting synergistically with other factors, has been described as a crucial transcription factor in various types of cancer. In meningiomas the expression of this marker has not yet been described. With our study, we aimed to identify and localize NANOG and other possible markers of pluripotency, stem cell properties and differentiation in meningioma tissue, to elucidate a possible effect on tumorigenesis. The gene expression levels of NANOG ( NANOG1 and NANOGP8 ), SOX2 , OCT4 , KLF4 , ABCG2 , CMYC , MSI1 , CD44 , NOTCH1 , NES , SALL4B , TP53 , and EPAS1 were quantitatively examined using RT‐qPCR in 33 surgical specimens of low‐ (WHO grade I) as well as in high‐grade (WHO grade II/III) meningiomas with dural tissue as reference. Immunofluorescence co‐localization analysis following confocal fluorescence microscopy for NANOG, OCT4, SOX2, Nestin, KI‐67, and CD44 was also performed. There was a significant overexpression of NANOG , MSI1 , and EPAS1 and a downregulation of NES in all examined tumors. Subgroup analysis (WHO grade I versus grade II/III) revealed differences in the expression of NANOG , CD44 , and MSI1 . We found 1% NANOG‐positive (NANOG+) cells in low‐grade and 2% in grade II/III meningiomas co‐expressing the other mentioned markers in various compositions. In particular, NANOG+ cells expressing SOX2 and OCT4 were successfully identified (26% low‐grade versus 20% high‐grade). Our data reveal an overexpression of NANOG and other markers of pluripotency and stemness in meningiomas. Such potentially pluripotent “stem cell‐like” cells may have an impact on tumorigenesis and progression in human meningiomas.
Homeobox protein NANOG
Rex1
KLF4
Stem cell marker
Nestin
Nanog Homeobox Protein
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Abstract The combined expression of a handful of pluripotency transcription factors (PluriTFs) in somatic cells can generate induced pluripotent stem cells (iPSCs). Here, we report the structural characterization of disordered regions contained in four important PluriTFs, namely Oct4, Sox2, Nanog and Esrrb. Moreover, many post-translational modifications (PTMs) have been detected on PluriTFs, whose roles are not yet characterized. To help in their study, we also present a method i) to produce well-characterized phosphorylation states of PluriTFs, using NMR analysis, and ii) to use them for pull-downs in stem cell extracts analyzed by quantitative proteomics to identify of Sox2 binders.
Homeobox protein NANOG
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To identify and characterize cancer stem cells (CSC) in glioblastoma multiforme (GBM).Four-micrometer thick formalin-fixed paraffin-embedded GBM samples from six patients underwent 3,3-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers NANOG, OCT4, SALL4, SOX2, and pSTAT3. IF IHC staining was performed to demonstrate co-expression of these markers with GFAP. The protein expression and the transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, and STAT3 were investigated using Western blotting (WB) and NanoString gene expression analysis, respectively.DAB and IF IHC staining demonstrated the presence of a CSC population expressing NANOG, OCT4, SOX2, SALL4, and pSTAT3 with the almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4, within GBM. The expression of NANOG, SOX2 and, pSTAT3 but, not OCT and SALL4, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of NANOG, OCT4, SALL4, STAT3, and SOX2 in GBM.This study demonstrated a population of CSCs within GBM characterized by the expression of the CSC markers NANOG, SALL4, SOX2, pSTAT3 and OCT4 at the protein and mRNA levels. The almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4 would support the putative existence of a stem cell hierarchy within GBM.
Homeobox protein NANOG
Nanog Homeobox Protein
Stem cell marker
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Citations (72)
The stemness-associated markers OCT4, NANOG, SOX2, KLF4 and c-MYC are expressed in numerous cancer types suggesting the presence of cancer stem cells (CSCs). Immunohistochemical (IHC) staining performed on 12 lung adenocarcinoma (LA) tissue samples showed protein expression of OCT4, NANOG, SOX2, KLF4 and c-MYC, and the CSC marker CD44. In situ hybridization (ISH) performed on six of the LA tissue samples showed mRNA expression of OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence staining performed on three of the tissue samples showed co-expression of OCT4 and c-MYC with NANOG, SOX2 and KLF4 by tumor gland cells, and expression of OCT4 and c-MYC exclusively by cells within the stroma. RT-qPCR performed on five LA-derived primary cell lines showed mRNA expression of all the markers except SOX2. Western blotting performed on four LA-derived primary cell lines demonstrated protein expression of all the markers except SOX2 and NANOG. Initial tumorsphere assays performed on four LA-derived primary cell lines demonstrated 0-80% of tumorspheres surpassing the 50 µm threshold. The expression of the stemness-associated markers OCT4, SOX2, NANOG, KFL4 and c-MYC by LA at the mRNA and protein level, and the unique expression patterns suggest a putative presence of CSC subpopulations within LA, which may be a novel therapeutic target for this cancer. Further functional studies are required to investigate the possession of stemness traits.
Homeobox protein NANOG
KLF4
Stem cell marker
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Citations (8)