REX1 mRNA Research Articles

KLF3 promotes the 8-cell-like transcriptional state in pluripotent stem cells.

ObjectivesMouse embryonic stem cell (mESC) culture contains various heterogeneous populations, which serve as excellent models to study gene regulation in early embryo development. The heterogeneity is typically defined by transcriptional activities, for example, the expression of Nanog or Rex1 mRNA. Our objectives were to identify mESC heterogeneity that are caused by mechanisms other than transcriptional control.Materials and methodsKlf3 mRNA and protein were analysed by RT‐qPCR, Western blotting or immunofluorescence in mESCs, C2C12 cells, early mouse embryos and various mouse tissues. An ESC reporter line expressing KLF3‐GFP fusion protein was made to study heterogeneity of KLF3 protein expression in ESCs. GFP‐positive mESCs were sorted for further analysis including RT‐qPCR and RNA‐seq.ResultsIn the majority of mESCs, KLF3 protein is actively degraded due to its proline‐rich sequence and highly disordered structure. Interestingly, KLF3 protein is stabilized in a small subset of mESCs. Transcriptome analysis indicates that KLF3‐positive mESCs upregulate genes that are initially activated in 8‐cell embryos. Consistently, KLF3 protein but not mRNA is dramatically increased in 8‐cell embryos. Forced expression of KLF3 protein in mESCs promotes the expression of 8‐cell‐embryo activated genes.ConclusionsOur study identifies previously unrecognized heterogeneity due to KLF3 protein expression in mESCs.

Effects of abundances of OCT-4 mRNA transcript on goat pre-implantation embryonic development

Unlike in mice, the function of pluripotent markers in early embryonic development of domestic animals remains to be elucidated and this may account for the failure to establish embryonic stem cell lines for these species. To study the functions of the OCT-4 protein which has important actions in maintenance of pluripotent and self-renewal processes during early embryonic development, there was induced reduction in relative abundance of OCT-4 mRNA transcript during goat early embryonic development by using RNA interference techniques. The injection of OCT-4 siRNA into goat IVF presumptive zygotes resulted in a decrease in the relative abundance of OCT-4 mRNA transcript; however, there was development of these embryos to the blastocyst stage at the same rate as there was in the control group. The blastocysts from the treated groups had a similar number of TE, ICM, and total cells compared to those from the control group. Although there was a greater relative abundance of NANOG, REX1, and CDX2 mRNA transcript in the embryos injected with siRNA at the 8–16 cell stage, the relative transcript abundances were similar for the control and treatment groups at the blastocyst stage. The relative abundance of SOX2 mRNA transcript was similar for the treatment and control group. It, therefore, is concluded that inhibition of abundances of OCT-4 mRNA transcript to about 20 % of that of the untreated control group did not affect blastocyst formation rate in goats. The functions of OCT-4 in maintaining ICM and TE integrity, however, remains to be assessed.

Generation of sepsis encephalopathy patient-specific inducible pluripotent stem cells with urine cells

To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.

Expression of pluripotency-related genes is highly dependent on trichostatin A-assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos.

The present study sought to examine whether trichostatin A (TSA)-assisted epigenetic transformation of porcine bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) affects the transcriptional activities of pluripotency-related genes (Oct4, Nanog, c-Myc, Sox2 and Rex1), multipotent stemness-related gene (Nestin) and anti-apoptotic/anti-senescence-related gene (Survivin). Epigenetically transformed or non-transformed BM-MSCs that had been transcriptionally profiled by qRT-PCR and had been analysed for different stages of apoptosis progression provided a source of nuclear donor cells for the in vitro production of cloned pig embryos. TSA-mediated epigenomic modulation has been found to enhance the multipotency extent, stemness and intracellular anti-ageing properties of porcine BM-MSCs. This has been confirmed by the relative abundances for Nanog, c-Myc Rex1, Sox2 and Survivin mRNAs in TSA-exposed BM-MSCs that turned out to be significantly higher than those of TSA-unexposed BM-MSCs. Additionally, TSA-assisted epigenomic modulation of BM-MSCs did not impact the caspase-8 activity, Bax protein expression and the incidence of TUNEL-positive cells. In conclusion, the considerably elevated quantitative profiles of Sox2, Rex1, c-Myc, Nanog and Survivin mRNA transcripts seem to trigger improved reprogrammability of TSA-treated BM-MSC nuclei in cloned pig embryos that thereby displayed remarkably increased blastocyst formation rates as compared to those noticed for embryos derived from TSA-untreated BM-MSCs.

Increased expression of stemness genes REX-1, OCT-4, NANOG, and SOX-2 in women with ovarian endometriosis versus normal endometrium: A case-control study.

BackgroundEndometriosis is a common, chronic inflammatory disease which is defined as an overgrowth of endometrial tissue outside the uterine cavity. The etiology of this disease is complex and multifactorial but there is a strong evidence that supports the presence of endometrial stem cells and their possible involvement in endometriosis.ObjectiveIn this study, we analyzed the mRNA expression of REX-1 stemness gene and reconsidered three other stemness genes SOX-2, NANOG, OCT-4 in women with endometriosis compared to normal endometrium.Materials and MethodsTen ectopic and ten eutopic tissue samples along with 23 normal endometrium specimens were recruited in this study. The expression levels of OCT-4, NANOG, SOX-2, and REX-1 genes were evaluated by the quantitative real-time polymerase chain reaction.ResultsThe transcription levels of OCT-4, NANOG, and SOX-2 mRNA were significantly increased in ectopic lesions compared with eutopic and control group (p = 0.041, p = 0.035, p = 0.048), although the REX-1 mRNA increase was not significant between endometriosis and control groups. Also, there were differences in the expression level of these genes in normal endometrium during the menstrual cycles (p = 0.031, p = 0.047, p = 0.031).ConclusionBased on our data, we confirm the dynamic role of stemness genes in proliferation and growth of normal endometrium during the menstrual cycle and conclude that differential expression levels of these genes may contribute to the pathophysiology of endometriosis.

Abstract 3315: Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis

Abstract The existence of a cancer stem cell (CSC) subset is well established in hepatocellular carcinoma (HCC). This subset can be characterized, among others, by its CD133 expression. Since the discovery of this marker, studies have revealed deregulated pathways and epigenetic alterations associated with this subset, but few have focused on changes in DNA methylation. This study aims to study the aberrant DNA methylation patterns governing CD133+ CSC-driven HCC. Results from Infinium HumanMethylation450 BeadChip (HuMet450 BeadChip) have led to the discovery of differential methylation patterns between CD133+ liver CSCs and matched CD133- differentiated samples. ZFP42/REX1 is one of the top-ranking differentially methylated candidates. Probes corresponding to its promoter are extensively hypermethylated in the CD133+ samples. We validated the down-regulation of REX1 mRNA levels in sorted CD133+ HCC cell lines (n=4) and HCC clinical samples (n=46, p&amp;lt;0.05), as compared to CD133- counterparts and non-tumor liver samples. REX1 and CD133 expressions are also significantly negatively correlated in HCC cell lines (n=8, R2=0.65). To explore the hypothesis that promoter DNA hypermethylation modulates REX1 expression, we treated HCC cells with 5-aza. Only low-REX1-expressing cells re-expressed REX1. Results of pyrosequencing of a CG island of interest located in the REX1 promoter show that REX1 expression appears to be negatively correlated with the degree of DNA methylation in HCC cell lines (n=4); and that HCC samples (n=15) exhibit a predominantly hypermethylated state compared to non-tumor samples (n=16). To add onto the clinical relevance of REX1, HuMet450 BeadChip datasets of HCC sample cohorts were obtained from Gene Expression Omnibus and the Cancer Genome Atlas. In these cohorts, the REX1 promoter region is significantly hypermethylated in HCC samples and this methylation is correlated to poor prognosis as well as increased expression of stemness markers. In addition to DNA methylation, we also discovered other regulatory mechanisms for REX1 expression involving histone methylation and acetylation. Functional studies with stable REX1 over-expressing and knock-down HCC cells suggest REX1 expression to negatively correlate with the cells' abilities to migrate, invade, and form foci in vitro; and initiate tumor formation, metastasize and self-renew in vivo. In summary, REX1 is a tumor-suppressor gene that is frequently down-regulated in human HCCs. The multiplicity of REX1 deactivating mechanisms together facilitates REX1 down-regulation to mediate various cancer and stem-like properties in the disease. Citation Format: Steve Tin Chi Luk, Man Tong, Kai Yu Ng, Kevin Yuk-Lap Yip, Xin Yuan Guan, Stephanie Ma. Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3315.

Abstract 4352: Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis

Abstract The existence of a cancer stem cell (CSCs) subset is well established in hepatocellular carcinoma (HCC). This subset can be characterized by its CD133 expression. Since the discovery of this marker, studies have revealed deregulated pathways and epigenetic alterations associated with this subset, but few have focused on changes in DNA methylation. This study aims to fill this research gap by discovering aberrant DNA methylation patterns governing CD133+ CSC-driven HCC. Results from Infinium HumanMethylation450 BeadChip (HuMet450 BeadChip) have led to the discovery of differential methylation patterns between CD133+ liver CSCs and matched CD133- differentiated samples. ZFP42/REX1 is one of the top-ranking differentially methylated candidates. Probes corresponding to its promoter are extensively hypermethylated in the CD133+ samples. To explore the hypothesis that promoter DNA hypermethylation modulates REX1 expression, REX1 mRNA levels were first validated to be down regulated in sorted CD133+ HCC cell lines (n=2) and HCC samples (n=46, p&amp;lt;0.05) by RT-qPCR. Upon 5-AZA treatment, only low-REX1-expressing cells re-expressed REX1. By in silico prediction, a CpG island of interest located in the REX1 promoter region was selected for further investigation. Results of pyrosequencing of this CpG island show that REX1 expression appears to be negatively correlated with the degree of DNA methylation in HCC cell lines (n=3); and that HCC samples (n=13) exhibit a predominantly hypermethylated state compared to the matched non-tumor samples. To add onto the clinical relevance of REX1, two HuMet450 BeadChip datasets of HCC sample cohorts (GSE54503 and GSE60753) were obtained from Gene Expression Omnibus (GEO). In these cohorts, the CpG probes corresponding to the REX1 promoter region are significantly hypermethylated (p&amp;lt;0.01) in HCC samples compared to the adjacent non-tumor tissues. Lentiviral-based approaches were adopted to establish stable REX1 overexpressing and knockdown cells in HCC cell lines. REX1 expression appears to be negatively correlated with the cells’ abilities to migrate, invade, and form foci in vitro; and tumorigenicity in vivo. ChIP-seq has been adopted to study the DNA-binding property of REX1 in HCC cells. These findings suggest that REX1 may play a part in mediating various cancer properties in HCC. Citation Format: Steve Tin-Chi Luk, Man Tong, Kai Yu Ng, Kevin Yuk-Lap Yip, Xin Yuan Guan, Stephanie Ma. Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2017-4352

Basic Fibroblast Growth Factor Regulates REX1 Expression Via IL-6 In Stem Cells Isolated From Human Exfoliated Deciduous Teeth.

Basic fibroblast growth factor (bFGF) regulates pluripotent marker expression and cellular differentiation in various cell types. However, the mechanism by which bFGF regulates REX1 expression in stem cells, isolated from human exfoliated deciduous teeth (SHEDs) remains unclear. The aim of the present study was to investigate the regulation of REX1 expression by bFGF in SHEDs. SHEDs were isolated and characterized. Their mRNA and protein expression levels were determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In some experiments, chemical inhibitors were added to the culture medium to impede specific signaling pathways. Cells isolated from human exfoliated deciduous tooth dental pulp tissue expressed mesenchymal stem cell surface markers (CD44, CD73, CD90, and CD105). These cells differentiated into osteogenic and adipogenic lineages, when appropriately induced. Treating SHEDs with bFGF induced REX1 mRNA expression and this effect was attenuated by pretreatment with FGFR or Akt inhibitors. Cycloheximide pretreatment also inhibited the bFGF-induced REX1 expression, implying the involvement of intermediate molecule(s). Further, the addition of an IL-6 neutralizing antibody attenuated the bFGF-induced REX1 expression by SHEDs. In conclusion, bFGF enhanced REX1 expression by SHEDs via the FGFR and Akt signaling pathways. Moreover, IL-6 participated in the bFGF-induced REX1 expression in SHEDs. J. Cell. Biochem. 118: 1480-1488, 2017. © 2016 Wiley Periodicals, Inc.

IL-6 regulates stress-induced REX-1 expression via ATP-P2Y1 signalling in stem cells isolated from human exfoliated deciduous teeth

ObjectiveTo investigate the relationship between ATP and IL-6 in mechanical stress-induced REX-1 expression in SHEDs. MethodsCells were stimulated with mechanical stress (0–2.5gcm−2), IL-6 (0.1–5ng/ml), or ATP (10–100μM) for 2h in serum-free media. IL-6 and REX-1 expression was examined by qualitative and quantitative polymerase chain reaction. ATP release was measured using a bioluminescence assay. The molecular mechanisms of the signalling pathways were investigated using chemical inhibitors. ResultsMechanical stress induced IL-6 and REX-1 mRNA expression and ATP release. JAK inhibitor I inhibited the increase in REX-1 expression and ATP release but not IL-6 induction. Furthermore, suramin inhibited the upregulation of REX-1 mRNA expression but not ATP release. Exogenous IL-6 promoted both ATP release and REX-1 expression. The IL-6-induced REX-1 expression was attenuated by a P2Y1-specific receptor antagonist. Moreover, REX-1 expression was upregulated in a dose-dependent manner by the addition of ATP or a P2Y1 agonist. This inductive effect was abolished by the P2Y1-specific receptor antagonist. ConclusionsATP-P2Y1 signalling is involved in IL-6-regulated stress-induced REX-1 expression in SHEDs. These results imply the participation of mechanical stress, IL-6, and ATP in regulating the expression of REX-1, a pluripotent stem cell marker.

Effect of basic fibroblast growth factor on pluripotent marker expression and colony forming unit capacity of stem cells isolated from human exfoliated deciduous teeth

Human dental pulp of exfoliated deciduous teeth contains the population of cells that exhibited mesenchymal stem cell (MSC) characters. Though, a cell amplification process is indeed required to secure an adequate cell number for such a potential employment. Several publications suggested the alteration of MSCs upon in vitro culture, for example, the decrease in proliferation and the loss of stem cell characters. Here, we investigated an influence of basic fibroblast growth factor (bFGF) on stem cells isolated from human exfoliated deciduous teeth (SHEDs) with respect to cell proliferation, colony forming unit efficiency and stem cell marker expression in both short- and long-term cultures. For short-term bFGF treatment, SHEDs were treated with bFGF for 48 h. While, in long-term bFGF supplementation, SHEDs were maintained in culture and continuous passage upon confluence in medium supplemented with bFGF. Cells at passage (P) 5 and 10 were employed for characterization. Our results showed that short-term bFGF treatment enhanced OCT4, REX1, and NANOG mRNA expression as well as colony forming unit ability. The FGFR inhibitor pretreatment was able to attenuate the influence of bFGF on pluripotent stem cell marker expression, confirming bFGF function. In addition, cells cultured in high passage number had decreased in cell proliferation, colony forming unit capacity, and pluripotent stem cell maker mRNA expression. However, bFGF supplementation in culture medium enhanced both pluripotent stem cell marker expression and colony forming unit capacity in later passage, though the effect was not robust. Together, these results indicate that high passage number may attenuate pluripotent properties of SHEDs and bFGF supplementation could be the beneficial approach to maintain SHEDs' stemness properties.

Fluid Flow Modulation of Murine Embryonic Stem Cell Pluripotency Gene Expression in the Absence of LIF

Fluid forces are strong modulators of cell fate and fundamental components of spinner flask bioreactors used for stem cell expansion and differentiation. Here, we investigated the effects of fluid forces on murine embryonic stem cells (mESCs) in the absence of Leukemia Inhibitory Factor (LIF) using parallel-plate flow chambers. Cells were seeded onto gelatin-coated glass slides and grown for 2.5 days before exposure to fluid forces. Pluripotency marker gene expression was quantified by qPCR. An average shear stress of 0.6 Pa applied for 24 h in the absence of LIF and presence of high molecular weight dextran increased Oct4 and Sox2, decreased Nanog, and did not change in Rex1 mRNA levels in comparison to statically cultured cells in the presence of LIF. At 0.3 Pa shear stress, Oct4 and Sox2 expression increased, with a reduction in Nanog and Rex1 levels. The presence of pulsation significantly increased expression of Rex1 and Nanog, but not expression of Oct4 or Sox2, compared to cells exposed to steady flow for 24 h. This study suggests incorporation of high cell–cell contact, viscosity elevation with dextran, moderate shear stress (0.6 Pa), and the presence of pulsatility in bioreactor expansion protocols for mESCs to support maintenance of pluripotency.

Knockdown of the Potential Cancer Stem-Like Cell Marker Rex-1 Improves Chemotherapeutic Effects in Gliomas

In the present study, we show that Rex-1 mRNA and protein are found at high levels in both 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant glioma cell subpopulations and malignant glioblastoma multiforme (GBM) tissue. We used a combination therapy of small interfering RNA (siRNA) against Rex-1 (siRex-1) and BCNU to target GBM cells. Rex-1 siRNA/BCNU treatment resulted in growth inhibition and a diminished S phase. The treatment efficiently induced P38/JNK and Akt/PI3K/GSK3β signaling and led to apoptosis both in vitro and in vivo. We also show that Rex-1/ABCG2 (ATP binding cassette transporter G2)-coexpressing subpopulations were chemoresistant; however, BCNU was not a substrate for ABCG2. siRex-1 treatment led to cell death in GBM subpopulations by promoting apoptosis. Moreover, siRex-1/BCNU combination therapy targeted both the major population and cancer stem cell-like subpopulations. Our findings are important for the development of clinical applications to treat GBM.

A novel, efficient method to derive bovine and mouse embryonic stem cells with in vivo differentiation potential by treatment with 5-azacytidine

Pluripotent embryonic stem cells (ESCs) were first isolated nearly three decades ago from mice, yet efficient ESC isolation has been limited to rodents and primates to date. We report a novel and robust technique for isolating ESCs from mammalian pre-implantation embryos by altering the epigenotype of embryonic explants and using pressed zona pellucida-free blastocysts. We first examined this technique for murine ESC derivation. Compared with controls, murine ESCs were efficiently derived when explants were exposed to 1μM 5-azacytidine, an epigenetic modifier that causes DNA demethylation (56.1% vs 31.6%; P < 0.01). Mouse ESCs stained positively for alkaline phosphatase, expressed markers of pluripotency including Oct4, Rex1 and SSEA1 and formed teratomas when injected into Severe Combined Immuno-Deficient (SCID) mice. The approach was subsequently used for bovine ESC derivation. In bovine a higher concentration of 5-azacytidine (5 μM) was required to elicit a response. This technique resulted in up to 18 times more efficient isolation of pluripotent cells than traditional methods (71.4% vs 4.0%; P < 0.001). These putative bovine ESCs expressed OCT4, REX1 mRNA and SSEA-1 and SSEA-4 proteins; and were able to form embryoid bodies in vitro and teratomas when injected in Severe Combined Immuno Deficient (SCID) mice. This is the first report on derivation of ESCs with both in vitro and in vivo differentiation potential in a livestock species.

Analysis of Rex1 (zfp42) function in embryonic stem cell differentiation

Rex1 (zfp42) is a zinc finger protein expressed primarily in undifferentiated stem cells, both in the embryo and the adult. Upon all-trans retinoic acid induced differentiation of murine embryonic stem (ES) cells, Rex1 mRNA levels decrease several fold. To characterize the function(s) of Rex1 more extensively, we generated Rex1 double knockout ES cell lines. The disruption of the Rex1 gene enhanced the expression of ectoderm, mesoderm, and endoderm markers as compared to wild-type (Wt) cells. We propose that Rex1 acts to reduce retinoic acid induced differentiation in ES cells. We performed microarray analyses on Wt and Rex1-/- cells cultured in the presence or absence of LIF to identify potential Rex1 targets. We also evaluated gene expression in a Wt line that overexpresses Rex1 and in a Rex1-/- line in which Rex1 expression was restored. These data, taken together, suggest that Rex1 influences differentiation, cell cycle regulation, and cancer progression.

Regulation of the Pluripotency Marker Rex-1 by Nanog and Sox2

Rex-1 (Zfp-42) is a known marker for undifferentiated embryonic stem cells and teratocarcinoma cells. However, the mechanism by which Rex-1 is regulated in pluripotent cells remains unresolved. Here we report that Nanog, an Nk-2 homeodomain protein known for its role in maintaining stem cell pluripotency, is a transcription activator for the Rex-1 promoter. Knockdown of Nanog in embryonic stem cells resulted in a reduction of Rex-1 expression, whereas forced expression of Nanog in P19 stimulated Rex-1 expression. Employing a Rex-1 reporter, we demonstrate that Nanog transactivates Rex-1 directly. Serial deletion studies mapped the Nanog-responsive element between -187 and -286 of the Rex-1 promoter. Although Oct-3/4 and Sox2 can both transactivate Rex-1 promoter, only Sox2 cooperates with Nanog in up-regulating Rex-1. Furthermore, we demonstrate that the C terminus of Nanog is responsible for transactivating the Rex-1 promoter, a function that can be substituted for by a viral transactivator Vp16 efficiently in NIH3T3 cells but less so in P19 cells. Taking these findings together, we conclude that Rex-1 is a direct target of Nanog, which is augmented by Sox2 and Oct-3/4.

Decreased expression of the human stem cell marker, Rex-1 (zfp-42) , in renal cell carcinoma

The Rex-1 (Zfp-42) gene encodes a zinc finger family transcription factor which is highly expressed in mouse and human embryonic stem cells. It is one of several gene markers used to identify human stem cells. While several organs are known to harbor adult human stem cells, the presence and distribution of stem cells in both the normal and neoplastic adult kidney remains largely unknown. In this study we evaluated Rex-1 mRNA and protein expression in normal and malignant kidney tissue specimens from human patients. Rex-1 mRNA expression was determined using both reverse transcription and real-time PCR. REX1 protein expression was assessed by western analysis and immunohistochemistry, using an affinity-purified, polyclonal antibody to the REX1 protein. We found that 14 of 15 (93%) non-tumor renal parenchymal specimens demonstrated Rex-1 mRNA, compared with 5 of 14 (36%) renal tumors (P < 0.005). REX1 protein expression was detected in 21 of 23 (91%) non-tumor and in 7 of 19 (37%) tumor specimens (P < 0.001). Furthermore, in six of these seven renal tumor specimens where REX1 protein expression was detected, the levels were at least 3-fold lower than those in adjacent, normal kidney tissue. There were no differences in Rex-1 mRNA or protein expression among the various histologic subtypes of renal tumors (clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and oncocytoma). Immunohistochemical staining confirmed the absence of REX1 in three renal tumor specimens (two clear cell and one papillary carcinoma), while the REX1 protein was detected in a small percentage of proximal tubular cells in normal renal tissue. Immunohistochemical staining of another stem cell marker, OCT4, demonstrated a similar pattern of protein expression in a small percentage of normal renal proximal tubular cells. In summary, we were able to detect Rex-1 mRNA and protein expression in over 90% of normal renal parenchymal specimens and we observed a significant reduction in REX1 expression in renal tumor specimens of all histologic subtypes.

The Effect of GSK3β Inhibitor on Murine Multipotent Adult Progenitor Cells (mMAPCs).

The Wnt/β-catenin pathway is important for maintenance of the undifferentiated state of human embryonic stem cells (ESCs), hematopoietic stem cells (HSCs) and other stem cells. We here tested if β-catenin activation, by inhibiting GSK3 β with BIO (6-Bromoindirubin-3′-oxime), affects the differentiation status of murine multipotent adult progenitor cells (mMAPCs). mMAPCs were cultured with or without 0.1–2 μ M BIO, LIF, EGF and PDGF-BB, at low density (plating density of 100/cm2 and cells passed every 2 days) or at high confluency (like ESCs). Quantitative RT-PCR (Q-RT-PCR) was done weekly for the ESC-specific pluripotency transcripts, Oct4, Rex1, Nanog, UTF1 and E-Ras, and lineage commitment genes, vWF, VE-cadherin, Sox1, Nestin, CK19, Albumin, HNF3β and AFP. After 2–4 weeks, cells were analyzed by FACS for ESC/MAPC specific antigens, and by immunohistochemsitry for Oct4, β-catenin and E-cadherin protein. Furthermore, cells were differentiated into endothelial cells and hepatocytes by using VEGF-A and HGF/FGF4, respectively for 2 weeks. Q-RT-PCR was used to demonstrate lineage differentiation.BIO caused a dose-dependent clustering of mMAPCs from 4 days after treatment at both cell densities. FACS phenotype of low- and high-density mMAPCs was not affected by BIO. Nanog mRNA was not detected in any of the cell populations. However, Oct4, Rex1, vWF, and CK19 mRNA levels decreased in a BIO dose-dependent manner in mMAPCs maintained at low density, while they acquired higher levels of Sox1 mRNA (P=NS). At high-density, Oct4 and Rex1 mRNA level decreased in a BIO dose-dependent manner for the first week. On the other hand, 0.1 μ M BIO-treatment induced higher levels of Oct4 and Rex1 in cells maintained at high density. β-catenin and E-cadherin protein was highly expressed in clusters of low-density mMAPCs induced by BIO as well as BIO-treated mMAPCs maintained at ESC densities. In both cell densities, only mMAPCs treated with 1 or 2 μM BIO contained large dense mMAPCs clusters that expressed very high levels of β-catenin and E-cadherin. There was no statistical difference of differentiation potential between BIO conditions and cell densities.BIO may not allow culture of mMAPCs without loss of Oct4 as it did for ESCs, but induces clusters like ESCs in a BIO-dose dependent manner. High concentrations of BIO enhances β-catenin and E-cadherin protein expression. BIO may induce lineage commitment of mMAPCs. Despite loss of Oct4 mRNA and protein, MAPCs treated with BIO continue to have the ability to differentiate. Ongoing studies are testing whether BIO treated cells maintained at high density also maintain in vivo engraftment and differentiation potential.

Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

Expression of REX-1, a Gene Containing Zinc Finger Motifs, Is Rapidly Reduced by Retinoic Acid in F9 Teratocarcinoma Cells

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.

Expression of REX-1, a gene containing zinc finger motifs, is rapidly reduced by retinoic acid in F9 teratocarcinoma cells.

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.