MAB4337 Sigma-AldrichAnti-UTF-1 Antibody, clone 5G10.2

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.

Undifferentiated cell Transcription Factor 1 (UTF1) is a chromatin-bound protein involved in stem cell differentiation. It was initially reported to be restricted to stem cells or germinal tissues. However, recent work suggests that UTF1 is also expressed in somatic cells and that its expression may increase during carcinogenesis. To further clarify the expression profile of UTF1, we evaluated UTF1 expression levels immunohistochemically in eight normal human epithelia (from breast, prostate, endometrium, bladder, colon, oesophagus, lung and kidney) and their corresponding tumours as well as in several epithelial cell lines. We showed UTF1 staining in normal and tumour epithelial tissues, but with varying intensities according to the tissue location. In vitro analyses also revealed that UTF1 is expressed in somatic epithelial cell lines even in the absence of Oct4A and Sox2, its two main known regulators. The comparison of UTF1 levels in normal and tumoral tissues revealed significant overexpression in endometrial and prostatic adenocarcinomas, whereas lower intensity of the staining was observed in renal and colic tumours, suggesting a potential tissue-specific function of UTF1. Altogether, these results highlight a potential dual role for UTF1, acting either as an oncogene or as a tumour suppressor depending on the tissue. These findings also question its role as a specific marker for stem cells.

Undifferentiated embryonic cell transcription factor-1 (UTF1) is an important transcription factor during development, which plays critical roles in cell fate determination. However, its expression and function in somatic tissues remain unclear. Here, we investigated the expression pattern of the UTF1 in the human normal and cancerous lesions of cervix and found that UTF1 was downregulated in cervical carcinogenesis, which was related to the hypermethylation of UTF1 promoter. Exogenous expression of UTF1 resulted in the significant inhibition of cell proliferation in vitro and tumorigenesis in vivo through attenuating cell cycle arrest via increasing the level of p27 (Kip1) . Luciferase reporter assay indicated that the region containing an intact activating transcription factor site between nucleotides -517 and -388 of the p27 (Kip1) promoter was indispensable for its activation by UTF1. Chromatin immunoprecipitation analysis confirmed the physical interaction between UTF1 and the p27 (Kip1) promoter. Taken together, our findings reveal that UTF1 attenuates cell proliferation and is inactivated in cervical carcinogenesis through epigenetic modification, which strongly supports that UTF1 is a potential tumor suppressor.

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression.

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.

We immunohistochemically evaluated UTF1 in 104 primary and 68 metastatic testicular germ cell tumors and 339 non-germ cell tumors. The percentage of tumor cells stained was semiquantitatively scored (0, no tumor cell staining; 1+, ≤30% of cells; 2+, 31%-60% of cells; 3+, 61%-90% of cells; 4+, >90% of cells). Staining intensity (nuclear) was scored as weak, moderate, or strong. UTF1 staining was seen in all 56 intratubular germ cell neoplasias, unclassified type (2+, 1; 3+, 2; 4+, 53; weak, 4; moderate, 49; strong, 3), all 72 seminomas (1+, 2; 2+, 4; 3+, 8; 4+, 58; weak, 10; moderate, 33; strong, 29), and 59 embryonal carcinomas (3+, 2; 4+, 57; moderate, 1; strong, 58). Weak UTF1 staining was seen in 15 of 37 yolk sac tumors (1+, 10; 2+, 2; 3+, 2; 4+, 1). All 34 teratomas, 9 choriocarcinomas, and 6 spermatocytic seminomas were negative for UTF1 staining. Among the 339 non-germ cell tumors, only 18 showed weak UTF1 staining (1+ to 4+). Normal prepubertal and postpubertal spermatogonia showed weak to strong UTF1 staining. UTF1 was differentially expressed in testicular germ cell tumors. Strong UTF1 staining can be used for diagnosing embryonal carcinoma and seminoma. UTF1 expression in spermatogonia suggests its possible role in spermatogenesis and renewal of spermatogonia.

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel α(1B)-deficient (α(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

The transcription factor OCT4 plays a crucial role in the earliest differentiation of the mammalian embryo and in self-renewal of embryonic stem cells. However, it remains controversial whether this gene is also expressed in somatic tissues. Here, we use a combination of RT-PCR on whole and microdissected tissues, in situ hybridization, immunohistochemistry and western blotting to show that OCT4 and SOX2 together with downstream targets, UTF1 and REX1/ZFP42, are expressed in the human male urogenital tract. We further support these results by the analysis of DNA methylation of a region in the OCT4 promoter. In culture, human primary epididymal cells formed spheres that continued to express the investigated genes for at least 20 days. Transcriptomic analysis of cultured cells showed up-regulation of CD29, CD44 and CD133 that are normally associated with sphere-forming cancer stem cells. Furthermore, stimulation with retinoic acid resulted in down-regulation of OCT4 expression, however, without multilineage differentiation. Our results show that OCT4 and associated genes are expressed in somatic epithelial cells from the urogenital tract and that these cells can form spheres, a general marker of stem cell behaviour.

Single-nucleotide polymorphisms (SNPs) are single-nucleotide sequence variations between individuals. Two missense SNPs are present in the human undifferentiated embryonic-cell transcription factor 1 (UTF1) gene and their consequences for UTF1 function are investigated in this study. Expression of the UTF1 gene is restricted to pluripotent cells and UTF1 is a chromatin-associated protein with core histone-like properties. UTF1 further acts as a transcriptional repressor and is required for proper differentiation of pluripotent cells. Two missense mutations in UTF1 are reported: rs11599284, which results in a glycine to an arginine change at amino acid 73, and rs4480453, resulting in a leucine to methionine change at amino acid 275. To study the effects of these two SNPs, P19CL6 mouse embryonic carcinoma cells stably expressing eGFP-hUTF1 constructs containing either one or both SNPs were generated. The single and double SNPs did not alter the localization or transcriptional repressor activity of the protein. Further, the single SNPs did not alter the chromatin association and mobility of hUTF1. However, the double mutant, G73R/L275M, demonstrated a decreased chromatin association, indicating a degree of protein malfunction.

In mice, during early embryonic development UTF1 (undifferentiated embryonic cell transcription factor 1) is expressed in the inner cell mass of blastocysts and in adult animals expression is restricted to the gonads. (Embryonic) Cells expressing UTF1 are generally considered pluripotent, meaning they can differentiate into all cell types of the adult body. In mouse it was shown that UTF1 is tightly associated with chromatin and that it is required for proper differentiation of embryonic carcinoma and embryonic stem cells. In this study we functionally characterized the human UTF1 protein. We show with localization, subnuclear fractionation, and strip-FRAP analyses that human UTF1 is a tightly DNA-associated protein with transcriptional repressor activity. Our data identify human UTF1 as a pluripotency-associated chromatin component with core histone-like characteristics.