The pluripotency transcription factor Nanog represses glutathione reductase gene expression in mouse embryonic stem cells.

OBJECTIVE: Redox homeostasis maintenance is essential to bring about cellular functions. Particularly, embryonic stem cells (ESCs) have high fidelity mechanisms for DNA repair, high activity of different antioxidant enzymes and low levels of oxidative stress. Although the expression and activity of antioxidant enzymes are reduced throughout the differentiation, the knowledge about the transcriptional regulation of genes involved in defense against oxidative stress is yet restricted. Since glutathione is a central component of a complex system involved in preserving cellular redox status, we aimed to study whether the expression of the glutathione reductase (Gsr) gene, which encodes an essential enzyme for cellular redox homeostasis, is modulated by the transcription factors critical for self-renewal and pluripotency of ESCs. RESULTS: We found that Gsr gene is expressed in ESCs during the pluripotent state and it was upregulated when these cells were induced to differentiate, concomitantly with Nanog decreased expression. Moreover, we found an increase in Gsr mRNA levels when Nanog was downregulated by a specific shRNA targeting this transcription factor in ESCs. Our results suggest that Nanog represses Gsr gene expression in ESCs, evidencing a role of this crucial pluripotency transcription factor in preservation of redox homeostasis in stem cells.

Superoxide dismutase 1 expression is modulated by the core pluripotency transcription factors Oct4, Sox2 and Nanog in embryonic stem cells.

Redox homeostasis is vital for cellular functions and to prevent the detrimental consequences of oxidative stress. Pluripotent stem cells (PSCs) have an enhanced antioxidant system which supports the preservation of their genome. Besides, reactive oxygen species (ROS) are proposed to be involved in both self-renewal maintenance and in differentiation in embryonic stem cells (ESCs). Increasing evidence shows that cellular systems related to the oxidative stress defense decline along differentiation of PSCs. Although redox homeostasis has been extensively studied for many years, the knowledge about the transcriptional regulation of the genes involved in these systems is still limited. In this work, we studied Sod1 gene modulation by the PSCs fundamental transcription factors Oct4, Sox2 and Nanog. We found that this gene, which is expressed in mouse ESCs (mESCs), was repressed when they were induced to differentiate. Accordingly, these factors induced Sod1 promoter activity in a trans-activation assay. Finally, Sod1 mRNA levels were reduced when Oct4, Sox2 and Nanog were down-regulated by a shRNA approach in mESCs. Taken together, we found that PSCs' key transcription factors are involved in the modulation of Sod1 gene, suggesting a relationship between the pluripotency core and redox homeostasis in these cells.

Inhibition of Cell Division and DNA Replication Impair Mouse-Naïve Pluripotency Exit.

The cell cycle has gained attention as a key determinant for cell fate decisions, but the contribution of DNA replication and mitosis in stem cell differentiation has not been extensively studied. To understand if these processes act as "windows of opportunity" for changes in cell identity, we established synchronized cultures of mouse embryonic stem cells as they exit the ground state of pluripotency. We show that initial transcriptional changes in this transition do not require passage through mitosis and that conversion to primed pluripotency is linked to lineage priming in the G1 phase. Importantly, we demonstrate that impairment of DNA replication severely blocks transcriptional switch to primed pluripotency, even in the absence of p53 activity induced by the DNA damage response. Our data suggest an important role for DNA replication during mouse embryonic stem cell differentiation, which could shed light on why pluripotent cells are only receptive to differentiation signals during G1, that is, before the S phase.

Protein arginine Methyltransferase 8 gene is expressed in pluripotent stem cells and its expression is modulated by the transcription factor Sox2.

Addition of methyl groups to arginine residues is catalyzed by a group of enzymes called Protein Arginine Methyltransferases (Prmt). Although Prmt1 is essential in development, its paralogue Prmt8 has been poorly studied. This gene was reported to be expressed in nervous system and involved in neurogenesis. In this work, we found that Prmt8 is expressed in mouse embryonic stem cells (ESC) and in induced pluripotent stem cells, and modulated along differentiation to neural precursor cells. We found that Prmt8 promoter activity is induced by the pluripotency transcription factors Oct4, Sox2 and Nanog. Moreover, endogenous Prmt8 mRNA levels were reduced in ESC transfected with Sox2 shRNA vector. As a whole, our results indicate that Prmt8 is expressed in pluripotent stem cells and its transcription is modulated by pluripotency transcription factors. These findings suggest that besides its known function in nervous system, Prmt8 could play a role in pluripotent stem cells.

Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription.

EDA-containing fibronectin increases proliferation of embryonic stem cells.

Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

[Science in Argentina. Where do we go from here?]. / ¿Hacia dónde va la ciencia argentina?

The Minister of Science described in detail his plans for the creation of closer bonds between science and society in a long term attempt to improve general quality of life. This will be accomplished by strengthening the infrastructure with diversification of the finances, multidisciplinary interrelations aiming to increase production, with special attention for social demands. One of these objectives includes the creation of research-private/public company interrelations, stressing the importance of multidisciplinary projects. Publication of results in high impact journals will always be a priority stressing the importance of basic research as a source of breakthroughs or technological inventions. The Minister also referred to the awarding of grants for scientific projects, the relation between research and production and the promotion of technological innovations. He defined three technological platforms, which are nanotechnology, biotechnology and communication. He also identified four problem/opportunity sectors, such as public health, energy sources, agro-industry and social development. Interaction between these has already led to an area of biotechnology applied to public health which will grow through translational medicine. He finally discussed the problem of patents and their importance in promoting successful business-research partnerships.

Induced pluripotent stem cells' self-renewal and pluripotency is maintained by a bovine granulosa cell line-conditioned medium.

Induced pluripotent stem cells (iPSCs) are a promising type of stem cells, comparable to embryonic stem cells (ESCs) in terms of self-renew and pluripotency, generated by reprogramming somatic cells. These cells are an attractive approach to supply patient-specific pluripotent cells, for producing in vitro models of disease, drug discovery, toxicology and potentially treating degenerative disease circumventing immune rejection. In spite of the great advance since iPSCs' establishment, their obtention and propagation is an increasing area of great interest. In a recent work, we have shown that the conditioned medium from a bovine granulosa cell line (BGC-CM) is able to preserve the basic properties of mESCs. Therefore, based on our previous results and the reported resemblance between iPSCs and ESCs, we hypothesized that BGC-CM could provide a favorable context to culturing iPSCs. In this work, we have reprogrammed mouse embryonic fibroblasts obtaining iPSC lines, and showed that they can be propagated in BGC-CM while maintaining self-renewal and pluripotency, evidenced by expression of specific gene markers and capability of in vitro and in vivo differentiation to cell types from the three germ layers. We believe that these findings may provide a novel context to propagate iPSCs to study the molecular mechanisms involved in self-renewal and pluripotency.

Modulation of chromatin modifying factors' gene expression in embryonic and induced pluripotent stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are a promising source of cells for regenerative medicine because of their potential of self renew and differentiation. Multiple evidences highlight the relationship of chromatin remodeling with stem cell properties, differentiation programs and reprogramming for iPSC obtention. With the purpose of finding chromatin modifying factors relevant to these processes, and based on ChIP on chip studies, we selected several genes that could be modulated by Oct4, Sox2 and Nanog, critical transcription factors in stem cells, and studied their expression profile along the differentiation in mouse and human ESCs, and in mouse iPSCs. In this work, we analyzed the expression of Gcn5l2, GTF3C3, TAF15, ATF7IP, Myst2, HDAC2, HDAC3, HDAC5, HDAC10, SUV39H2, Jarid2, and Bmi-1. We found some genes from different functional groups that were highly modulated, suggesting that they could be relevant both in the undifferentiated state and during differentiation. These findings could contribute to the comprehension of molecular mechanisms involved in pluripotency, early differentiation and reprogramming. We believe that a deeper knowledge of the epigenetic regulation of ESC will allow improving somatic cell reprogramming for iPSC obtention and differentiation protocols optimization.

Maintenance of murine embryonic stem cells' self-renewal and pluripotency with increase in proliferation rate by a bovine granulosa cell line-conditioned medium.

Murine embryonic stem cells (mESCs) are pluripotent cells that can be propagated in an undifferentiated state in continuous culture on a feeder layer or without feeders in the presence of leukemia inhibitory factor (LIF). Although there has been a great advance since their establishment, ESC culture is still complex and expensive. Therefore, finding culture conditions that maintain the self-renewal of ESCs, preventing their differentiation and promoting their proliferation, is still an area of great interest. In this work, we studied the effects of the conditioned medium from a bovine granulosa cell line (BGC-CM) on the maintenance of self-renewal and pluripotency of mESCs. We found that this medium is able to maintain mESCs' self-renewal while preserving its critical properties without LIF addition. mESCs cultured in BGC-CM expressed the stem cell markers Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. Moreover, mESCs cultured in BGC-CM gave rise to embryoid bodies and teratomas that differentiated effectively to diverse cell populations from endoderm, mesoderm, and ectoderm. Further, we found that mESCs cultured in BGC-CM have an increased proliferation rate compared with cells grown in the mESC standard culture medium supplemented with LIF. These findings may provide a powerful tool to culture mESCs for long periods of time with high proliferation rate while preserving its basic characteristics, contributing to the application of these cells to assess potential tissue engineering and cellular therapy applications.

STAT5 transcriptional activity is impaired by LIF in a mammary epithelial cell line.

Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation. We have also found that STAT5 activity is negatively modulated by this cytokine, both on a gene whose expression is induced, as well as on a promoter repressed by STAT5. Besides, our results show that lactogenic hormones increase LIF effect on gene induction without modifying STAT3 phosphorylation state. Our findings strongly suggest that there is crosstalk between STAT5 and STAT3 pathways that could modulate their ability to regulate gene expression.

High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.