Maria I. Sousa

From gametogenesis and stem cells to cancer: common metabolic themes

BACKGROUND Both pluripotent stem cells (PSCs) and cancer cells have been described as having similar metabolic pathways, most notably a penchant for favoring glycolysis even under aerobiosis, suggesting common themes that might be explored for both stem cell differentiation and anti-oncogenic purposes. METHODS A search of the scientific literature available in the PubMed/Medline was conducted for studies on metabolism and mitochondrial function related to gametogenesis, early development, stem cells and cancers in the reproductive system, notably breast, prostate, ovarian and testicular cancers. RESULTS Both PSCs and some types of cancer cells, particularly reproductive cancers, were found to obtain energy mostly by glycolysis, often reducing mitochondrial activity and oxidative phosphorylation. This strategy links proliferating cells, allowing for the biosynthesis reactions necessary for cell division. Interventions that affect metabolic pathways, and force cells to change their preferences, can lead to shifts in cell status, increasing either pluripotency or differentiation of stem cells, and causing cancer cells to become more or less aggressive. Interestingly metabolic changes in many cases seemed to lead to cell transformation, not necessarily follow it, suggesting a direct role of metabolic choices in influencing the (epi)genetic program of different cell types. CONCLUSIONS There are uncanny similarities between PSCs and cancer cells at the metabolic level. Furthermore, metabolism may also play a direct role in cell status and targeting metabolic pathways could therefore be a promising strategy for both the control of cancer cell proliferation and the regulation of stem cell physiology, in terms of manipulating stem cells toward relevant phenotypes that may be important for tissue engineering, or making cancer cells become less tumorigenic.

Sirtuins in metabolism, stemness and differentiation.

BACKGROUND Pluripotent stem cells promise innovative approaches for enduring diseases, including disease modeling and drug screens. Accordingly, efforts have been undertaken in order to efficiently reprogram somatic cells to pluripotency, and then differentiate them into pure cultures of specific cell lineages. However, the latter step remains mostly elusive, and, in order to better control differentiation and design more efficient differentiation strategies, the cellular mechanisms behind different pluripotency stages that mimic embryonic development are being actively addressed. SCOPE OF REVIEW Metabolism is one of many cellular processes that are in constant adjustment during mammalian embryo development, as well as in pluripotent stem cell establishment and differentiation. Thus, the role of molecular pathways known to be involved in metabolic control has been recently addressed as potential modulators of pluripotency. Notably, mammalian sirtuins have emerged as master regulators of many cellular processes, including epigenetics and metabolism. In this review we address the potential developmental role of sirtuins, with a particular focus on sirtuin 1. MAJOR CONCLUSIONS This review focuses on the most recent studies implying sirtuins as regulators of pluripotency and differentiation of pluripotent stem cells, highlighting metabolic control as associated with the control of pluripotency. It notably stresses the role of sirtuin 1 in these processes, creating parallels between in vitro manipulations and developmental cues. GENERAL SIGNIFICANCE Using metabolic control in order to determine cellular fate, both in terms of somatic cell reprogramming to pluripotency and pluripotent stem cell differentiation, is a topic of increasing interest, and sirtuins are key players in these efforts.

Dichloroacetate, the Pyruvate Dehydrogenase Complex and the Modulation of mESC Pluripotency.

Introduction The pyruvate dehydrogenase (PDH) complex is localized in the mitochondrial matrix catalyzing the irreversible decarboxylation of pyruvate to acetyl-CoA and NADH. For proper complex regulation the E1-α subunit functions as an on/off switch regulated by phosphorylation/dephosphorylation. In different cell types one of the four-pyruvate dehydrogenase kinase isoforms (PDHK1-4) can phosphorylate this subunit leading to PDH inactivation. Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells. Therefore, we wondered if metabolic modulation, via inexpensive pharmacological inhibition of PDHK, could impact metabolism and pluripotency. Methods/Results In order to assess the importance of the PDH cycle in mouse Embryonic Stem Cells (mESC), we incubated cells with the PDHK inhibitor dichloroacetate (DCA) and observed that in its presence ESC started to differentiate. Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored. Interestingly, we were also able to describe a possible pathway that involves Hif-1α and p53 during DCA-induced loss of pluripotency. Results with ESCs treated with DCA were comparable to those obtained for cells grown without Leukemia Inhibitor Factor (LIF), used in this case as a positive control for differentiation. Conclusions DCA negatively affects ESC pluripotency by changing cell metabolism and elements related to the PDH cycle, suggesting that PDHK could function as a possible metabolic gatekeeper in ESC, and may be a good target to modulate metabolism and differentiation. Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic profile, by using some of the signaling pathways found in the latter cells.

Concentration-dependent Sildenafil citrate (Viagra) effects on ROS production, energy status, and human sperm function

Abstract Literature regarding the effects of sildenafil citrate on sperm function remains controversial. In the present study, we specifically wanted to determine if mitochondrial dysfunction, namely membrane potential, reactive oxygen species production, and changes in energy content, are involved in in vitro sildenafil-induced alterations of human sperm function. Sperm samples of healthy men were incubated in the presence of 0.03, 0.3, and 3 μM sildenafil citrate in a phosphate buffered saline (PBS)-based medium for 2, 3, 12, and 24 hours. Sperm motility and viability were evaluated and mitochondrial function, i.e., mitochondrial membrane potential and mitochondrial superoxide production were assessed using flow-cytometry. Additionally, adenosine triphosphate (ATP) levels were determined by high performance liquid chromatography (HPLC) analysis. Results show a decrease in sperm motility correlated with the level of mitochondria-generated superoxide, without a visible effect on mitochondrial membrane potential or viability upon exposure to sildenafil. The effect on both motility and superoxide production was higher for the intermediate concentration of sildenafil (0.3 µM) indicating that the in vitro effects of sildenafil on human sperm do not vary linearly with drug concentration. Adenosine triphosphate levels also decreased following sildenafil exposure, but this decrease was only detected after a decrease in motility was already evident. These results suggest that along with the level of ATP and mitochondrial function other factors are involved in the early sildenafil-mediated decline in sperm motility. However, the further decrease in ATP levels and increase in mitochondria-generated reactive oxygen species after 24 hours of exposure might further contribute towards declining sperm motility.

Pluri-IQ: Quantification of Embryonic Stem Cell Pluripotency through an Image-Based Analysis Software

Summary Image-based assays, such as alkaline phosphatase staining or immunocytochemistry for pluripotent markers, are common methods used in the stem cell field to assess pluripotency. Although an increased number of image-analysis approaches have been described, there is still a lack of software availability to automatically quantify pluripotency in large images after pluripotency staining. To address this need, we developed a robust and rapid image processing software, Pluri-IQ, which allows the automatic evaluation of pluripotency in large low-magnification images. Using mouse embryonic stem cells (mESC) as a model, we combined an automated segmentation algorithm with a supervised machine-learning platform to classify colonies as pluripotent, mixed, or differentiated. In addition, Pluri-IQ allows the automatic comparison between different culture conditions. This efficient user-friendly open-source software can be easily implemented in images derived from pluripotent cells or cells that express pluripotent markers (e.g., OCT4-GFP) and can be routinely used, decreasing image assessment bias.

High glucose levels affect spermatogenesis: an in vitro approach

Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitus may further exacerbate a decline in male fertility. This metabolic disease, clinically characterised by a hyperglycaemic phenotype, has devastating consequences in terms of human health, with reproductive dysfunction being one of the associated clinical complications. Nonetheless, the mechanisms responsible for such alterations are still poorly understood due to the multiplicity of factors involved in the induced pathophysiological changes. With this in mind, we focused on the main mediator of diabetes-associated alterations and performed an in vitro approach to address the effects of high glucose conditions on spermatogenesis, avoiding other confounding in vivo factors. Mouse (5 days post partum) testis fragments were cultured on agar gel stands at a gas-liquid interface with either 5, 25 or 50mM D-glucose for 3 weeks. Stereological analysis revealed that high D-glucose levels increased Sertoli cell number (P<0.05) and decreased tubular luminal area (P<0.01), suggesting an impairment of this somatic cell type. Moreover, higher proliferative activity in a TM4 Sertoli cell line exposed to high D-glucose was found (P<0.05) without compromising cell viability (P>0.05), further suggesting altered Sertoli cell maturation. Overall, high D-glucose concentrations may lead to impairment of Sertoli cell function, which, given their significant role in spermatogenic control, may compromise male fertility.

Mitochondrial functionality and chemical compound action on sperm function.

During the last decade, several studies have shown that mitochondrial parameters, such as integrity, respiratory activity, membrane potential and ROS production are intimately linked with sperm quality. Given the limitations of conventional semen analyses in terms of predicting male fertility, an increasing number of studies are focusing on the characterization of sperm mitochondria in order to more accurately assess sperm functionality. Moreover, mitochondria from several organs, such as the liver, have been described as a powerful screening tool for drug safety, being an easy in vitro model to assess the toxicity of distinct families of compounds. Given that mitochondrial functionality is intimately related to sperm homeostasis, it has become important to understand how compounds, ranging from dietary supplements, environmental pollutants, dependency-inducing drugs to pharmacological agents (such as erectile dysfunction-targeted drugs and male contraceptives) affect sperm mitochondrial function. In this review, we discuss studies describing the effects of various chemical agents on spermatozoa, with particular emphasis on mitochondrial function. From the extensive literature analyzed, we conclude that in some cases the role of sperm mitochondria as putative predictors of sperm functionality is very obvious, while in others further studies are needed to clarify this issue.

Erratum: Pluri-IQ: Quantification of Embryonic Stem Cell Pluripotency through an Image-Based Analysis Software (Stem Cell Reports (2017) 9(2) (697–709) (S2213671117302692) (10.1016/j.stemcr.2017.06.006))

(Stem Cell Reports 9, 697–709; August 8, 2017) In the originally published version of this Resource, an error appeared in Equation 4 in the Experimental Procedures. The error does not affect the conclusions of the paper; all of the calculations with the Dice index were done with the correct formula. Equation 4 has been corrected in the paper online, and appears correctly below. The authors apologize for any confusion this mistake may have caused.

Data on the potential impact of food supplements on the growth of mouse embryonic stem cells

The use of new compounds as dietary supplements is increasing, but little is known in terms of possible consequences of their use. Pluripotent stem cells are a promising research tool for citotoxicological research for evaluation of proliferation, cell death, pluripotency and differentiation. Using the mouse embryonic stem cell (mESC) model, we present data on three different compounds that have been proposed as new potential supplements for co-adjuvant disease treatments: kaempferol, berberine and Tauroursodeoxycholic acid (TUDCA). Cell number and viability were monitored following treatment with increased concentrations of each drug in pluripotent culture conditions.