Characterisation of encapsulated embryonic stem cells using silac-based proteomics

The embryonic stem cells have two hallmarks characters, the ability to reproduce self-renewal and generate other cell lineages. Despite the excessive advance in stem cell research, their clinical applications delay owing lack of an optimal culture condition in vitro. Combining biometrical scaffold with stem cells provides a promising way to cellular delivery and tissue transplant. In vitro 3D culture offers both a model to understand self-renewal and stem cell behaviour in vivo as a route to industrial production. We have used a variety of analytical tools including proteomics and quantitative RT-PCR to compare 3D (both static & dynamic) with 2D cultures. We further show that 3D dynamic culture increases expression of the master gene regulators (Oct4, Nanog and Sox2). Consistent with this Rex1, an inner-cell mass (ICM) associated marker, is over-expressed in 3D dynamic culture whereas Fgf5, an epiblast transition marker is down-regulated. Using SILAC-based proteomics to compare 2D vs. 3D dynamic culture, we show that encapsulated stem cell are characterized by glycolytic pathways down-regulation and increased mitochondrial respiratory proteins. Additionally, ECM proteins expression (laminin, fibronectin, heparan sulfate (HS) proteoglycans, agrin, and nidogen-2) were up-regulated. Cells shortly after encapsulation in 3D constructs (both static &dynamic) showed high oxygen uptake rates compared to 2D culture. After 9 days of encapsulation glucose uptake increased in both static & dynamic 3D cultures and was combined with a pronounced increase in cell density and up-regulation of hypoxia induce-factors (HIf-1α and Hif-2α). This shift in metabolism toward anaerobic glycolysis is associated with high expression of hexokinas2 (Hk2) and increased lactate production. Despite extensive cell proliferation at day 18, where cell numbers reached up to 80k/ bead, cells in dynamic culture switched back from anaerobic to aerobic metabolism. This alteration in energy consumption was not associated with nutrient depletion since both glucose and glutamate were not depleted in the culture medium. By monitoring the oxygen consumption, hypoxia-induced factors expression as well as Oct4 levels at different time points, we demonstrate that the stem cells self-renewal status in 3D condition is regulated despite the metabolism transitions. We postulate that the 3D culture environment provides the niche required sensing and responding to external and internal stimuli different from recognised in vitro embryonic stem cell behaviour.Open Acces

An Insight into the Impact of Serum Tellurium, Thallium, Osmium and Antimony on the Antioxidant/Redox Status of PCOS Patients: A Comprehensive Study

Humans exploit heavy metals for various industrial and economic reasons. Although some heavy metals are essential for normal physiology, others such as Tellurium (Te), Thallium (TI), antimony (Sb), and Osmium (Os) are highly toxic and can lead to Polycystic Ovarian Syndrome (PCOS), a common female factor of infertility. The current study was undertaken to determine levels of the heavy metals TI, Te, Sb and Os in serum of PCOS females (n = 50) compared to healthy non-PCOS controls (n = 56), and to relate such levels with Total Antioxidant Capacity (TAC), activity of key antioxidant enzymes, oxidative stress marker levels and redox status. PCOS serum samples demonstrated significantly higher levels of TI, Te, Sb and Os and diminished TAC compared to control (p < 0.001). Furthermore, there was significant inhibition of SOD, CAT and several glutathione-related enzyme activities in sera of PCOS patients with concurrent elevations in superoxide anions, hydrogen and lipid peroxides, and protein carbonyls, along with disrupted glutathione homeostasis compared to those of controls (p < 0.001 for all parameters). Additionally, a significant negative correlation was found between the elevated levels of heavy metals and TAC, indicative of the role of metal-induced oxidative stress as a prominent phenomenon associated with the pathophysiology of the underlying PCOS. Data obtained in the study suggest toxic metals as risk factors causing PCOS, and thus protective measures should be considered to minimize exposure to prevent such reproductive anomalies

An Insight into the Impact of Serum Tellurium, Thallium, Osmium and Antimony on the Antioxidant/Redox Status of PCOS Patients: A Comprehensive Study

Humans exploit heavy metals for various industrial and economic reasons. Although some heavy metals are essential for normal physiology, others such as Tellurium (Te), Thallium (TI), antimony (Sb), and Osmium (Os) are highly toxic and can lead to Polycystic Ovarian Syndrome (PCOS), a common female factor of infertility. The current study was undertaken to determine levels of the heavy metals TI, Te, Sb and Os in serum of PCOS females (n = 50) compared to healthy non-PCOS controls (n = 56), and to relate such levels with Total Antioxidant Capacity (TAC), activity of key antioxidant enzymes, oxidative stress marker levels and redox status. PCOS serum samples demonstrated significantly higher levels of TI, Te, Sb and Os and diminished TAC compared to control (p p < 0.001 for all parameters). Additionally, a significant negative correlation was found between the elevated levels of heavy metals and TAC, indicative of the role of metal-induced oxidative stress as a prominent phenomenon associated with the pathophysiology of the underlying PCOS. Data obtained in the study suggest toxic metals as risk factors causing PCOS, and thus protective measures should be considered to minimize exposure to prevent such reproductive anomalies