Sergiy Kyrylenko

Adaptation to Robust Monolayer Expansion Produces Human Pluripotent Stem Cells With Improved Viability

The generation of human pluripotent stem cells (hPSCs) of sufficient quantity and quality remains a major challenge for biomedical application. Here we present an efficient feeder‐free, high‐density monolayer system in which hPSCs become SSEA‐3‐high and gradually more viable than their feeder‐dependent counterparts without changes attributed to culture adaptation. As a consequence, monolayer hPSCs possess advantages over their counterparts in embryoid body development, teratoma formation, freezing as a single‐cell suspension, and colony‐forming efficiency. Importantly, this monolayer culture system is reversible, preserving the competence of hPSCs to gradually reacquire features of colony growth, if necessary. Therefore, the monolayer culture system is highly suitable for long‐term, large‐scale propagation of hPSCs, which is necessary in drug development and pluripotent stem cell‐based therapies.

GSM 900 MHz cellular phone radiation can either stimulate or depress early embryogenesis in Japanese quails depending on the duration of exposure

Abstract Purpose: Our study was designed to assess the effects of low intensity radiation of a GSM (Global System for Mobile communication) 900 MHz cellular phone on early embryogenesis in dependence on the duration of exposure. Materials and methods: Embryos of Japanese Quails were exposed in ovo to GSM 900 MHz cellular phone radiation during initial 38 h of brooding or alternatively during 158 h (120 h before brooding plus initial 38 h of brooding) discontinuously with 48 sec ON (average power density 0.25 μW/cm2, specific absorption rate 3 μW/kg) followed by 12 sec OFF intervals. A number of differentiated somites were assessed microscopically. Possible DNA damage evoked by irradiation was assessed by an alkaline comet assay. Results: Exposure to radiation from a GSM 900 MHz cellular phone led to a significantly altered number of differentiated somites. In embryos irradiated during 38 h the number of differentiated somites increased (p < 0.001), while in embryos irradiated during 158 h this number decreased (p < 0.05). The lower duration of exposure led to a significant (p < 0.001) decrease in a level of DNA strand breaks in cells of 38-h embryos, while the higher duration of exposure resulted in a significant (p < 0.001) increase in DNA damage as compared to the control. Conclusion: Effects of GSM 900 MHz cellular phone radiation on early embryogenesis can be either stimulating or deleterious depending on the duration of exposure.

MEK and TGF-beta Inhibition Promotes Reprogramming without the Use of Transcription Factor

The possibility of replacing the originally discovered and widely used DNA reprogramming transcription factors is stimulating enormous effort to identify more effective compounds that would not alter the genetic information. Here, we describe the generation of induced pluripotent stem cells (iPSc) from head-derived primary culture of mouse embryonic cells using small chemical inhibitors of the MEK and TGF-beta pathways without delivery of exogenous transcription factors. These iPSc express standard pluripotency markers and retain their potential to differentiate into cells of all germ layers. Our data indicate that head-derived embryonic neural cells might have the reprogramming potential while neither the same primary cells cultivated over five passages in vitro nor a cell population derived from adult brain possesses this capacity. Our results reveal the potential for small molecules to functionally replace routinely used transcription factors and lift the veil on molecular regulation controlling pluripotency. The conditions described here could provide a platform upon which other genome non integrative and safer reprogramming processes could be developed. This work also shows novel potential for developing embryonic neural cells.

GSM 900 MHz microwave radiation affects embryo development of Japanese quails

A wide range of non thermal biological effects of microwave radiation (MW) was revealed during the last decades. A number of reports showed evident hazardous effects of MW on embryo development in chicken. In this study, we aimed at elucidating the effects of MW emitted by a commercial model of GSM 900 MHz cell phone on embryo development in quails (Coturnix coturnix japonica) during both short and prolonged exposure. For that, fresh fertilized eggs were irradiated during the first 38 h or 14 days of incubation by a cell phone in “connecting” mode activated continuously through a computer system. Maximum intensity of incident radiation on the eggs surface was 0.2 μW/cm2.The irradiation led to a significant (p < 0.001) increase in numbers of differentiated somites in 38-hour exposed embryos and to a significant (p < 0.05) increase in total survival of embryos from exposed eggs after 14 days exposure. We hypothesized that observed facilitating effect was due to enhancement of metabolism in exposed embryos provoked via peroxidation mechanisms. Indeed, a level of thiobarbituric acid (TBA) reactive substances was significantly (p < 0.05–0.001) higher in brains and livers of hatchlings from exposed embryos. Thus, observed effects of radiation from commercial GSM 900 MHz cell phone on developing quail embryos signify a possibility for non-thermal impact of MW on embryogenesis. We suggest that the facilitating effect of low doses of irradiation on embryo development can be explained by a hormesis effect induced by reactive oxygen species (ROS). Future studies need to be done to clarify this assumption.

Transgenic human embryonic stem cells overexpressing FGF2 stimulate neuroprotection following spinal cord ventral root avulsion.

&NA; Ventral root avulsion (VRA) triggers a strong glial reaction which contributes to neuronal loss, as well as to synaptic detachment. To overcome the degenerative effects of VRA, treatments with neurotrophic factors and stem cells have been proposed. Thus, we investigated neuroprotection elicited by human embryonic stem cells (hESC), modified to overexpress a human fibroblast growth factor 2 (FGF‐2), on motoneurons subjected to VRA. Lewis rats were submitted to VRA (L4‐L6) and hESC/FGF‐2 were applied to the injury site using a fibrin scaffold. The spinal cords were processed to evaluate neuronal survival, synaptic stability, and glial reactivity two weeks post lesion. Then, qRT‐PCR was used to assess gene expression of &bgr;2‐microglobulin (&bgr;2m), TNF&agr;, IL1&bgr;, IL6 and IL10 in the spinal cord in vivo and FGF2 mRNA levels in hESC in vitro. The results indicate that hESC overexpressing FGF2 significantly rescued avulsed motoneurons, preserving synaptic covering and reducing astroglial reactivity. The cells were also shown to express BDNF and GDNF at the site of injury. Additionally, engraftment of hESC led to a significant reduction in mRNA levels of TNF&agr; at the spinal cord ventral horn, indicating their immunomodulatory properties. Overall, the present data suggest that hESC overexpressing FGF2 are neuroprotective and can shift gene expression towards an anti‐inflammatory environment. HighlightsHuman embryonic stem cell (hESC) therapy is neuroprotective.hESCs produce BDNF and GDNF in vitro and in vivo.hESCs overexpressing FGF2 lead to synapse preservation following ventral root avulsion (VRA).hESCs overexpressing FGF2 reduce astroglial reaction after VRA.

Nonlinear Regression Models for Determination of Nicotinamide Adenine Dinucleotide Content in Human Embryonic Stem Cells

Recent evidence suggests that energy metabolism contributes to molecular mechanisms controlling stem cell identity. For example, human embryonic stem cells (hESCs) receive their metabolic energy mostly via glycolysis rather than mitochondrial oxidative phosphorylation. This suggests a connection of metabolic homeostasis to stemness. Nicotinamide adenine dinucleotide (NAD) is an important cellular redox carrier and a cofactor for various metabolic pathways, including glycolysis. Therefore, accurate determination of NAD cellular levels and dynamics is of growing importance for understanding the physiology of stem cells. Conventional analytic methods for the determination of metabolite levels rely on linear calibration curves. However, in actual practice many two-enzyme cycling assays, such as the assay systems used in this work, display prominently nonlinear behavior. Here we present a diaphorase/lactate dehydrogenase NAD cycling assay optimized for hESCs, together with a mechanism-based, nonlinear regression models for the determination of NAD+, NADH, and total NAD. We also present experimental data on metabolic homeostasis of hESC under various physiological conditions. We show that NAD+/NADH ratio varies considerably with time in culture after routine change of medium, while the total NAD content undergoes relatively minor changes. In addition, we show that the NAD+/NADH ratio, as well as the total NAD levels, vary between stem cells and their differentiated counterparts. Importantly, the NAD+/NADH ratio was found to be substantially higher in hESC-derived fibroblasts versus hESCs. Overall, our nonlinear mathematical model is applicable to other enzymatic amplification systems.

Monochromatic red light of LED protects embryonic cells from oxidative stress caused by radiofrequency radiation

Objective: Oxidative mechanisms of the mutagenic and carcinogenic potential of radiofrequency radiation (RFR) have been demonstrated recently. This opens the need for antioxidative approach for protection of living cells from harmful effects of RFR. In this study, we aimed to assess the antioxidant potential of monochromatic red light of light-emitting diodes (LED) in RFR-exposed embryonic cells. Methods: One group of Japanese quail embryos was exposed in ovo to GSM 900 MHz RFR (I = 1 14 μW/cm2; SAR = 0.17 mW/kg; t = 158 h; discontinuously) before and during the first hours of incubation. The second group of embryos was exposed to RFR in the same regimen and additionally to LED red light (λmax = 630-650 nm; I = 0.1 mW/cm2; t = 180 c; discontinuously). The third group of embryos were served as control. The rate of somitogenesis, level of lipid peroxidation, activity of superoxide dismutase (SOD) and catalase in tissues of 38-h embryos were assessed. Results: Red light of LED exposure resulted in statistically significant reversion of the rate of somitogenesis decreased under RFR exposure; as well as in reversion of significantly increased level of lipid peroxidation and decreased catalase activity in tissues of RFR exposed embryos. In vitro significant suppression of SOD and catalase activities by short-term RFR exposure were partially reversed by LED red light treatment. Conclusion: Red light of LED can protect embryonic cells from oxidative stress caused by low intensity RFR exposure. This is of particularly importance in terms of potential mutagenicity and carcinogenicity of low intensity RFR, which in turn depends on the oxidative potential of RFR.