A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1.

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

Ferroptosis: The functions of Nrf2 in human embryonic stem cells.

Human embryonic stem cells (hESCs) have the capacity of self-renewal as well as differentiation towards three germ layer derivatives which makes them as a source of therapeutic application. hESCs are tremendously prone to cell death after dissociation into single cells. Therefore, it technically hinders their applications. Our recent study has revealed that hESCs can be prone to ferroptosis which differs from those in earlier explorations reporting that cellular detachment results in a process cited as anoikis. Ferroptosis occurs via increasing intracellular iron. Therefore, this form of programmed cell death is distinct from other cell deaths in terms of biochemistry, morphology, and genetics. Ferroptosis is found by excessive iron which plays an important part role in reactive oxygen species (ROS) generation through the Fenton reaction as a cofactor. Many genes are related to ferroptosis under the control of nuclear factor erythroid 2-related factor 2 (Nrf2) which is a transcription factor regulating the expression of genes to protect cells from oxidative stress. Nrf2 was demonstrated to take a perilous role in the suppression of ferroptosis by regulating the iron, antioxidant defense enzymes, usage, and restoration of glutathione, thioredoxin, and NADPH. Mitochondrial function is another target of Nrf2 to control cell homeostasis through the modulation of ROS production. In this review, we will give a succinct overview of lipid peroxidation and discuss the major players in the ferroptotic cascade. Additionally, we discussed the important role of the Nrf2 signaling pathway in mediating lipid peroxidation and ferroptosis, with a focus on known Nrf2 target genes that inhibit these processes and their possible role in hESCs.

Potential therapeutic strategies for photoreceptor degeneration: the path to restore vision.

Photoreceptors (PRs), as the most abundant and light-sensing cells of the neuroretina, are responsible for converting light into electrical signals that can be interpreted by the brain. PR degeneration, including morphological and functional impairment of these cells, causes significant diminution of the retina's ability to detect light, with consequent loss of vision. Recent findings in ocular regenerative medicine have opened promising avenues to apply neuroprotective therapy, gene therapy, cell replacement therapy, and visual prostheses to the challenge of restoring vision. However, successful visual restoration in the clinical setting requires application of these therapeutic approaches at the appropriate stage of the retinal degeneration. In this review, firstly, we discuss the mechanisms of PR degeneration by focusing on the molecular mechanisms underlying cell death. Subsequently, innovations, recent developments, and promising treatments based on the stage of disorder progression are further explored. Then, the challenges to be addressed before implementation of these therapies in clinical practice are considered. Finally, potential solutions to overcome the current limitations of this growing research area are suggested. Overall, the majority of current treatment modalities are still at an early stage of development and require extensive additional studies, both pre-clinical and clinical, before full restoration of visual function in PR degeneration diseases can be realized.

Monitoring the induction of ferroptosis following dissociation in human embryonic stem cells.

Human embryonic stem cells (hESCs) are vulnerable to cell death upon dissociation. Thus, dissociation is an obstacle in culturing, maintaining, and differentiating of hESCs. To date, apoptosis has become the focus of research into the nature of cell death triggered by cellular detachment; it remains baffling whether another form of cell death can occur upon dissociation in hESCs. Here, we demonstrate that iron accumulation and subsequently lipid peroxidation are responsible for dissociation-mediated hESC death. Moreover, we found that a decrease of glutathione peroxidase 4 because of iron accumulation promotes ferroptosis. Inhibition of lipid peroxidation (ferrostatin-1) or chelating iron (deferoxamine) largely suppresses iron accumulation-induced ferroptosis in dissociated hESCs. The results show that P53 mediates the dissociation-induced ferroptosis in hESCs, which is suppressed by pifithrin α. Multiple genes involved in ferroptosis are regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2). In this study, solute carrier family 7 member 11 and glutathione peroxidase 4 are involved in GSH synthesis decreased upon dissociation as a target of Nrf2. In conclusion, our study demonstrates that iron accumulation as a consequence of cytoskeleton disruption appears as a pivotal factor in the initiation of ferroptosis in dissociated hESCs. Nrf2 inhibits ferroptosis via its downstream targets. Our study suggests that the antiferroptotic target might be a good candidate for the maintenance of hESCs.

The Role of Endoplasmic Reticulum and Mitochondria in Maintaining Redox Status and Glycolytic Metabolism in Pluripotent Stem Cells.

Pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells (iPSCs), can be applicable for regenerative medicine. They strangely rely on glycolysis metabolism akin to aerobic glycolysis in cancer cells. Upon differentiation, PSCs undergo a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). The metabolic shift depends on organelles maturation, transcriptome modification, and metabolic switching. Besides, metabolism-driven chromatin regulation is necessary for cell survival, self-renewal, proliferation, senescence, and differentiation. In this respect, mitochondria may serve as key organelle to adapt environmental changes with metabolic intermediates which are necessary for maintaining PSCs identity. The endoplasmic reticulum (ER) is another organelle whose role in cellular identity remains under-explored. The purpose of our article is to highlight the recent progress on these two organelles' role in maintaining PSCs redox status focusing on metabolism. Topics include redox status, metabolism regulation, mitochondrial dynamics, and ER stress in PSCs. They relate to the maintenance of stem cell properties and subsequent differentiation of stem cells into specific cell types.

Stable Knockdown of Adenosine Kinase by Lentiviral Anti-ADK miR-shRNAs in Wharton's Jelly Stem Cells.

OBJECTIVES: In this study, we describe an efficient approach for stable knockdown of adenosine kinase (ADK) using lentiviral system, in an astrocytoma cell line and in human Wharton's jelly mesenchymal stem cells (hWJMSCs). These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy. MATERIALS AND METHODS: In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA (shRNAs). First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction (qRT-PCR) was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker (CD44+) and lack of expression of hematopoietic lineage marker (CD45-). Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs. RESULTS: Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miRshRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miRshRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level. CONCLUSIONS: Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors (AAV) might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy.