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.

Cellular rejuvenation protects neurons from inflammation mediated cell death.

In multiple sclerosis (MS), the invasion of the central nervous system by peripheral immune cells is followed by the activation of resident microglia and astrocytes. This cascade of events results in demyelination, which triggers neuronal damage and death. The molecular signals in neurons responsible for this damage are not yet fully characterized. In MS, retinal ganglion cell neurons (RGCs) of the central nervous system (CNS) undergo axonal injury and cell death. This phenomenon is mirrored in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. To understand the molecular landscape, we isolated RGCs from mice subjected to the EAE protocol. RNA-sequencing and ATAC-sequencing analyses were performed. Pathway analysis of the RNA-sequencing data revealed that RGCs displayed a molecular signature, similar to aged neurons, showcasing features of senescence. Single-nucleus RNA-sequencing analysis of neurons from human MS patients revealed a comparable senescence-like phenotype., which was supported by immunostaining RGCs in EAE mice. These changes include alterations to the nuclear envelope, modifications in chromatin marks, and accumulation of DNA damage. Transduction of RGCs with an Oct4 - Sox2 - Klf4 transgene to convert neurons in the EAE model to a more youthful epigenetic and transcriptomic state enhanced the survival of RGCs. Collectively, this research uncovers a previously unidentified senescent-like phenotype in neurons under pathological inflammation and neurons from MS patients. The rejuvenation of this aged transcriptome improved visual acuity and neuronal survival in the EAE model supporting the idea that age rejuvenation therapies and senotherapeutic agents could offer a direct means of neuroprotection in autoimmune disorders.

Overview of the Therapeutic Applications of Stem Cell-Derived Exosomes: A Research and Commercial Perspective.

Progress in extracellular vesicle (EV) research over the past two decades has generated significant interest in using EVs in the biomedical field. Exosomes are a subgroup of EVs that comprise endocytic membrane-bound nanovesicles of 40 to 160 nm diameter. These vesicles have been shown to facilitate intercellular communication via the delivery of cellular molecules. There are currently several exciting applications for exosomes being developed in therapeutics, diagnostics, drug delivery, and cellular reprogramming. Stem cell-derived exosomes present the opportunity to harness the power of stem cells while circumventing several of the risks associated with their use. This review summarizes the recent developments in exosome technology and lends a prospective view to the future of exosome use and application in research and medicine. Through a review of relevant patent filings, recent literature, and ongoing clinical trials, a valuable overview of the field of exosomes is provided. © 2021 Wiley Periodicals LLC.

C-terminal Tail of ß-Tubulin and its Role in the Alterations of Dynein Binding Mode.

Dynein is a cytoskeletal molecular motor protein that moves along the microtubule (MT) and transports various cellular cargos during its movement. Using standard Molecular Dynamics (MD) simulation, Principle Component Analysis (PCA), and Normal Mode Analysis (NMA) methods, this investigation studied large-scale movements and local interactions of dynein's Microtubule Binding Domain (MTBD) when bound to tubulin heterodimer subunits. Examination of the interactions between the MTBD segments, and their adjustments in terms of intra- and intermolecular distances at the interfacial area with tubulin heterodimer, particularly at α-H16, ß-H18, and ß-tubulin C-terminal tail (CTT), was the main focus of this study. The specific intramolecular interactions, electrostatic forces, and the salt bridge residue pairs were shown to be the dominating factors in orchestrating movements of the MTBD and MT interfacial segments in the dynein's low-high-affinity binding modes. Important interactions included ß-Glu447 and ß-Glu449 (CTT) with Arg3469 (MTBD-H6), Lys3472 (MTBD-H6-H7 loop) and Lys3479 (MTBD-H7); ß-Glu449 with Lys3384 (MTBD-H8), Lys3386 and His3387 (MTBD-H1). The structural and precise position, orientation, and functional effects of the CTTs on the MT-MTBD, within reasonable cut-off distance for non-bonding interactions and under physiological conditions, are unavailable from previous studies. The absence of the residues in the highly flexible MT-CTTs in the experimentally solved structures is perhaps in some cases due to insufficient data from density maps, but these segments are crucial in protein binding. The presented work contributes to the information useful for the MT-MTBD structure refinement.

Interdomain twists of human thymidine phosphorylase and its active-inactive conformations: Binding of 5-FU and its analogues to human thymidine phosphorylase versus dihydropyrimidine dehydrogenase.

5-fluorouracil (5-FU) is an anticancer drug, which inhibits human thymidine phosphorylase (hTP) and plays a key role in maintaining the process of DNA replication and repair. It is involved in regulating pyrimidine nucleotide production, by which it inhibits the mechanism of cell proliferation and cancerous tumor growth. However, up to 80% of the administered drug is metabolized by dihydropyrimidine dehydrogenase (DPD). This work compares binding of 5-FU and its analogues to hTP and DPD, and suggests strategies to reduce drug binding to DPD to decrease the required dose of 5-FU. An important feature between the proteins studied here was the difference of charge distribution in their binding sites, which can be exploited for designing drugs to selectively bind to the hTP. The 5-FU presence was thought to be required for a closed conformation. Comparison of the calculation results pertaining to unliganded and liganded protein showed that hTP could still undergo open-closed conformations in the absence of the ligand; however, the presence of a positively charged ligand better stabilizes the closed conformation and rigidifies the core region of the protein more than unliganded or neutral liganded system. The study has also shown that one of the three hinge segments linking the two major α and α/ß domains of the hTP is an important contributing factor to the enzyme's open-close conformational twist during its inactivation-activation process. In addition, the angle between the α/ß-domain and the α-domain has shown to undergo wide rotations over the course of MD simulation in the absence of a phosphate, suggesting that it contributes to the stabilization of the closed conformation of the hTP.

Mechanism of Off-Target Interactions and Toxicity of Tamoxifen and Its Metabolites.

Tamoxifen is an estrogen modulator that acts to competitively inhibit the binding of endogenous estrogens. It is widely used for treatment of breast cancer; however, analogous with many antineoplastic agents, tamoxifen is associated with numerous adverse effects, most prominently nausea. We have identified several off-target receptors of tamoxifen and 22 of its metabolites that include histamine H1 and H3, and muscarinic M1, M4, and M5 subtypes, and dopamine D2 receptor. We have shown how they are associated with tamoxifen and its metabolites' toxicity through a comprehensive computational analysis of their interaction modes, which were also compared to that of the related endogenous substrates of each receptor. The results were further evaluated using available in vivo and in vitro data. The presented work provides foundational knowledge toward the determination of the precise mechanism of nausea induction, and in particular, interactions of tamoxifen and its metabolites with the receptors involved in that biomolecular pathway. This study can assist in predicting the potential undesired effects of the chemicals with common pharmacophores or similar fragments to that of tamoxifen and its metabolites and serve drug discovery research in developing more effective and tolerable tamoxifen analogues or chemotherapeutic agents.