Reprogramming human somatic cells to primed or naive induced pluripotent stem cells (iPSC) recapitulates the different stages of early human embryonic development [1][2][3][4][5][6] . The molecular mechanism underpinning the reprogramming of human somatic cells to primed or naive induced pluripotency remains largely unexplored, impeding our understanding and limiting rational improvements to reprogramming protocols. To address this, we reconstructed molecular reprogramming trajectories using single-cell transcriptomics. This revealed that reprogramming into primed and naive human pluripotency follows diverging and distinct trajectories. Moreover, genome-wide accessible chromatin analyses showed key changes in regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets unveiled an unexpected role of trophectoderm (TE) lineage-associated transcription factors and the existence of a subpopulation of cells that enter a TE-like state during reprogramming. Furthermore, this TE-like state could be captured, allowing the derivation of induced Trophoblast Stem Cells (iTSCs). iTSCs are molecularly and functionally similar to TSCs derived from human blastocysts or first-trimester placental trophoblasts 7 . Altogether, these results provide a high-resolution roadmap for transcription factor-mediated human 3 reprogramming, revealing an unanticipated role of the TE-lineage specific regulatory program during this process and facilitating the direct reprogramming of somatic cells into iTSCs.

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Lysosomes are the stomachs of the cells that degrade endocytosis and intracellular biomacromolecules and participate in various other cellular processes, such as apoptosis and cell migration. The ability of long-term tracking of lysosomes is very important to understand the details of lysosomal functions and to evaluate drug and gene delivery systems. For studying lysosomes, we designed and synthesized a water-soluble triscyclometalated iridium(III) complex (Ir-lyso) attaching morpholine moieties. The phosphorescent intensity of Ir-lyso is responsive to pH and decreases with an increase in the pH but not quenching in high pH. With excellent two-photon properties, Ir-lyso was used to light up the lysosomes in living cells and 3D tumor spheroids. Moreover, Ir-lyso could label lysosomes more than 4 days, so we developed this complex to act as a long-term probe for tracking lysosomes during cell migration and apoptosis. To the best of our knowledge, this is the first paradigm of metal complexes as the two-photon phosphorescent probe for long-term lysosomes tracking.

Antiseizure medications (ASMs) are frequently implicated in T cell-mediated drug hypersensitivity reactions and cause skin tropic pathologies that range in severity from mild rashes to life-threatening systemic syndromes. During the acute stages of the more severe manifestations of these reactions, drug responsive proinflammatory CD8+ T cells display classical features of Th1 cytokine production (e.g. IFNγ) and cytolysis (e.g. granzyme B, perforin). These T cells may be found locally at the site of pathology (e.g. blister cells/fluid), as well as systemically (e.g. blood, organs). What is less understood are the long-lived immunological effects of the memory T cell pool following T cell-mediated drug hypersensitivity reactions. In this study, we examine the ASM carbamazepine (CBZ) and the CBZ-reactive memory T cell pool in patients who have a history of either Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) from 3-to-20 years following their initial adverse reaction. We show that in vitro drug restimulation of CBZ-reactive CD8+ T cells results in a proinflammatory profile and produces a mainly focused, yet private, T cell receptor (TCR) usage amongst human leukocyte antigen (HLA)-B*15:02-positive SJS or TEN patients. Additionally, we show that expression of these CBZ-reactive TCRs in a reporter cell line, lacking endogenous αβTCR, recapitulates the features of TCR activation reported for ASM-treated T cell lines/clones, providing a useful tool for further functional validations. Finally, we conduct a comprehensive evaluation of the HLA-B*15:02 immunopeptidome following ASM (or a metabolite) treatment of a HLA-B*15:02-positive B-lymphoblastoid cell line (C1R.B*15:02) and minor perturbation of the peptide repertoire. Collectively, this study shows that the CBZ-reactive T cells characterized require both the drug and HLA-B*15:02 for activation and that reactivation of memory T cells from blood results in a focused private TCR profile in patients with resolved disease.

Characterisation of mouse pluripotent stem cells has revealed two distinct pluripotent states, naive and primed, that maintain characteristics of the pre and post implanted epiblast respectively. Recent studies have developed several culture systems that seek to recapitulate the naive phenomenon in human pluripotent stem cells. Therefore, robust methods to isolate these cells will be fundamental to assess their potential in modelling human development and disease. Here we review current methods for human naive pluripotent culture and collate a list of cell surface antigens that have been identified as markers to differentiate naive from primed human pluripotent stem cells. While these culture systems do display marker variability, and not all antigens mentioned were assessed in all methods, this review provides a resource for researchers of the human naive pluripotent stem cell state. SSEA-4, SSEA-3, CD24, CD75, CD7, CD77, CD130/GP130, CD57, CD90 and NLGN4X were all found to have a +/- expression profile in at least 2 methods, while +/- expression of Tra-1-81, CDH3, CD172a, CD107b, CD229 was reported in one method. Often it was reported that naive and primed cells could be defined using a low/medium/high expression of the following antigens TRA-1-60, PCDH1, GPR64, MHC Class I, however these markers were more likely to display expression pattern differences between methods. Studies using mouse naive cells indicate that they may have benefits over primed cells in modelling development and disease, and while it is yet to be determined if the same can be said about a human naive state, tools to identify this population should greatly further the field.

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