γδ T cells are a rare and unique prototype of T cells that share properties with natural killer cells in secondary lymphoid organs. Although many studies have revealed the function and importance of adult-derived γδ T cells in cancer biology and regenerative medicine, the low numbers of these cells hamper their application as therapeutic cell sources in the clinic. To solve this problem, pluripotent stem cell-derived γδ T cells are considered alternative cell sources; however, few studies have reported the generation of human pluripotent stem cell-derived γδ T cells. In the present study, we investigated whether lymphoid lineage γδ T cells were successfully generated from human pluripotent stem cells via hemogenic endothelium under defined culture conditions. Our results revealed that pluripotent stem cells successfully generated γδ T cells with an overall increase in transcriptional activity of lymphoid lineage genes and cytolytic factors, indicating the importance of the optimization of culture conditions in generating lymphoid lineage γδ T cells. We uncovered an initial step in differentiating γδ T cells that could be applied to basic and translational investigations in the field of cancer biology. Based on our result, we will develop an appropriate method to purify γδ T cells with functionality and it helpful for the study of basic mechanism of γδ T cells in pathophysiologic condition as well as clinic application.

Hemogenic endothelium (HE) plays a pivotal and inevitable role in haematopoiesis and can generate all blood and endothelial lineage cells in the aorta-gonad-mesonephros of mouse embryos. Whether definitive HE can prospectively isolate pure HE from human pluripotent stem cells that can spontaneously differentiate into heterogeneous cells remains unknown. Here, we identified and validated a CD34dim subpopulation with hemogenic potential. We also purified CD34 cells with a CXCR4- CD73- phenotype as a definitive HE population that generated haematopoietic stem cells and lymphocytes. The frequency of CXCR4- CD73- CD34dim was evidently increased by bone morphogenetic protein 4, and purified HE cells differentiated into haematopoietic cells with myeloid and T lymphoid lineages including Vδ2+ subset of γ/δ T cells. We developed a simple method to purify HE cells that were enriched in CD34dim cells. We uncovered an initial step in differentiating haematopoietic lineage cells that could be applied to basic and translational investigations into regenerative medicine.
© 2022 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.

Human adipose tissue-derived stem cells (hASC) secretome display various therapeutically relevant effects in regenerative medicine, such as induction of angiogenesis and tissue repair. The benefits of hASC secretome are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in host cells. However, the composition and the innate characteristics of hASC secretome can be highly variable depending on the culture conditions. Here, we evaluated the combined effect of serum-free media and hypoxia preconditioning on the hASCs secretome composition and biological effects on angiogenesis and wound healing. The hASCs were cultured in serum-free media under normoxic (NCM) or hypoxic (HCM) preconditioning. The proteomic profile showed that pro- and anti-antiangiogenic factors were detected in NCM and HCM secretomes. In vitro studies demonstrated that hASCs secretomes enhanced endothelial proliferation, survival, migration, in vitro tube formation, and in vivo Matrigel plug angiogenesis. In a full-thickness skin-wound mouse model, injection of either NCM or HCM significantly accelerated the wound healing. Finally, hASC secretomes were potent in increasing endothelial density and vascular coverage of resident pericytes expressing NG2 and nestin to the lesion site, potentially contributing to blood vessel maturation. Overall, our data suggest that serum-free media or hypoxic preconditioning enhances the vascular regenerative effects of hASC secretome in a preclinical wound healing model.
Copyright: © 2022 Silveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.
Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

Mesenchymal stromal cells (MSCs) are known to have immunosuppressive ability and have been used in clinical treatment of acute graft-versus-host disease, one of severe complications of the hematopoietic stem cell transplantation. However, MSCs are activated to suppress the immune system only after encountering an inflammatory stimulation. Thus, it will be ideal if MSCs are primed to be activated and ready to suppress the immune reaction before being administered. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb-Tripterygium wilfordii Hook.f. It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aimed to use TPL to prime umbilical cord-derived MSCs (TPL-primed UC-MSCs) to enter a stronger immunosuppressive status. UC-MSCs were primed with TPL, which was washed out thoroughly, and the TPL-primed UC-MSCs were resuspended in fresh medium. Although TPL inhibited the proliferation of UC-MSCs, 0.01 μM TPL for 24 h was tolerable. The surface markers of TPL-primed UC-MSCs were identical to those of non-primed UC-MSCs. TPL-primed UC-MSCs exhibited stronger anti-proliferative effect for activated CD4+ and CD8+ T cells in the allogeneic mixed lymphocyte reaction assay than the non-primed UC-MSCs. TPL-primed UC-MSCs promoted the expression of IDO-1 in the presence of IFN-γ, but TPL alone was not sufficient. Furthermore, TPL-primed UC-MSCs showed increased expression of PD-L1. Conclusively, upregulation of IDO-1 in the presence of IFN-γ and induction of PD-L1 enhances the immunosuppressive potency of TPL-primed UC-MSCs on the proliferation of activated T cells. Thus, TPL- primed MSCs may provide a novel immunosuppressive cell therapy.
Copyright © 2021 He, Takahashi, Mukai, Hori, Narita, Tojo, Yang and Nagamura-Inoue.