Human T memory stem (TSCM) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long‐lived T‐cell memory and are thus considered an ...attractive population to be used in adoptive transfer‐based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T‐cell receptor stimulation curbs human effector CD8+ T‐cell differentiation and allows the generation of CD45RO–CD45RA+CCR7+CD27+CD95+ ‐phenotype cells from highly purified naïve T‐cell precursors, resembling naturally‐occurring human TSCM. These cells proliferate extensively in vitro and in vivo, express low amounts of effector‐associated genes and transcription factors and undergo considerable self‐renewal in response to IL‐15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly‐activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long‐lived memory T cells with potential application in adoptive cell transfer immunotherapy.
T memory stem cells (TSCM) with increased persistence are most suitable cell type for adoptive cell transfer immunotherapy. Weak TCR stimulation of human naïve CD8+ T cells induces a CD45RO– CD45RA+ TSCM phenotype. The proliferating TSCM display decreased T‐cell activation, effector molecules and mitochondrial content compared to more differentiated T effectors.
The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we ...describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling "signet ring cells" was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.
Various diseases and toxic factors easily impair cellular and organic functions in mammals. Organ transplantation is used to rescue organ function, but is limited by scarce resources. Mesenchymal ...stem cell (MSC)‐based therapy carries promising potential in regenerative medicine because of the self‐renewal and multilineage potency of MSCs; however, MSCs may lose biological functions after isolation and cultivation for a long time in vitro. Moreover, after they are injected in vivo and migrate into the damaged tissues or organs, they encounter a harsh environment coupled with death signals due to the inadequate tensegrity structure between the cells and matrix. Preconditioning, genetic modification and optimization of MSC culture conditions are key strategies to improve MSC functions in vitro and in vivo, and all of these procedures will contribute to improving MSC transplantation efficacy in tissue engineering and regenerative medicine. Preconditioning with various physical, chemical and biological factors is possible to preserve the stemness of MSCs for further application in studies and clinical tests. In this review, we mainly focus on preconditioning and the corresponding mechanisms for improving MSC activities in vitro and in vivo; we provide a glimpse into the promotion of MSC‐based cell therapy development for regenerative medicine. As a promising consequence, MSC transplantation can be applied for the treatment of some terminal diseases and can prolong the survival time of patients in the near future.
Derivation of naïve human embryonic stem cells Ware, Carol B.; Nelson, Angelique M.; Mecham, Brigham ...
Proceedings of the National Academy of Sciences - PNAS,
03/2014, Letnik:
111, Številka:
12
Journal Article
Recenzirano
Odprti dostop
The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded ...derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state Hanna J, et al. (2010) Proc Natl Acad Sci USA 107 (20):9222—9227. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.
Summary
Severe acquired aplastic anaemia (AA) is a serious disease characterised by autoreactive T cells attacking haematopoietic stem cells, leading to marrow hypoplasia and pancytopenia. ...Immunosuppressive therapy combined with antithymocyte globulin and ciclosporin can rescue most patients with AA. However, the relapse after ciclosporin withdrawal and the severe side effects of long‐term ciclosporin administration remain unresolved. As such, new strategies should be developed to supplement current therapeutics and treat AA. In this study, the possibility of all‐trans‐retinoic acid (ATRA) as an alternative AA treatment was tested by using an immune‐mediated mouse model of AA. Results revealed that ATRA inhibited T‐cell proliferation, activation and effector function. It also restrained the Fas/Fasl pathway, shifted Th1 towards Th2 cell development, rebalanced T‐cell subsets at a relatively high level and corrected the Th1/Th2 ratio by targeting NFAT1 signalling. In addition, ATRA inhibited Th17 cell differentiation and promoted regulatory T‐cell development. Therefore, ATRA was an effective agent to improve AA treatment outcomes.
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral ...vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.
Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, ...using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.
Systemic lupus erythematosus (SLE) is a potentially life-threatening autoimmune disease characterized by altered balance of activity between effector and regulatory CD4(+) T cells. The homeostasis of ...CD4(+) T cell subsets is regulated by interleukin (IL)-2, and reduced production of IL-2 by T cells is observed in individuals with SLE. Here we report that treatment with low-dose recombinant human IL-2 selectively modulated the abundance of regulatory T (Treg) cells, follicular helper T (TFH) cells and IL-17-producing helper T (TH17) cells, but not TH1 or TH2 cells, accompanied by marked reductions of disease activity in patients with SLE.
The interaction of mesenchymal stromal cells (MSCs) with natural killer (NK) cells is traditionally thought of as a static inhibitory model, whereby resting MSCs inhibit NK cell effector function. ...Here, we use a dynamic in vitro system of poly(I:C) stimulation to model the interaction of NK cells and tissue-resident MSCs in the context of infection or tissue injury. The experiments suggest a time-dependent system of regulation and feedback, where, at early time points, activated MSCs secrete type I interferon to enhance NK cell effector function, while at later time points TGF-β and IL-6 limit NK cell effector function and terminate inflammatory responses by induction of a regulatory senescent-like NK cell phenotype. Importantly, feedback of these regulatory NK cells to MSCs promotes survival, proliferation, and pro-angiogenic properties. Our data provide additional insight into the interaction of stromal cells and innate immune cells and suggest a model of time-dependent MSC polarization and licensing.
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•Poly(I:C) activation of tissue-resident MSC leads to a time-dependent polarization•MSC early inflammatory response, via IFN-α/β, activates NK cells•MSC late regulatory response induces senescent-like NK cells via TGF-β and IL-6•Senescent-like NK cell feedback to MSCs promotes survival and pro-angiogenic activity
Brandau et al. stimulated MSCs of the nasal mucosa with poly(I:C), a compound which mimics viral RNA. Although resting MSCs are thought to inhibit NK cell function, stimulated MSCs exerted a time-dependent biphasic response, which either promoted NK cell effector function (early response) or induced a senescent-like process (late response). The data suggest a model of time-dependent MSC polarization and innate immune interaction.
The function of somatic stem cells declines with age. Understanding the molecular underpinnings of this decline is key to counteract age-related disease. Here, we report a dramatic drop in the neural ...stem cells (NSCs) number in the aging murine brain. We find that this smaller stem cell reservoir is protected from full depletion by an increase in quiescence that makes old NSCs more resistant to regenerate the injured brain. Once activated, however, young and old NSCs show similar proliferation and differentiation capacity. Single-cell transcriptomics of NSCs indicate that aging changes NSCs minimally. In the aging brain, niche-derived inflammatory signals and the Wnt antagonist sFRP5 induce quiescence. Indeed, intervention to neutralize them increases activation of old NSCs during homeostasis and following injury. Our study identifies quiescence as a key feature of old NSCs imposed by the niche and uncovers ways to activate NSCs to repair the aging brain.
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•Time-dependent stem cell depletion levels off in the old brain via increased quiescence•Age minimally changes the neural stem cell transcriptome•Once-activated neural stem cells perform similar in the old and young brain•The old niche keeps stem cells quiescent via inflammation and Wnt activity regulation
Aged brains have a small number of highly quiescent neural stem cells that, upon activation, functionally resemble young stem cells; it is the niche itself that becomes more inflammatory and drives this quiescence.
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