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We thank the Cellular Genetics wet lab support team, Cellular Genetics IT team, Sanger Sequencing operations and Sanger Cytometry Core facility for their essential help. We thank the Gene Editing team for providing iPSC knock-out lines. We would also like to thank Ruxandra Tesloianu and Luz Garcia-Alonso for their help setting up the scATAC-seq computational analysis. We thank Jana Eliasova for her help with figure design and Christina Usher and Aidan Maartens for their edits in the text. This work was mainly funded by the Open Targets consortium (OTAR026 and OTAR032 project) and the Wellcome Sanger core funding (WT206194) with additional support from Open Targets projects OTAR037, OTAR2065, OTAR2071. The authors are grateful to the funders for their support and additional care given to their members during the COVID-19 pandemic. D.A.-E. thanks CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. This study makes use of cell lines and data generated by the HiPSci Consortium, funded by The Wellcome Trust and the MRC (Medical Research Council). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This publication is part of the Human Cell Atlas- www.humancellatlas.org/publications. We thank the Cellular Genetics wet lab support team, Cellular Genetics IT team, Sanger Sequencing operations and Sanger Cytometry Core facility for their essential help. We thank the Gene Editing team for providing iPSC knock-out lines. We would also like to thank Ruxandra Tesloianu and Luz Garcia-Alonso for their help setting up the scATAC-seq computational analysis. We thank Jana Eliasova for her help with figure design and Christina Usher and Aidan Maartens for their edits in the text. This work was mainly funded by the Open Targets consortium (OTAR026 and OTAR032 project) and the Wellcome Sanger core funding (WT206194) with additional support from Open Targets projects OTAR037, OTAR2065, OTAR2071. The authors are grateful to the funders for their support and additional care given to their members during the COVID-19 pandemic. D.A.-E. thanks CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. This study makes use of cell lines and data generated by the HiPSci Consortium, funded by The Wellcome Trust and the MRC (Medical Research Council). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This publication is part of the Human Cell Atlas-www.humancellatlas.org/publications. Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

A major clinical challenge of diffuse large B-cell lymphoma (DLBCL) is that up to 40% of patients have refractory disease or relapse after initial response to therapy as a result of drug-specific molecular resistance. The purpose of the present study was to investigate microRNA (miRNA) involvement in vincristine resistance in DLBCL, which was pursued by functional in vitro analysis in DLBCL cell lines and by outcome analysis of patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Differential miRNA expression analysis identified miR-155 as highly expressed in vincristine-sensitive DLBCL cell lines compared with resistant ones. Ectopic upregulation of miR-155 sensitized germinal-center B-cell-like (GCB)–DLBCL cell lines to vincristine, and consistently, reduction and knockout of miR-155 induced vincristine resistance, documenting that miR-155 functionally induces vincristine sensitivity. Target gene analysis identified miR-155 as inversely correlated with Wee1, supporting Wee1 as a target of miR-155 in DLBCL. Chemical inhibition of Wee1 sensitized GCB cells to vincristine, suggesting that miR-155 controls vincristine response through Wee1. Outcome analysis in clinical cohorts of DLBCL revealed that high miR-155 expression level was significantly associated with superior survival for R-CHOP-treated patients of the GCB subclass, independent of international prognostic index, challenging the commonly accepted perception of miR-155 as an oncomiR. However, miR-155 did not provide prognostic information when analyzing the entire DLBCL cohort or activated B-cell–like classified patients. In conclusion, we experimentally confirmed a direct link between high miR-155 expression and vincristine sensitivity in DLBCL and documented an improved clinical outcome of GCB-classified patients with high miR-155 expression level.

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology., Summary: A cloned porcine disease model to advance topical treatment in the debilitating skin disorder psoriasis.

Cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. MicroRNAs are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. A characteristic microRNA profile in inflammatory skin suggests putative functions of microRNAs in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. It remains unclear, however, why certain microRNAs are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. In this report, we focus on cytokine regulation by microRNA-203 (miR-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. By screening a panel of cytokines that are upregulated in psoriatic skin for regulation by miR-203, we identify the genes encoding the pro-inflammatory cytokines TNFα and IL24 as direct targets of miR-203. Studies of miR-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of TNFα and IL24 by miR-203 in cell lines and primary keratinocytes. Our findings suggest that miR-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key pro-inflammatory cytokines.

Post-transcriptional managing of RNA plays a key role in the intricate network of cellular pathways that regulate our genes. Numerous small RNA species have emerged as crucial regulators of RNA processing and translation. Among these, microRNAs (miRNAs) regulate protein synthesis through specific interactions with target RNAs and are believed to play a role in almost any cellular process and tissue. Skin is no exception, and miRNAs are intensively studied for their role in skin homoeostasis and as potential triggers of disease. For use in skin and many other tissues, therapeutic RNA managing by small RNA technologies is now widely explored. Despite the easy accessibility of skin, the natural barrier properties of skin have challenged genetic intervention studies, and unique tools for studying gene expression and the regulatory role of small RNAs, including miRNAs, in human skin are urgently needed. Human immunodeficiency virus (HIV)-derived lentiviral vectors (LVs) have been established as prominent carriers of foreign genetic cargo. In this review, we describe the use of HIV-derived LVs for efficient gene transfer to skin and establishment of long-term transgene expression in xenotransplanted skin. We outline the status of engineered LVs for delivery of small RNAs and their in vivo applicability for expression of genes and small RNA effectors including small hairpin RNAs, miRNAs and miRNA inhibitors. Current findings suggest that LVs may become key tools in experimental dermatology with particular significance for cutaneous RNA managing and in vivo genetic intervention.

Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness.

MicroRNAs (miRNAs) are key regulators of gene expression and modulators of diverse biological pathways. Analyses of miRNA function as well as therapeutic managing of miRNAs rely on cellular administration of miRNA inhibitors which may be achieved by the use of viral vehicles. This study explores the miRNA-suppressive capacity of inhibitors expressed intracellularly from lentivirus-derived gene vectors. Superior activity of two decoy-type inhibitors, a “Bulged Sponge” with eight miRNA recognition sites and a hairpin-shaped “Tough Decoy” containing two miRNA recognition sites, is demonstrated in a side-by-side comparison of seven types of miRNA inhibitors transcribed as short RNAs from an RNA Pol III promoter. We find that lentiviral vectors expressing Tough Decoy inhibitors are less vulnerable than Bulged Sponge-encoding vectors to targeting by the cognate miRNA and less prone, therefore, to reductions in transfer efficiency. Importantly, it is demonstrated that Tough Decoy inhibitors retain their miRNA suppression capacity in the context of longer RNA transcripts expressed from an RNA Pol II promoter. Such RNA Pol II-transcribed Tough Decoy inhibitors are new tools in managing of miRNAs and may have potential for temporal and spatial regulation of miRNA activity as well as for therapeutic targeting of miRNAs that are aberrantly expressed in human disease.

Post-transcriptional managing of RNA plays a key role in the intricate network of cellular pathways that regulate our genes. Numerous small RNA species have emerged as crucial regulators of RNA processing and translation. Among these, microRNAs (miRNAs) regulate protein synthesis through specific interactions with target RNAs and are believed to play a role in almost any cellular process and tissue. Skin is no exception, and miRNAs are intensively studied for their role in skin homoeostasis and as potential triggers of disease. For use in skin and many other tissues, therapeutic RNA managing by small RNA technologies is now widely explored. Despite the easy accessibility of skin, the natural barrier properties of skin have challenged genetic intervention studies, and unique tools for studying gene expression and the regulatory role of small RNAs, including miRNAs, in human skin are urgently needed. Human immunodeficiency virus (HIV)-derived lentiviral vectors (LVs) have been established as prominent carriers of foreign genetic cargo. In this review, we describe the use of HIV-derived LVs for efficient gene transfer to skin and establishment of long-term transgene expression in xenotransplanted skin. We outline the status of engineered LVs for delivery of small RNAs and their in vivo applicability for expression of genes and small RNA effectors including small hairpin RNAs, miRNAs and miRNA inhibitors. Current findings suggest that LVs may become key tools in experimental dermatology with particular significance for cutaneous RNA managing and in vivo genetic intervention.