Murine haematopoiesis: studies using X chromosome-inactivation mosaics

Blood erythrocytes and leukocytes were serially sampled over many months from female mice that were heterozygous at the X-chromosomal locusencoding the glycolytic enzyme phosphoglycerate kinase (PGK-1). PGK-1A andPGK-1B alloenzymes were identified and quantified electrophoretically. There was little variation in PGK-1 phenotype between serial samples from individual mice. This small amount of variation was discussed in terms of the number of clones participating in haematopoiesis and the contribution of technical factors.Similar studies were performed using radiation chimaeras, repopulated with either a high dose (107 cells) or a low dose (105 cells) of PGK-1AB bonemarrow. The variation in PGK-1 phenotype between serial samples taken fromthe animals repopulated with a high dose of bone marrow was comparable to that seen in normal animals. In contrast, the variation observed in the low- dose chimaeras was. relatively large. These animals were used to study the clonal organisation of the haematopoietic system.The development of B lymphocytes carrying the X-linked immunodeficiency mutation (xid) was studied in mice that were heterozygous at both the x[d and the Pgk-1 loci.An abnormallly large population of B lymphocytes, possessing an characteristic membrane phenotype, was observed in the peripheral blood of a group of experimental mice. This behaved as a transplantable neoplasia. Subsequently, similar populations were found in several aged (>2 years) CBA/Ca mice. A preliminary characterisation of these cells was carried out and their possible relevance to human chronic lymphocytic leukaemia (CLL) was discussed

Progress in the production of haematopoietic stem and progenitor cells from human pluripotent stem cells

Cell therapies are currently used to treat many haematological diseases. These treatments range from the long-term reconstitution of the entire haematopoietic system using the most potent haematopoietic stem cells (HSCs) to the short-term rescue with mature functional end cells such as oxygen-carrying red blood cells and cells of the immune system that can fight infection and repair tissue. Limitations in supply and the risk of transmitting infection has prompted the design of protocols to produce some of these cell types from human pluripotent stem cells (hPSCs). Although it has proven challenging to generate the most potent HSCs directly from hPSCs, significant progress has been made in the development of differentiation protocols that can successfully produce haematopoietic progenitor cells and most of the mature cell lineages. We review the key steps used in the production of haematopoietic stem and progenitor cells (HSPCs) from hPSCs and the cell surface markers and reporter strategies that have been used to define specific transitions. Most studies have relied on the use of known markers that define HSPC production in vivo but more recently single cell RNA sequencing has allowed a less biased approach to their characterisation. Transcriptional profiling has identified new markers for naïve and committed hPSC-derived HSPC populations and trajectory analyses has provided novel insights into their lineage potential. Direct comparison of in vitro- and in vivo-derived RNA single cell sequencing datasets has highlights similarities and differences between the two systems and this deeper understanding will be key to the design and the tracking of improved and more efficient differentiation protocols

Pluripotent Stem Cell Derived Macrophages: Current Applications and Future Perspectives

The ability to derive macrophages from human-induced pluripotent stem cells (iPSCs) provides an unlimited source of genotype-specific cells with the potential to play a role in advancing our understanding of macrophage biology in both homeostasis and disease. While sharing many of the functional characteristics of monocyte-derived macrophages, iPSC-derived macrophages have also been shown to have phenotypical and functional features associated with tissue resident macrophages. These features present new opportunities to develop models of human disease and to understand the role of developmental or tissue context in innate immune cell function. iPSCs-derived macrophages have also been identified as a highly attractive source for cell and gene therapy in the treatment of diverse degenerative diseases based on their anti-inflammatory activity, their ability to clear scarred cells by phagocytosis, and providing extracellular matrices. We review and present a concise discussion on macrophage differentiation from stem cells highlighting their advantages over classical monocyte-derived macrophages in modelling organ specific macrophages. We summarize the various disease models utilizing iPSCs-derived macrophages including hereditary syndromes and host-pathogen interactions in tissue repair and the strategies used to mimic pathological phenotypes. Finally, we describe the pre-clinical studies that have addressed the application of iPSCs-derived macrophages as a therapeutic intervention

An all-in-one UniSam vector system for efficient gene activation

AbstractWe have generated a drug-free, all-in-one dCAS9-SAM vector that can activate endogenous gene expression with the potential to modify cell fate. We demonstrate that this strategy can be used in a number of cell lines and avoids exceptionally high levels of gene expression that are observed in standard transgenic approaches. Compared to the multi-plasmid system, this all-in-one vector activates gene expression to a comparable level but the reduced overall DNA content results in significantly higher viability of transfected cells. This allowed us to use the RUNX1C-GFP human embryonic stem cell reporter cell line to monitor gene activation in individual cells and to show that activation could occur at all stages of the cell cycle.</jats:p

A murine specific expansion of the Rhox cluster involved in embryonic stem cell biology is under natural selection

BACKGROUND: The rodent specific reproductive homeobox (Rhox) gene cluster on the X chromosome has been reported to contain twelve homeobox-containing genes, Rhox1-12. RESULTS: We have identified a 40 kb genomic region within the Rhox cluster that is duplicated eight times in tandem resulting in the presence of eight paralogues of Rhox2 and Rhox3 and seven paralogues of Rhox4. Transcripts have been identified for the majority of these paralogues and all but three are predicted to produce full-length proteins with functional potential. We predict that there are a total of thirty-two Rhox genes at this genomic location, making it the most gene-rich homoeobox cluster identified in any species. From the 95% sequence similarity between the eight duplicated genomic regions and the synonymous substitution rate of the Rhox2, 3 and 4 paralogues we predict that the duplications occurred after divergence of mouse and rat and represent the youngest homoeobox cluster identified to date. Molecular evolutionary analysis reveals that this cluster is an actively evolving region with Rhox2 and 4 paralogues under diversifying selection and Rhox3 evolving neutrally. The biological importance of this duplication is emphasised by the identification of an important role for Rhox2 and Rhox4 in regulating the initial stages of embryonic stem (ES) cell differentiation. CONCLUSION: The gene rich Rhox cluster provides the mouse with significant biological novelty that we predict could provide a substrate for speciation. Moreover, this unique cluster may explain species differences in ES cell derivation and maintenance between mouse, rat and human

A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and in vivo tracking of therapeutic cells

We describe the production of a human induced pluripotent stem cell (iPSC) line, SFCi55-ZsGr, that has been engineered to express the fluorescent reporter gene, ZsGreen, in a constitutive manner. The CAG-driven ZsGreen expression cassette was inserted into the AAVS1 locus and a high level of expression was observed in undifferentiated iPSCs and in cell lineages derived from all three germ layers including haematopoietic cells, hepatocytes and neurons. We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsGreen iPSC line and show that they are indistinguishable from those generated from their parental SFCi55 iPSC line in terms of gene expression, cell surface marker expression and phagocytic activity. The high level of ZsGreen expression had no effect on the ability of macrophages to be activated to an M(LPS + IFNγ), M(IL10) or M(IL4) phenotype nor on their plasticity, assessed by their ability to switch from one phenotype to another. Thus, targeting of the AAVS1 locus in iPSCs allows for the production of fully functional, fluorescently tagged human macrophages that can be used for in vivo tracking in disease models. The strategy also provides a platform for the introduction of factors that are predicted to modulate and/or stabilize macrophage function. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’

Single cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs

Haematopoietic stem and progenitor cells (HSPCs) develop through distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) is able to recapitulate some of these processes, however, it has proven difficult to generate functional haematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we exploited single cell RNA sequencing (scRNAseq) in combination with single cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors as well as erythroid, megakaryocyte and leukocyte-committed progenitors and we identified CD44, CD326, ICAM2/CD9 and CD18 as markers of these progenitors, respectively. Using an artificial neural network (ANN), that we trained on a scRNAseq derived from human fetal liver, we were able to identify a wide range of hPSCs-derived HPSC phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded and was completely missing in the dataset that had been generated using cells selected on the basis of CD43expression. By comparing the single cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver we identified transcription factors and molecular pathways that can be exploited in the future to improve the in vitro production of HSCs

Alternatively activated macrophages promotes necrosis resolution following acute liver injury

Background & Aim Following acetaminophen (APAP) overdose, acute liver injury (ALI) can occur in patients that present too late for N-acetylcysteine treatment, potentially leading to acute liver failure, systemic inflammation, and death. Macrophages influence the progression and resolution of ALI due to their innate immunological function and paracrine activity. Syngeneic primary bone marrow-derived macrophages (BMDMs) were tested as a cell-based therapy in a mouse model of APAP-induced ALI (APAP-ALI). Methods Several phenotypically distinct BMDM populations were delivered intravenously to APAP-ALI mice when hepatic necrosis was established, and then evaluated based on their effects on injury, inflammation, immunity, and regeneration. In vivo phagocytosis assays were used to interrogate the phenotype and function of alternatively activated BMDMs (AAMs) post-injection. Finally, primary human AAMs sourced from healthy volunteers were evaluated in immunocompetent APAP-ALI mice. Results BMDMs rapidly localised to the liver and spleen within 4 h of administration. Injection of AAMs specifically reduced hepatocellular necrosis, HMGB1 translocation, and infiltrating neutrophils following APAP-ALI. AAM delivery also stimulated proliferation in hepatocytes and endothelium, and reduced levels of several circulating proinflammatory cytokines within 24 h. AAMs displayed a high phagocytic activity both in vitro and in injured liver tissue post-injection. Crosstalk with the host innate immune system was demonstrated by reduced infiltrating host Ly6Chi macrophages in AAM-treated mice. Importantly, therapeutic efficacy was partially recapitulated using clinical-grade primary human AAMs in immunocompetent APAP-ALI mice, underscoring the translational potential of these findings. Conclusion We identify that AAMs have value as a cell-based therapy in an experimental model of APAP-ALI. Human AAMs warrant further evaluation as a potential cell-based therapy for APAP overdose patients with established liver injury. Lay summary After an overdose of acetaminophen (paracetamol), some patients present to hospital too late for the current antidote (N-acetylcysteine) to be effective. We tested whether macrophages, an injury-responsive leukocyte that can scavenge dead/dying cells, could serve as a cell-based therapy in an experimental model of acetaminophen overdose. Injection of alternatively activated macrophages rapidly reduced liver injury and reduced several mediators of inflammation. Macrophages show promise to serve as a potential cell-based therapy for acute liver injury