Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System.

Apelin receptor (APLNR/AGTRLl1/APJ) marks a transient cell population during the differentiation of hematopoietic stem and progenitor cells (HSPCs) from pluripotent stem cells, but its function during the production and maintenance of hematopoietic stem cells is not clear. We generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line and showed that HSPCs are generated exclusively from mesodermal cells that express Aplnr-tdTomato. HSPC production from mESCs was impaired when Aplnr was deleted, implying that this pathway is required for their production. To address the role of APLNR signaling in HSPC maintenance, we added APELIN ligands to ex vivo AGM cultures. Activation of the APLNR pathway in this system impaired the generation of long-term reconstituting HSPCs and appeared to drive myeloid differentiation. Our data suggest that the APLNR signaling is required for the generation of cells that give rise to HSCs, but that its subsequent downregulation is required for their maintenance.

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

Hematopoietic stem and progenitor cells (HSPCs) develop in distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates some of these processes; however, it has proven difficult to generate functional hematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we explored single-cell RNA sequencing (scRNAseq) in combination with single-cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors and erythroid-, megakaryocyte-, and leukocyte-committed progenitors, and we identified CD44, CD326, ICAM2/CD9, and CD18, respectively, as markers of these progenitors. Using an artificial neural network that we trained on scRNAseq derived from human fetal liver, we identified a wide range of hPSC-derived HSPCs phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded, and was completely missing in the data set that had been generated using cells selected on the basis of CD43 expression. 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 explored in the future to improve the in vitro production of HSCs.

Genetic programming of macrophages generates an in vitro model for the human erythroid island niche.

Red blood cells mature within the erythroblastic island (EI) niche that consists of specialized macrophages surrounded by differentiating erythroblasts. Here we establish an in vitro system to model the human EI niche using macrophages that are derived from human induced pluripotent stem cells (iPSCs), and are also genetically programmed to an EI-like phenotype by inducible activation of the transcription factor, KLF1. These EI-like macrophages increase the production of mature, enucleated erythroid cells from umbilical cord blood derived CD34+ haematopoietic progenitor cells and iPSCs; this enhanced production is partially retained even when the contact between progenitor cells and macrophages is inhibited, suggesting that KLF1-induced secreted proteins may be involved in this enhancement. Lastly, we find that the addition of three secreted factors, ANGPTL7, IL-33 and SERPINB2, significantly enhances the production of mature enucleated red blood cells. Our study thus contributes to the ultimate goal of replacing blood transfusion with a manufactured product.

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'.

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

We 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.

Injection of embryonic stem cell derived macrophages ameliorates fibrosis in a murine model of liver injury.

Chronic liver injury can be caused by viral hepatitis, alcohol, obesity, and metabolic disorders resulting in fibrosis, hepatic scarring, and cirrhosis. Novel therapies are urgently required and previous work has demonstrated that treatment with bone marrow derived macrophages can improve liver regeneration and reduce fibrosis in a murine model of hepatic injury and fibrosis. Here, we describe a protocol whereby pure populations of therapeutic macrophages can be produced in vitro from murine embryonic stem cells on a large scale. Embryonic stem cell derived macrophages display comparable morphology and cell surface markers to bone marrow derived macrophages but our novel imaging technique revealed that their phagocytic index was significantly lower. Differences were also observed in their response to classical induction protocols with embryonic stem cell derived macrophages having a reduced response to lipopolysaccharide and interferon gamma and an enhanced response to IL4 compared to bone marrow derived macrophages. When their therapeutic potential was assessed in a murine, carbon tetrachloride-induced injury and fibrosis model, embryonic stem cell derived macrophages significantly reduced the amount of hepatic fibrosis to 50% of controls, down-regulated the number of fibrogenic myofibroblasts and activated liver progenitor cells. To our knowledge, this is the first study that demonstrates a therapeutic effect of macrophages derived in vitro from pluripotent stem cells in a model of liver injury. We also found that embryonic stem cell derived macrophages repopulated the Kupffer cell compartment of clodronate-treated mice more efficiently than bone marrow derived macrophages, and expressed comparatively lower levels of Myb and Ccr2, indicating that their phenotype is more comparable to tissue-resident rather than monocyte-derived macrophages.

Erratum: Author Correction: Injection of embryonic stem cell-derived macrophages ameliorates fibrosis in a murine model of liver injury.

[This corrects the article DOI: 10.1038/s41536-017-0017-0.].

Activation of KLF1 Enhances the Differentiation and Maturation of Red Blood Cells from Human Pluripotent Stem Cells.

Blood transfusion is widely used in the clinic but the source of red blood cells (RBCs) is dependent on donors, procedures are susceptible to transfusion-transmitted infections and complications can arise from immunological incompatibility. Clinically-compatible and scalable protocols that allow the production of RBCs from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been described but progress to translation has been hampered by poor maturation and fragility of the resultant cells. Genetic programming using transcription factors has been used to drive lineage determination and differentiation so we used this approach to assess whether exogenous expression of the Erythroid Krüppel-like factor 1 (EKLF/KLF1) could augment the differentiation and stability of iPSC-derived RBCs. To activate KLF1 at defined time points during later stages of the differentiation process and to avoid transgene silencing that is commonly observed in differentiating pluripotent stem cells, we targeted a tamoxifen-inducible KLF1-ERT2 expression cassette into the AAVS1 locus. Activation of KLF1 at day 10 of the differentiation process when hematopoietic progenitor cells were present, enhanced erythroid commitment and differentiation. Continued culture resulted the appearance of more enucleated cells when KLF1 was activated which is possibly due to their more robust morphology. Globin profiling indicated that these conditions produced embryonic-like erythroid cells. This study demonstrates the successful use of an inducible genetic programing strategy that could be applied to the production of many other cell lineages from human induced pluripotent stem cells with the integration of programming factors into the AAVS1 locus providing a safer and more reproducible route to the clinic. Stem Cells 2017;35:886-897.

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells.

UNLABELLED: : We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ER(T2) expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43(+)CD34(+) progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a(+)/CD71(+) erythroid cells. However, this did not result in a significant increase in more mature CD235a(+)/CD71(-) cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (ß) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs. SIGNIFICANCE: As worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.

A Role for MOSPD1 in Mesenchymal Stem Cell Proliferation and Differentiation.

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1-null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self-renewal capacity of MOSPD1-null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)-like cells expressing CD73, CD90, and CD105 were generated from MOSPD1-null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1-null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA-sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre-MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation.

Haematopoietic differentiation is inhibited when Notch activity is enhanced in FLK1(+) mesoderm progenitors.

Notch signalling has been implicated during haematopoietic development in vivo and in the differentiation of haematopoietic cells from pluripotent cells in vitro. However interpretation of data from many of these studies has been complicated by the heterogeneous nature of cell populations under study and by the fact that the Notch pathway is active during embryogenesis prior to the development of the haematopoietic system. To define the role of Notch signalling in more precise cell populations during the early stages of haematopoietic development within the aorta-gonad-mesonephros (AGM) microenvironment we co-cultured differentiating ESCs on a stromal cell line derived from this region of the embryo. Our co-culture system had no effect on the production of FLK1(+) mesoderm progenitor cells but promoted their subsequent haematopoietic differentiation. We assessed the role of Notch signalling on haematopoietic differentiation of isolated FLK1(+) cells. Notch activity is dynamic and drops to basal levels as FLK1(+) cells commit to a haematopoietic fate. Further reduction of Notch activity by the inducible expression of dominant negative MAML had no functional consequences. In contrast, induction of Notch activity using an inducible NotchIC expression system had an inhibitory effect on haematopoietic differentiation. We used a Cre-mediated recombination strategy whereby NotchIC-expressing cells were marked with the hCD2 receptor and observed a reduction in the number of multi-lineage and myeloid colonies derived from NotchIC(+) compared to NotchIC(-) FLK1(+) cells isolated from the same culture. We believe that our culture system represents a good model for haematopoietic development within the AGM microenvironment and our data suggest that haematopoietic commitment of FLK1(+) cells in this setting occurs when Notch activity is below a specific threshold.

SCF/KIT inhibition has a cumulative but reversible effect on the self-renewal of embryonic stem cells and on the survival of differentiating cells.

The receptor tyrosine kinase c-KIT is expressed in embryonic stem cells (ESCs) and adult stem cells, and many functional studies have demonstrated the importance of the SCF/KIT signaling pathway in adult stem cell maintenance. In this study, we show that a high level of KIT expression in wild-type ESCs correlates with an enhanced self-renewal and that inhibition of KIT signaling in ESCs for extended periods of time has a cumulative but reversible effect on self-renewal. Together these data suggest that continued KIT signaling in some cells within a self-renewing ESC population is required for optimal ESC function. Using a KIT blocking antibody, we recapitulated the phenotype we previously reported for genetically deficient KIT-null cells, demonstrating that SCF/KIT signaling is essential for the survival of differentiating ESCs. Here we show that this phenotype is also reversible. Pharmacological inhibition of JNK also had a cumulative but reversible detrimental effect on the survival of differentiating cells, thus recapitulating the Kit null phenotype and implicating JNK as a downstream mediator of KIT signaling. In contrast, the self-renewal of ESCs was unaffected by prolonged exposure to the JNK inhibitor, suggesting that JNK-independent downstream pathways are involved in KIT-mediated ESC self-renewal whereas KIT-mediated survival of differentiating ESC is likely to be JNK dependent.

Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance.

Embryonic stem cells provide a potentially convenient source of macrophages in the laboratory. Given the propensity of macrophages for plasticity in phenotype and function, standardised culture and differentiation protocols are required to ensure consistency in population output and activity in functional assays. Here we detail the development of an optimised culture protocol for the production of murine embryonic stem cell-derived macrophages (ESDM). This protocol provides improved yields of ESDM and we demonstrate that the cells are suitable for application to the study of macrophage responses to apoptotic cells. ESDM so produced were of higher purity than commonly used primary macrophage preparations and were functional in chemotaxis assays and in phagocytosis of apoptotic cells. Maturation of ESDM was found to be associated with reduced capacity for directed migration and increased capacity for phagocytic clearance of apoptotic cells. These results show ESDM to be functionally active in sequential phases of interaction with apoptotic cells and establish these macrophage populations as useful models for further study of molecular mechanisms underlying the recognition and removal of apoptotic cells.

HOXB4 can enhance the differentiation of embryonic stem cells by modulating the hematopoietic niche.

Hematopoietic differentiation of embryonic stem cells (ESCs) in vitro has been used as a model to study early hematopoietic development, and it is well documented that hematopoietic differentiation can be enhanced by overexpression of HOXB4. HOXB4 is expressed in hematopoietic progenitor cells (HPCs) where it promotes self-renewal, but it is also expressed in the primitive streak of the gastrulating embryo. This led us to hypothesize that HOXB4 might modulate gene expression in prehematopoietic mesoderm and that this property might contribute to its prohematopoietic effect in differentiating ESCs. To test our hypothesis, we developed a conditionally activated HOXB4 expression system using the mutant estrogen receptor (ER(T2)) and showed that a pulse of HOXB4 prior to HPC emergence in differentiating ESCs led to an increase in hematopoietic differentiation. Expression profiling revealed an increase in the expression of genes associated with paraxial mesoderm that gives rise to the hematopoietic niche. Therefore, we considered that HOXB4 might modulate the formation of the hematopoietic niche as well as the production of hematopoietic cells per se. Cell mixing experiments supported this hypothesis demonstrating that HOXB4 activation can generate a paracrine as well as a cell autonomous effect on hematopoietic differentiation. We provide evidence to demonstrate that this activity is partly mediated by the secreted protein FRZB.

The culture of mouse embryonic stem cells and formation of embryoid bodies.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of the pre-implantation blastocyst. They have the capacity to undergo indefinite rounds of self-renewing cell division and differentiate into all the cell lineages of the developing embryo. In suspension culture, ES cells will differentiate into aggregates known as embryoid bodies in a manner similar to the early embryo. This culture system therefore provides a useful model to study the relatively inaccessible stages of mammalian development. We describe methods for the routine maintenance of mouse embryonic stem cells in culture, assays of stem cell self-renewal potential in monolayer culture and the generation of embryoid bodies to study differentiation pathways.

The survival of differentiating embryonic stem cells is dependent on the SCF-KIT pathway.

The stem cell factor (SCF)-KIT signal transduction pathway plays a role in the proliferation, differentiation and survival of a range of stem and progenitor cell types but little is known about its function in embryonic stem (ES) cells. We generated ES cells carrying a null allele of Kit as well as a knock-in allele that encodes an SCF-independent hybrid KIT receptor that can be activated by the FKBP binding drug, AP20187. KIT null ES cells die when induced to differentiate upon withdrawal of leukaemia inhibitory factor in monolayer culture. This phenotype is recapitulated in wild-type ES cells treated with a KIT-neutralising antibody and reversed in mutant cells by activation of the hybrid KIT receptor. Differentiating KIT null ES cells exhibit elevated levels of DNA laddering and reduced BCL2 expression, indicative of apoptosis. We conclude that mouse ES cell differentiation in vitro is dependent on the SCF-KIT pathway contrasting with the apparently normal differentiation of KIT null inner cell mass or epiblast cells in vivo. This discrepancy could be explained by the presence of compensatory signals in the embryo or it could lend support to the idea of a phenotypic relationship between ES cells and early germ cells.