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.

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'.

FANTOM5 CAGE profiles of human and mouse samples.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

SplitAx: A novel method to assess the function of engineered nucleases.

Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1-158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization.

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.

Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

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.

Induction of hematopoietic differentiation of mouse embryonic stem cells by an AGM-derived stromal cell line is not further enhanced by overexpression of HOXB4.

Hematopoietic differentiation of embryonic stem (ES) cells can be enhanced by co-culture with stromal cells derived from hematopoietic tissues and by overexpression of the transcription factor HOXB4. In this study, we compare the hematopoietic inductive effects of stromal cell lines derived from different subregions of the embryonic aorta-gonad-mesonephros tissue with the commonly used OP9 stromal cell line and with HOXB4 activation. We show that stromal cell lines derived from the aorta and surrounding mesenchyme (AM) act at an earlier stage of the differentiation process compared with the commonly used OP9 stromal cells. AM stromal cells were able to promote the further differentiation of isolated brachyury-GFP(+) mesodermal cells into hematopoietic progenitors, whereas the OP9 stromal cells could not support the differentiation of these cells. Co-culture and analyses of individual embryoid bodies support the hypothesis that the AM stromal cell lines could enhance the de novo production of hematopoietic progenitors, lending support to the idea that AM stromal cells might act on prehematopoietic mesoderm. The induction level observed for AM stromal cells was comparable to HOXB4 activation, but no additive effect was observed when these 2 inductive strategies were combined. Addition of a γ-secretase inhibitor reduced the inductive effects of both the stromal cell line and HOXB4, providing clues to possible shared molecular mechanisms.

Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells.

Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5' intron. We also show that this relative positional independence is linked to the human beta-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting.

Aminopeptidase O contains a functional nucleolar localization signal and is implicated in vascular biology.

We have identified a gene trap integration into Aminopeptidase O, the gene encoding a member of the M1 family of metalloproteases. Using the betagal reporter of the gene trap vector, we have revealed that at least some ApO isoforms are expressed predominantly in embryonic and adult blood vessels leading us to propose that ApO plays a role in vascular cell biology. The protein produced from an engineered Gfp-ApO fusion cDNA localises to the nucleolus in transfected COS7 cells. We confirm that indeed the APO protein contains a functional nucleolar localisation domain by demonstrating that GFP-APO fusion proteins that lack the predicted nucleolar localisation signal are retained in the cytoplasm. We report the existence of multiple alternatively spliced Apo isoforms that differ with respect to the presence of exons encoding important functional domains. Alternative splicing predictably produces protein products with or without the catalytic domain and/or a nucleolar localisation signal and therefore likely represents an important mechanism in regulating the biological activity of APO that has been reported to cleave one of the peptides of the renin angiotensin pathway.

A novel transmembrane MSP-containing protein that plays a role in right ventricle development.

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.