ABSTRACT
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.
Subject(s)
Antigens, Differentiation , Hematopoietic Stem Cells , Machine Learning , Pluripotent Stem Cells , RNA-Seq , Single-Cell Analysis , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Fetus/cytology , Fetus/metabolism , Gene Expression Regulation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Liver/cytology , Liver/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolismABSTRACT
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.
Subject(s)
Cell Differentiation , Erythrocytes/cytology , Erythrocytes/metabolism , Induced Pluripotent Stem Cells/cytology , Kruppel-Like Transcription Factors/metabolism , Cell Nucleus/metabolism , Cell Proliferation , Erythropoiesis/genetics , Gene Expression Regulation , Globins/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , K562 Cells , Protein Transport , Recombinant Fusion Proteins/metabolismABSTRACT
Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion-transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long-term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future.
Subject(s)
Blood Cells/cytology , Cellular Reprogramming Techniques/methods , Pluripotent Stem Cells/cytology , Cell Differentiation , Hematologic Diseases/therapy , Hematopoietic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Transcription Factors/physiologyABSTRACT
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.
Subject(s)
Cell Differentiation/genetics , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Membrane Proteins/genetics , Mesenchymal Stem Cells , Adipocytes/metabolism , Bone Marrow/metabolism , Cell Lineage/genetics , Embryonic Stem Cells/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Osteoblasts/metabolism , RNA, Messenger/biosynthesisABSTRACT
Glucocorticoids are vital for the structural and functional maturation of foetal organs, yet excessive foetal exposure is detrimental to adult cardiovascular health. To elucidate the role of glucocorticoid signalling in late-gestation cardiovascular maturation, we have generated mice with conditional disruption of glucocorticoid receptor (GR) in cardiomyocytes and vascular smooth muscle cells using smooth muscle protein 22-driven Cre recombinase (SMGRKO mice) and compared them with mice with global deficiency in GR (GR(-/-)). Echocardiography shows impaired heart function in both SMGRKO and GR(-/-) mice at embryonic day (E)17.5, associated with generalized oedema. Cardiac ultrastructure is markedly disrupted in both SMGRKO and GR(-/-) mice at E17.5, with short, disorganized myofibrils and cardiomyocytes that fail to align in the compact myocardium. Failure to induce critical genes involved in contractile function, calcium handling and energy metabolism underpins this common phenotype. However, although hearts of GR(-/-) mice are smaller, with 22% reduced ventricular volume at E17.5, SMGRKO hearts are normally sized. Moreover, while levels of mRNA encoding atrial natriuretic peptide are reduced in E17.5 GR(-/-) hearts, they are normal in foetal SMGRKO hearts. These data demonstrate that structural, functional and biochemical maturation of the foetal heart is dependent on glucocorticoid signalling within cardiomyocytes and vascular smooth muscle, though some aspects of heart maturation (size, ANP expression) are independent of GR at these key sites.
Subject(s)
Fetal Heart/growth & development , Glucocorticoids/metabolism , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Signal Transduction , Animals , Corticosterone/blood , Corticosterone/physiology , Fetal Heart/physiology , Heart/embryology , Heart/physiology , Mice , Mice, Transgenic , Muscle, Smooth, Vascular/embryology , Muscle, Smooth, Vascular/metabolism , Myocardial Contraction , Myocardium/ultrastructure , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Myofibrils/ultrastructureABSTRACT
Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages. Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration. Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.
ABSTRACT
Pluripotent stem cells can be differentiated into hematopoietic lineages in vitro and hold promise for the future treatment of hematological disease. Differentiation strategies involving defined factors in serum-free conditions have been successful in producing hematopoietic progenitors and some mature cell types from mouse and human embryonic stem cells and induced pluripotent cells. However, these precisely defined protocols are relatively inefficient and have not been used successfully to produce hematopoietic stem cells capable of multilineage long-term reconstitution of the hematopoietic system. More complex differentiation induction strategies including coculture with stromal cells derived from sites of hematopoietic activity in vivo and enforced expression of reprogramming transcription factors, such as HOXB4, have been required to increase the efficiency of the differentiation procedure and to produce these most potent hematopoietic stem cells. We review the studies that have used HOXB4 to improve hematopoietic differentiation from pluripotent cells focusing on studies that have provided some insight into its mechanism of action. A better understanding of the molecular pathways involved in the action of HOXB4 might lead to more defined culture systems and safer protocols for clinical translation.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/physiology , Homeodomain Proteins/physiology , Transcription Factors/physiology , Animals , Embryonic Stem Cells/metabolism , Gene Expression Regulation , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/physiology , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Stem Cell Niche , Transcription Factors/metabolism , Animals , Cells, Cultured , Embryo, Mammalian/metabolism , Embryonic Stem Cells/metabolism , Gene Expression , Gene Expression Profiling , Gene Expression Regulation , Glycoproteins/genetics , Glycoproteins/metabolism , Hematopoiesis , Homeodomain Proteins/genetics , Humans , Intracellular Signaling Peptides and Proteins , Mice , NIH 3T3 Cells , Oligonucleotide Array Sequence Analysis , Paracrine Communication , Transcription Factors/genetics , beta Catenin/metabolismABSTRACT
Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.
ABSTRACT
To generate sufficient numbers of transplantable hematopoietic stem cells (HSCs) in vitro, a detailed understanding of how this process takes place in vivo is essential. The endothelial-to-hematopoietic transition (EHT), which culminates in the production of the first HSCs, is a highly complex process during which key regulators are switched on and off at precise moments, and that is embedded into a myriad of microenvironmental signals from surrounding cells and tissues. We have previously demonstrated an HSC-supportive function for GATA3 within the sympathetic nervous system and the sub-aortic mesenchyme, but show here that it also plays a cell-intrinsic role during the EHT. It is expressed in hemogenic endothelial cells and early HSC precursors, where its expression correlates with a more quiescent state. Importantly, endothelial-specific deletion of Gata3 shows that it is functionally required for these cells to mature into HSCs, placing GATA3 at the core of the EHT regulatory network.
Subject(s)
Hemangioblasts , Hematopoietic Stem Cells , Cell Differentiation/genetics , Endothelium , Gonads , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Mesoderm , MesonephrosABSTRACT
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.
ABSTRACT
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.
Subject(s)
Apelin Receptors/metabolism , Hematopoiesis , Signal Transduction , Animals , Apelin/metabolism , Apelin Receptors/genetics , Cell Aggregation , Cell Differentiation , Cells, Cultured , Gene Deletion , Gene Expression Regulation , Genes, Reporter , Hemangioblasts/metabolism , Hematopoiesis/genetics , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Ligands , Mesoderm/cytology , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Peptide Hormones/metabolismABSTRACT
Erythropoiesis is one of the most demanding processes in the body, with more than 2 million red blood cells produced every second. Multiple hereditary and acquired red blood cell disorders arise from this complex system, with existing treatments effective in managing some of these conditions but few offering a long-term cure. Finding new treatments relies on the full understanding of the cellular and molecular interactions associated with the production and maturation of red blood cells, which take place within the erythroblastic island niche. The elucidation of processes associated within the erythroblastic island niche in health and during stress erythropoiesis has relied on in vivo modeling in mice, with complexities dissected using simple in vitro systems. Recent progress using state-of-the-art stem cell technology and gene editing has enabled a more detailed study of the human niche. Here, we review these different models and describe how they have been used to identify and characterize the cellular and molecular pathways associated with red blood cell production and maturation. We speculate that these systems could be applied to modeling red blood cell diseases and finding new druggable targets, which would prove especially useful for patients resistant to existing treatments. These models could also aid in research into the manufacture of red blood cells in vitro to replace donor blood transfusions, which is the most common treatment of blood disorders.
Subject(s)
Disease Models, Animal , Erythroblasts/cytology , Erythropoiesis/physiology , Models, Biological , Stem Cell Niche/physiology , Stress, Physiological/physiology , Animals , Cell Adhesion Molecules/deficiency , Cell Communication , Cells, Cultured , Coculture Techniques , Drug Evaluation, Preclinical , Erythropoiesis/drug effects , Erythropoiesis/genetics , Hematinics/therapeutic use , Hematologic Diseases/drug therapy , Hematologic Diseases/physiopathology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Janus Kinase 2/genetics , Janus Kinase 2/physiology , Macrophages/classification , Macrophages/physiology , Mice , Mice, Transgenic , Stem Cell Niche/drug effects , Stress, Physiological/geneticsABSTRACT
Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.
Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/cytology , Macrophages/cytology , Biomarkers/metabolism , Cell Count , Cell Differentiation , Cell Membrane/metabolism , Cell Polarity , Cell Shape , Cells, Cultured , Embryoid Bodies/cytology , Humans , Macrophages/metabolism , Phagocytosis , PhenotypeABSTRACT
Tumor-associated macrophages (TAMs) are becoming a promising target for cancer immunotherapy. Significant efforts have been made to study the detrimental role of TAMs both in vivo and in vitro. However, it remains challenging to isolate these macrophages to study their function in human cancers and there is the need to seek alternatives to address these limitations. In this review, we will focus on the three most relevant approaches to obtain in vitro fully differentiated macrophages i.e. peripheral blood, immortalized cell lines such as THP-1 or human induced pluripotent stem cells. We will also provide protocols for the polarization of human macrophages to a TAM-like cells in vitro.
Subject(s)
Tumor-Associated Macrophages/cytology , Cell Culture Techniques/methods , Cell Differentiation , Cell Line , Cell Separation/methods , Humans , Immunophenotyping/methods , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/immunology , Monocytes/cytology , Monocytes/immunology , Tumor-Associated Macrophages/immunologyABSTRACT
Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.
ABSTRACT
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.
Subject(s)
Erythroblasts/cytology , Erythrocytes/cytology , Erythropoiesis/physiology , Induced Pluripotent Stem Cells/cytology , Kruppel-Like Transcription Factors/metabolism , Macrophages/cytology , Angiopoietin-Like Protein 7 , Angiopoietin-like Proteins/metabolism , Antigens, CD34/metabolism , Blood Substitutes/therapeutic use , Blood Transfusion , Hematopoietic Stem Cells/cytology , Humans , Interleukin-33/metabolism , Kruppel-Like Transcription Factors/genetics , Plasminogen Activator Inhibitor 2/metabolismABSTRACT
The roles of tumor-associated macrophages (TAMs) and circulating monocytes in human cancer are poorly understood. Here, we show that monocyte subpopulation distribution and transcriptomes are significantly altered by the presence of endometrial and breast cancer. Furthermore, TAMs from endometrial and breast cancers are transcriptionally distinct from monocytes and their respective tissue-resident macrophages. We identified a breast TAM signature that is highly enriched in aggressive breast cancer subtypes and associated with shorter disease-specific survival. We also identified an auto-regulatory loop between TAMs and cancer cells driven by tumor necrosis factor alpha involving SIGLEC1 and CCL8, which is self-reinforcing through the production of CSF1. Together these data provide direct evidence that monocyte and macrophage transcriptional landscapes are perturbed by cancer, reflecting patient outcomes.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Cellular Reprogramming , Macrophages/metabolism , Monocytes/metabolism , Paracrine Communication , Transcription, Genetic , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Chemokine CCL8/genetics , Chemokine CCL8/metabolism , Endometrial Neoplasms/genetics , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Induced Pluripotent Stem Cells/metabolism , Macrophage Colony-Stimulating Factor/genetics , Macrophages/pathology , Molecular Targeted Therapy , Monocytes/pathology , Sialic Acid Binding Ig-like Lectin 1/genetics , Sialic Acid Binding Ig-like Lectin 1/metabolism , Signal Transduction , THP-1 Cells , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Aminopeptidases/metabolism , Endothelial Cells/enzymology , Endothelium, Vascular/enzymology , Nuclear Localization Signals/metabolism , Alternative Splicing , Aminopeptidases/genetics , Animals , COS Cells , Cell Nucleolus/metabolism , Chlorocebus aethiops , Embryo, Mammalian/metabolism , Endothelium, Vascular/embryology , Endothelium, Vascular/growth & development , Green Fluorescent Proteins/genetics , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Organ Specificity , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Umbilical Veins/cytologyABSTRACT
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'.