Generation of heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2/COUP-TFII knockout human iPSC lines.

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.

Modeling human skeletal development using human pluripotent stem cells.

Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring in vivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.

Lymphoid cell development from fetal hematopoietic progenitors and human pluripotent stem cells.

Lymphoid cells encompass the adaptive immune system, including T and B cells and Natural killer T cells (NKT), and innate immune cells (ILCs), including Natural Killer (NK) cells. During adult life, these lineages are thought to derive from the differentiation of long-term hematopoietic stem cells (HSCs) residing in the bone marrow. However, during embryogenesis and fetal development, the ontogeny of lymphoid cells is both complex and multifaceted, with a large body of evidence suggesting that lymphoid lineages arise from progenitor cell populations antedating the emergence of HSCs. Recently, the application of single cell RNA-sequencing technologies and pluripotent stem cell-based developmental models has provided new insights into lymphoid ontogeny during embryogenesis. Indeed, PSC differentiation platforms have enabled de novo generation of lymphoid immune cells independently of HSCs, supporting conclusions drawn from the study of hematopoiesis in vivo. Here, we examine lymphoid development from non-HSC progenitor cells and technological advances in the differentiation of human lymphoid cells from pluripotent stem cells for clinical translation.

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development.

Precursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA+) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-ß and WNT signaling (SB/CHIR). Microfluidic and orbital culture promoted the formation of proliferative CD34+RUNX1C-GFP+SOX17-mCHERRY+ precursor cells that were released into the artificial circulation from SOX17+ arterial-like structures. Single-cell transcriptomic analysis delineated extra-embryonic (yolk sac) and HOXA+ embryonic blood differentiation pathways. SB/CHIR and circulatory flow enhance hematopoiesis by the formation of proliferative HOXA+RUNX1C+CD34+ precursor cells that differentiate into monocyte/macrophage, granulocyte, erythrocyte, and megakaryocyte progenitors.

Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection.

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.

Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

PLCG1 is required for AML1-ETO leukemia stem cell self-renewal.

In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

VEGF, FGF2, and BMP4 regulate transitions of mesoderm to endothelium and blood cells in a human model of yolk sac hematopoiesis.

Exogenous growth factors play an important role in mediating hematopoietic differentiation of human pluripotent stem cells. We explored the role of different factors in early human blood cell production using blast colony formation in methylcellulose as a surrogate assay for yolk sac hematopoiesis. A reporter cell line that read out endothelial (SOX17+) and hematopoietic (RUNX1C+) progenitors facilitated the identification of basic fibroblast growth and vascular endothelial growth factor as critical signals for the progression of mesoderm into endothelium. Bone morphogenetic protein 4 was needed for the subsequent generation of blood from hemogenic endothelium, and this was antagonized by Activin A or high concentrations of the WNT agonist CHIR-99021. Manipulations of the Hedgehog pathway or inhibition of Notch signaling reduced blast colony frequency but did not perturb cell differentiation. These data help to define distinct roles for prerequisite growth factors that commit mesoderm to hemogenic endothelium and subsequently allocate cells to blood lineages.

Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI1B-dependent blood and SOX17-positive endothelium.

The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17-CD34+CD43- endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17-CD34+CD43+ blood cells and SOX17+CD34+CD43- endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.

Expression of RUNX1-ETO Rapidly Alters the Chromatin Landscape and Growth of Early Human Myeloid Precursor Cells.

Acute myeloid leukemia (AML) is a hematopoietic malignancy caused by recurrent mutations in genes encoding transcriptional, chromatin, and/or signaling regulators. The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO (RUNX1-RUNX1T1), which by itself is insufficient to cause disease. t(8;21) AML patients show extensive chromatin reprogramming and have acquired additional mutations. Therefore, the genomic and developmental effects directly and solely attributable to RUNX1-ETO expression are unclear. To address this, we employ a human embryonic stem cell differentiation system capable of forming definitive myeloid progenitor cells to express RUNX1-ETO in an inducible fashion. Induction of RUNX1-ETO causes extensive chromatin reprogramming by interfering with RUNX1 binding, blocks differentiation, and arrests cellular growth, whereby growth arrest is reversible following RUNX1-ETO removal. Single-cell gene expression analyses show that RUNX1-ETO induction alters the differentiation of early myeloid progenitors, but not of other progenitor types, indicating that oncoprotein-mediated transcriptional reprogramming is highly target cell specific.

Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids.

Defining the ontogeny of the human adaptive immune system during embryogenesis has implications for understanding childhood diseases including leukaemias and autoimmune conditions. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent-stem-cell-derived haematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively upregulated a cohort of recognized T-cell-associated genes, arresting development at the CD4+CD8+ stage. Sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Flow cytometry and single-cell-RNA-sequencing data showed that early RAG1+ cells co-expressed the endothelial/haematopoietic progenitor markers CD34, VECAD and CD90, whereas imaging studies identified RAG1+ cells within CD31+ endothelial structures that co-expressed SOX17+ or the endothelial marker CAV1. Collectively, these observations provide evidence for a wave of human T-cell development that originates directly from haemogenic endothelium via a RAG1+ intermediate with multilineage potential.

In the absence of apoptosis, myeloid cells arrest when deprived of growth factor, but remain viable by consuming extracellular glucose.

Withdrawal of the growth factor interleukin-3 (IL-3) from IL-3-dependent myeloid cells causes them to undergo Bax/Bak1-dependent apoptosis, whereas factor-deprived Bax-/-Bak1-/- cells remain viable, but arrest and shrink. It was reported that withdrawal of IL-3 from Bax-/-Bak1-/- cells caused decreased expression of the glucose transporter Glut1, leading to reduced glucose uptake, so that arrested cells required Atg5-dependent autophagy for long-term survival. In other cell types, a decrease in Glut1 is mediated by the thioredoxin-interacting protein (Txnip), which is induced in IL-3-dependent myeloid cells when growth factor is removed. We mutated Atg5 and Txnip by CRISPR/Cas9 and found that Atg5-dependent autophagy was not necessary for the long-term viability of cycling or arrested Bax-/-Bak1-/- cells, and that Txnip was not required for the decrease in Glut1 expression in response to IL-3 withdrawal. Surprisingly, Atg5-deficient Bax/Bak1 double mutant cells survived for several weeks in medium supplemented with 10% fetal bovine serum (FBS), without high concentrations of added glucose or glutamine. When serum was withdrawn, the provision of an equivalent amount of glucose present in 10% FBS (~0.5 mM) was sufficient to support cell survival for more than a week, in the presence or absence of IL-3. Thus, Bax-/-Bak1-/- myeloid cells deprived of growth factor consume extracellular glucose to maintain long-term viability, without a requirement for Atg5-dependent autophagy.

NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.

WNT9A Is a Conserved Regulator of Hematopoietic Stem and Progenitor Cell Development.

Hematopoietic stem cells (HSCs) differentiate into all cell types of the blood and can be used therapeutically to treat hematopoietic cancers and disorders. Despite decades of research, it is not yet possible to derive therapy-grade HSCs from pluripotent precursors. Analysis of HSC development in model organisms has identified some of the molecular cues that are necessary to instruct hematopoiesis in vivo, including Wnt9A, which is required during an early time window in zebrafish development. Although bona fide HSCs cannot be derived in vitro, it is possible to model human hematopoietic progenitor development by differentiating human pluripotent stem cells to hematopoietic cells. Herein, we modulate WNT9A expression during the in vitro differentiation of human embryonic stem cells to hematopoietic progenitor cells and demonstrate that WNT9A also regulates human hematopoietic progenitor cell development in vitro. Overexpression of WNT9A only impacts differentiation to CD34⁺/CD45⁺ cells during early time windows and does so in a dose-dependent manner. The cells that receive the Wnt signal-not the cells that secrete WNT9A-differentiate most efficiently to hematopoietic progenitors; this mimics the paracrine action of Wnt9a during in vivo hematopoiesis. Taken together, these data indicate that WNT9A is a conserved regulator of zebrafish and human hematopoietic development.

Human haematopoietic stem cell development: from the embryo to the dish.

Haematopoietic stem cells (HSCs) emerge during embryogenesis and give rise to the adult haematopoietic system. Understanding how early haematopoietic development occurs is of fundamental importance for basic biology and medical sciences, but our knowledge is still limited compared with what we know of adult HSCs and their microenvironment. This is particularly true for human haematopoiesis, and is reflected in our current inability to recapitulate the development of HSCs from pluripotent stem cells in vitro In this Review, we discuss what is known of human haematopoietic development: the anatomical sites at which it occurs, the different temporal waves of haematopoiesis, the emergence of the first HSCs and the signalling landscape of the haematopoietic niche. We also discuss the extent to which in vitro differentiation of human pluripotent stem cells recapitulates bona fide human developmental haematopoiesis, and outline some future directions in the field.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34(Low)CD31(Pos)VEGFR-3(Pos)PODOPLANIN(Pos) LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

UNLABELLED: Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. SIGNIFICANCE: This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold.