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.

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.

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.

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.

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.

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.

NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

APELIN promotes hematopoiesis from human embryonic stem cells.

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells, identified by binding of the fluoresceinated peptide ligand, APELIN (APLN), or an anti-APLNR mAb, were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs), increased the expression of hematoendothelial genes in differentiating hESCs, and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore, APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells.

Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview.

Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

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.

Fourier transform infrared microspectroscopy reveals that tissue culture conditions affect the macromolecular phenotype of human embryonic stem cells.

We employed Fourier transform infrared (FTIR) microspectroscopy to investigate the effects of different tissue culture environments on the FTIR spectra of undifferentiated human embryonic stem cells (hESCs) and their differentiated progeny. First we tested whether there were any possible spectral artifacts resulting from the use of transflectance measurements by comparing them with transmission measurements and found no evidence of these concluding that the lack of any differences resulted from the homogeneity of the dried cytospun cellular monolayers. We found that hESCs that were enzymatically passaged onto mouse embryonic fibroblasts (MEFs) in KOSR based hESC medium, hESCs enzymatically passaged onto Matrigel in mTESR medium and hESCs mechanically passaged onto MEFs in KOSR-based hESC medium, possessed unique FTIR spectroscopic signatures that reflect differences in their macromolecular chemistry. Further, these spectroscopic differences persisted even upon differentiation towards mesendodermal lineages. Our results suggest that FTIR microspectroscopy is a powerful, objective, measurement modality that complements existing methods for studying the phenotype of hESCs and their progeny, particularly changes induced by the cellular environment.

The characterisation of pluripotent and multipotent stem cells using Fourier transform infrared microspectroscopy.

Fourier transform infrared (FTIR) microspectroscopy shows potential as a benign, objective and rapid tool to screen pluripotent and multipotent stem cells for clinical use. It offers a new experimental approach that provides a holistic measurement of macromolecular composition such that a signature representing the internal cellular phenotype is obtained. The use of this technique therefore contributes information that is complementary to that acquired by conventional genetic and immunohistochemical methods.

ErythRED, a hESC line enabling identification of erythroid cells.

A human embryonic stem cell (hESC) line that enabled globin-expressing cells to be easily recognized would facilitate optimization of erythroid differentiation in vitro and aid in the identification of hESC-derived erythroid cells in transplanted animals. We describe a genetically modified hESC line, ErythRED, in which expression of RFP, controlled by regulatory sequences from the human beta-globin locus control region, is restricted to maturing erythroid cells.

A targeted NKX2.1 human embryonic stem cell reporter line enables identification of human basal forebrain derivatives.

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.

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.

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.

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.

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.