Hans-Willem Snoeck

Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells

Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-β and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.

Efficient Derivation of Purified Lung and Thyroid Progenitors from Embryonic Stem Cells

Two populations of Nkx2-1(+) progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1(GFP) knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.

Efficient generation of lung and airway epithelial cells from human pluripotent stem cells

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP—agonists to signaling pathways critical for early lung development in the mouse—recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.

Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency

A genetic cause for severe influenza Although chicken soup and plenty of rest get most kids through an influenza virus infection, some require hospitalization. Ciancanelli et al. report on one child who suffered severely from influenza because of null mutations in the gene for transcription factor IRF7. Cells isolated from this patient could not make enough secreted antiviral proteins, called interferons, to halt viral replication. The requirement for IRF7 seems quite specific, because this patient recovers normally from other common childhood viral infections. Science, this issue p. 448 A mutation that reduces antiviral interferons underlies certain cases of severe influenza in children. Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient’s leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient’s dermal fibroblasts and induced pluripotent stem cell (iPSC)–derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.

Prdm16 is a physiologic regulator of hematopoietic stem cells

Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.

Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential

Haematopoietic stem cells (HSCs), which sustain production of all blood cell lineages, rely on glycolysis for ATP production, yet little attention has been paid to the role of mitochondria. Here we show in mice that the short isoform of a critical regulator of HSCs, Prdm16 (refs 4, 5), induces mitofusin 2 (Mfn2), a protein involved in mitochondrial fusion and in tethering of mitochondria to the endoplasmic reticulum. Overexpression and deletion studies, including single-cell transplantation assays, revealed that Mfn2 is specifically required for the maintenance of HSCs with extensive lymphoid potential, but not, or less so, for the maintenance of myeloid-dominant HSCs. Mfn2 increased buffering of intracellular Ca2+, an effect mediated through its endoplasmic reticulum–mitochondria tethering activity, thereby negatively regulating nuclear translocation and transcriptional activity of nuclear factor of activated T cells (Nfat). Nfat inhibition rescued the effects of Mfn2 deletion in HSCs, demonstrating that negative regulation of Nfat is the prime downstream mechanism of Mfn2 in the maintenance of HSCs with extensive lymphoid potential. Mitochondria therefore have an important role in HSCs. These findings provide a mechanism underlying clonal heterogeneity among HSCs and may lead to the design of approaches to bias HSC differentiation into desired lineages after transplantation.

Quantitative Trait Analysis Reveals Transforming Growth Factor-β2 as a Positive Regulator of Early Hematopoietic Progenitor and Stem Cell Function

Elucidation of pathways involved in mouse strain–dependent variation in the hematopoietic stem cell (HSC) compartment may reveal novel mechanisms relevant in vivo. Here, we demonstrate genetically determined variation in the proliferation of lin−Sca1++kit+ (LSK) primitive hematopoietic progenitor cells in response to transforming growth factor-β (TGF-β) 2, the dose response of which was biphasic with a stimulatory effect at low concentrations. In contrast, the dose responses of TGF-β1 or -β3 were inhibitory and did not show mouse strain–dependent variation. A quantitative trait locus (QTL) for the effect of TGF-β2 was identified on chromosome 4 overlapping with a QTL regulating the frequency of LSK cells. These overlapping QTL were corroborated by the observation that the frequency of LSK cells is lower in adult Tgfb2 +/− mice than in wild-type littermates, indicating that TGF-β2 is a genetically determined positive regulator LSK number in vivo. Furthermore, adult Tgfb2 +/− mice have a defect in competitive repopulation potential that becomes more pronounced upon serial transplantation. In fetal TGF-β2–deficient HSCs, a defect only appears after serial reconstitution. These data suggest that TGF-β2 can act cell autonomously and is important for HSCs that have undergone replicative stress. Thus, TGF-β2 is a novel, genetically determined positive regulator of adult HSCs.

A three-dimensional model of human lung development and disease from pluripotent stem cells

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

Lin−Sca1+Kit− Bone Marrow Cells Contain Early Lymphoid-Committed Precursors That Are Distinct from Common Lymphoid Progenitors

The significance of a population in mouse bone marrow of lineage-negative (Lin−), Sca1-positive, c-kit-negative (LSK−) cells, which is reported to be devoid of long-term repopulation capacity or myeloid potential, is unknown. In this study, we show that the LSK− population is composed of several subsets defined by the expression of flt3, CD25, and IL-7Rα. The first subset was CD25− and more than 90% expressed either flt3, IL-7Rα, or both. The CD25−LSK− population had T cell, B cell, and NK cell potential in vivo, and most of this activity was localized in the flt3+ subset, irrespective of the expression of IL-7Rα. Although lymphoid potential of flt3+LSK− cells in vivo was 3-fold lower than that of lin−Sca1lowkitlowIL7Rα+ common lymphoid progenitors (CLPs), their cloning efficiency in vitro was 10-fold lower than that of CLPs. Furthermore, although the myeloid potential of flt3+LSK− cells was 10-fold lower than that of CLPs in the absence of M-CSF, the relative myeloid potential of both populations was similar in its presence. These observations suggest differential growth factor requirements of both populations. The second subset of LSK− cells was homogeneously CD25+flt3−IL7Rα+ and could be generated from both CD25−LSK− cells and from CLPs, but did not engraft in immunodeficient Rag1−/− or Rag1−/−γc−/− hosts. This population, of which the significance is unclear, was increased in Rag1−/− mice and in old mice. Thus, the LSK− population is phenotypically and functionally heterogeneous and contains early lymphoid-committed precursors. Our findings imply that the early stages of lymphoid commitment are more complex than was thus far assumed.

The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes ∼50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.