Building a human lung from pluripotent stem cells to model respiratory viral infections.

To protect against the constant threat of inhaled pathogens, the lung is equipped with cellular defenders. In coordination with resident and recruited immune cells, this defence is initiated by the airway and alveolar epithelium following their infection with respiratory viruses. Further support for viral clearance and infection resolution is provided by adjacent endothelial and stromal cells. However, even with these defence mechanisms, respiratory viral infections are a significant global health concern, causing substantial morbidity, socioeconomic losses, and mortality, underlining the need to develop effective vaccines and antiviral medications. In turn, the identification of new treatment options for respiratory infections is critically dependent on the availability of tractable in vitro experimental models that faithfully recapitulate key aspects of lung physiology. For such models to be informative, it is important these models incorporate human-derived, physiologically relevant versions of all cell types that normally form part of the lungs anti-viral response. This review proposes a guideline using human induced pluripotent stem cells (iPSCs) to create all the disease-relevant cell types. iPSCs can be differentiated into lung epithelium, innate immune cells, endothelial cells, and fibroblasts at a large scale, recapitulating in vivo functions and providing genetic tractability. We advocate for building comprehensive iPSC-derived in vitro models of both proximal and distal lung regions to better understand and model respiratory infections, including interactions with chronic lung diseases.

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.

RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266.

PGC-1α and reactive oxygen species regulate human embryonic stem cell-derived cardiomyocyte function.

Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is normally induced during development of cardiomyocytes, decreased mitochondrial content and activity and decreased the capacity for coping with energetic stress. Yet, concurrently, reactive oxygen species (ROS) levels were lowered, and the amplitude of the action potential and the maximum amplitude of the calcium transient were in fact increased. Importantly, in control cardiomyocytes, lowering ROS levels emulated this beneficial effect of PGC-1α knockdown and similarly increased the calcium transient amplitude. Our results suggest that controlling ROS levels may be of key physiological importance for recapitulating mature cardiomyocyte phenotypes, and the combination of bioassays used in this study may have broad application in the analysis of cardiac physiology pertaining to disease.

Germ layer induction in ESC--following the vertebrate roadmap.

Controlled differentiation of pluripotential cells takes place routinely and with great success in developing vertebrate embryos. It therefore makes sense to take note of how this is achieved and use this knowledge to control the differentiation of embryonic stem cells (ESCs). An added advantage is that the differentiated cells resulting from this process in embryos have proven functionality and longevity. This unit reviews what is known about the embryonic signals that drive differentiation in one of the most informative of the vertebrate animal models of development, the amphibian Xenopus laevis. It summarizes their identities and the extent to which their activities are dose-dependent. The unit details what is known about the transcription factor responses to these signals, describing the networks of interactions that they generate. It then discusses the target genes of these transcription factors, the effectors of the differentiated state. Finally, how these same developmental programs operate during germ layer formation in the context of ESC differentiation is summarized.