Glycogen synthase kinase 3 induces multilineage maturation of human pluripotent stem cell-derived lung progenitors in 3D culture.

Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development.

Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation.

The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility, but the mechanisms by which this occurs are poorly understood. Here, we analyze the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). Our results show that early enhancers are characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. We envision that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner.

Organ regeneration does not require a functional stem cell niche in plants.

Plants rely on the maintenance of stem cell niches at their apices for the continuous growth of roots and shoots. However, although the developmental plasticity of plant cells has been demonstrated, it is not known whether the stem cell niche is required for organogenesis. Here we explore the capacity of a broad range of differentiating cells to regenerate an organ without the activity of a stem cell niche. Using a root-tip regeneration system in Arabidopsis thaliana to track the molecular and functional recovery of cell fates, we show that re-specification of lost cell identities begins within hours of excision and that the function of specialized cells is restored within one day. Critically, regeneration proceeds in plants with mutations that fail to maintain the stem cell niche. These results show that stem-cell-like properties that mediate complete organ regeneration are dispersed in plant meristems and are not restricted to niches, which nonetheless seem to be necessary for indeterminate growth. This regenerative reprogramming of an entire organ without transition to a stereotypical stem cell environment has intriguing parallels to recent reports of induced transdifferentiation of specific cell types in the adult organs of animals.

The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila.

In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

A distal lung organoid model to study interstitial lung disease, viral infection and human lung development.

Organoids have been an exciting advancement in stem cell research. Here we describe a strategy for directed differentiation of human pluripotent stem cells into distal lung organoids. This protocol recapitulates lung development by sequentially specifying human pluripotent stem cells to definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm, lung bud organoids and finally lung organoids. The organoids take ~40 d to generate and can be maintained more than 180 d, while progressively maturing up to a stage consistent with the second trimester of human gestation. They are unique because of their branching morphology, the near absence of non-lung endodermal lineages, presence of mesenchyme and capacity to recapitulate interstitial lung diseases. This protocol can be performed by anyone familiar with cell culture techniques, is conducted in serum-free conditions and does not require lineage-specific reporters or enrichment steps. We also provide a protocol for the generation of single-cell suspensions for single-cell RNA sequencing.

Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation.

Chromatin accessibility is integral to the process by which transcription factors (TFs) read out cis-regulatory DNA sequences, but it is difficult to differentiate between TFs that drive accessibility and those that do not. Deep learning models that learn complex sequence rules provide an unprecedented opportunity to dissect this problem. Using zygotic genome activation in Drosophila as a model, we analyzed high-resolution TF binding and chromatin accessibility data with interpretable deep learning and performed genetic validation experiments. We identify a hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, whereas patterning TFs augment chromatin accessibility in sequence contexts where they mediate enhancer activation. We conclude that chromatin accessibility occurs in two tiers: one through pioneering, which makes enhancers accessible but not necessarily active, and the second when the correct combination of TFs leads to enhancer activation.

Engraftment and injury repair in regionally conditioned rat lung in vivo by lung progenitors derived from human pluripotent stem cells.

Although lung disease is a major cause of mortality, the mechanisms involved in human lung regeneration are unclear because of the lack of experimental models. Here we report a novel model where human pluripotent stem cell-derived expandable cell lines sharing features of airway secretory and basal cells engraft in the distal rat lung after conditioning by locoregional de-epithelialization followed by irradiation and immunosuppression. The engrafting cells, which we named distal lung epithelial progenitors (DLEPs), contributed to alveolar epithelial cells and generated 'KRT5-pods', structures involved in distal lung repair after severe injury, but only rarely to distal airways. Most strikingly, however, injury induced by the conditioning regimen was largely prevented by the engrafting DLEPs. The approach described here provides a model to study mechanisms involved in human lung regeneration, and potentially lays the foundation for the preclinical development of cell therapy to treat lung injury and disease.

Knowledge, Intention, and Self-Efficacy Associated with Breastfeeding: Impact of These Factors on Breastfeeding during Postpartum Hospital Stays in Taiwanese Women.

Breastfeeding knowledge, intention, and self-efficacy affect breastfeeding rates during the postpartum period. Insufficient knowledge, lack of intention, and poor breastfeeding self-efficacy reduce the likelihood of breastfeeding postpartum. The purposes of this study were to (1) assess women's intention to breastfeed and knowledge and self-efficacy regarding breastfeeding following childbirth, and to (2) identify the factors associated with postpartum breastfeeding during women's hospital stays. This longitudinal study with a pretest and posttest design study recruited pregnant women from the gynecology and obstetrics outpatient departments and inpatient wards at a medical center in northern Taiwan. Demographic and obstetric characteristics were recorded, and participants were assessed using the Numeric Rating Scale, the Breastfeeding Knowledge Questionnaire, the Breastfeeding Self-Efficacy Scale-Short Form, and breastfeeding status postpartum. Of the 120 participants, 25% reported breastfeeding during the postpartum hospital stay. Postpartum breastfeeding was associated with lower levels of education and higher prenatal levels of breastfeeding intention. Establishing a breastfeeding-friendly environment in the family and workplace may effectively increase continued breastfeeding.

The in vitro multilineage differentiation and maturation of lung and airway cells from human pluripotent stem cell-derived lung progenitors in 3D.

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 mature lung and airway epithelial cells obtained through maturation of NKX2.1+ hPSC-derived lung progenitors in a 3D matrix of collagen I in the absence of glycogen synthase kinase 3 inhibition. This protocol is an extension of our previously published protocol on the directed differentiation of lung and airway epithelium from hPSCs that modifies the technique and offers additional applications. This protocol is conducted in defined media conditions, has a duration of 50-80 d, does not require reporter lines and results in cultures containing mature alveolar type II and I cells as well as airway basal, ciliated, club and neuroendocrine cells. We also present a flow cytometry strategy to assess maturation in the cultures. Several of these populations, including mature NGFR+ basal cells, can be prospectively isolated by cell sorting and expanded for further investigation.

Perovskite Quantum Dots with Near Unity Solution and Neat-Film Photoluminescent Quantum Yield by Novel Spray Synthesis.

In this study, a novel perovskite quantum dot (QD) spray-synthesis method is developed by combining traditional perovskite QD synthesis with the technique of spray pyrolysis. By utilizing this new technique, the synthesis of cubic-shaped perovskite QDs with a homogeneous size of 14 nm is demonstrated, which shows an unprecedented stable absolute photoluminescence quantum yield ≈100% in the solution and even in the solid-state neat film. The highly emissive thin films are integrated with light emission devices (LEDs) and organic light emission displays (OLEDs). The color conversion type QD-LED (ccQD-LED) hybrid devices exhibit an extremely saturated green emission, excellent external quantum efficiency of 28.1%, power efficiency of 121 lm W-1 , and extraordinary forward-direction luminescence of 8 500 000 cd m-2 . The conceptual ccQD-OLED hybrid display also successfully demonstrates high-definition still images and moving pictures with a 119% National Television System Committee 1931 color gamut and 123% Digital Cinema Initiatives-P3 color gamut. These very-stable, ultra-bright perovskite QDs have the properties necessary for a variety of useful applications in optoelectronics.