Search

Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in which human induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) form polarized duct-like lumens alongside engineered perfused vessels lined with human umbilical vein endothelium, all within a 3D, physiologically relevant microenvironment. Using this model, we investigated the morphologic and signaling consequences of the KRASG12D mutation, a commonly identified oncogene in human lung adenocarcinoma (LUAD). We show that expression of the mutant KRASG12D isoform in iAT2s leads to a hyperproliferative response and morphologic dysregulation in the epithelial monolayer. Interestingly, the mutant epithelia also drive an angiogenic response in the adjacent vasculature that is mediated by enhanced secretion of the pro-angiogenic factor soluble uPAR. These results demonstrate the functionality of a multi-cellular in vitro platform capable of modeling mutation-specific behavioral and signaling changes associated with lung adenocarcinoma.

Abstract Alveolar type 2 epithelial cells (AT2s) derived from human induced pluripotent stem cells (iAT2s) have rapidly contributed to our understanding of AT2 function and disease. However, while iAT2s are primarily cultured in three-dimensional (3D) Matrigel, a matrix derived from cancerous mouse tissue, it is unclear how a physiologically relevant matrix will impact iAT2s phenotype. As extracellular matrix (ECM) is recognized as a vital component in directing cellular function and differentiation, we sought to derive hydrogels from decellularized human lung alveolar-enriched ECM (aECM) to provide an ex vivo model to characterize the role of physiologically relevant ECM on iAT2 phenotype. We demonstrate aECM hydrogels retain critical in situ ECM components, including structural and basement membrane proteins. While aECM hydrogels facilitate iAT2 proliferation and alveolosphere formation, a subset of iAT2s rapidly change morphology to thin and elongated ring-like cells. This morphological change correlates with upregulation of recently described iAT2-derived transitional cell state genetic markers. As such, we demonstrate a potentially underappreciated role of physiologically relevant aECM in iAT2 differentiation.

Abstract While the generation of many lineages from pluripotent stem cells has resulted in basic discoveries and clinical trials, the derivation of tissue-specific mesenchyme via directed differentiation has markedly lagged. The derivation of lung-specific mesenchyme is particularly important since this tissue plays crucial roles in lung development and disease. Here we generate a mouse induced pluripotent stem cell (iPSC) line carrying a lung-specific mesenchymal reporter/lineage tracer. We identify the pathways (RA and Shh) necessary to specify lung mesenchyme and find that mouse iPSC-derived lung mesenchyme (iLM) expresses key molecular and functional features of primary developing lung mesenchyme. iLM recombined with engineered lung epithelial progenitors self-organizes into 3D organoids with juxtaposed layers of epithelium and mesenchyme. Co-culture increases yield of lung epithelial progenitors and impacts epithelial and mesenchymal differentiation programs, suggesting functional crosstalk. Our iPSC-derived population thus provides an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single-cell resolution. Here, we performed head-to-head comparisons among the transcriptomes of primary (1°) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell–derived AEC2s (iAEC2s). We found each population occupied a distinct transcriptomic space with cultured AEC2s (1° and iAEC2s) exhibiting similarities to and differences from freshly purified 1° cells. Across each cell type, we found an inverse relationship between proliferative and maturation states, with preculture 1° AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2s did not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s cocultured with fibroblasts acquired a transitional cell state described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1° and engineered AEC2s, 2 in vitro models that can be harnessed to study human lung health and disease.

Enhancers for endodermal organs are primed at the chromatin level prior to lineage induction by FOXA pioneer transcription factors; how pervasive this is, is not well known. Here the authors show that only a small subset of organ-specific enhancers are bound and primed by FOXA prior to lineage induction, whereas the majority do not undergo chromatin priming and engage FOXA upon lineage induction.

Human-induced pluripotent stem cells (hiPSCs) cells have the proliferative potential and ability to differentiate into numerous cell types [...]

Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell–derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.

The identity of the earliest murine in vivo lung epithelial progenitors (marked by NKX2-1 expression) is unclear. Here, the authors use single-cell RNA sequencing to define the genetic program of these lung primordial progenitors, which will improve in vitro lung specification of pluripotent stem cells.

Human induced pluripotent stem cell-derived intestinal organoids (HIOs) are powerful tools to study development and diseases of the gastrointestinal tract. Here, the authors develop a directed differentiation protocol to generate mesenchyme-free HIOs that can be patterned towards proximal small intestine or colonic epithelium, and demonstrated their utility in modeling CFTR function.

Summary: Airway basal cells play an essential role in the maintenance of the airway epithelium. Here, we provide a detailed directed differentiation protocol to generate ‘‘induced basal cells (iBCs)’’ from human pluripotent stem cells. iBCs recapitulate biological and functional properties of airway basal cells including mucociliary differentiation in vitro or in vivo in tracheal xenografts, facilitating the study of inherited and acquired diseases of the airway, as well as potential use in regenerative medicine.For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2021).

Summary: Induced pluripotent stem cells (iPSCs) provide a potential source for the derivation of smooth muscle cells (SMCs); however, current approaches are limited by the production of heterogeneous cell types and a paucity of tools or markers for tracking and purifying candidate SMCs. Here, we develop murine and human iPSC lines carrying fluorochrome reporters (Acta2hrGFP and ACTA2eGFP, respectively) that identify Acta2+/ACTA2+ cells as they emerge in vitro in real time during iPSC-directed differentiation. We find that Acta2hrGFP+ and ACTA2eGFP+ cells can be sorted to purity and are enriched in markers characteristic of an immature or synthetic SMC. We characterize the resulting GFP+ populations through global transcriptomic profiling and functional studies, including the capacity to form engineered cell sheets. We conclude that these reporter lines allow for generation of sortable, live iPSC-derived Acta2+/ACTA2+ cells highly enriched in smooth muscle lineages for basic developmental studies, tissue engineering, or future clinical regenerative applications. : In this article, Wong, Kotton, and colleagues developed murine and human iPSC lines with fluorochrome reporters for ACTA2, allowing for isolation of putative iPSC-derived smooth muscle-like populations (iPSC-SMC) with transcriptomic profiles reminiscent of immature or synthetic SMCs. The iPSC-SMCs were used to generate cell sheets with mechanical properties within physiological ranges to serve as building blocks for multi-layered tissue constructs. Keywords: induced pluripotent stem cells, directed differentiation, smooth muscle cells, cell sheets, fluorochrome reporters

Stem cell-based therapies to reconstitute in vivo organ function hold great promise for future clinical applications to a variety of diseases. Hypothyroidism resulting from congenital lack of functional thyrocytes, surgical tissue removal, or gland ablation, represents a particularly attractive endocrine disease target that may be conceivably cured by transplantation of long-lived functional thyroid progenitors or mature follicular epithelial cells, provided a source of autologous cells can be generated and a variety of technical and biological challenges can be surmounted. Here we review the emerging literature indicating that thyroid follicular epithelial cells can now be engineered in vitro from the pluripotent stem cells (PSCs) of mice, normal humans, or patients with congenital hypothyroidism. We review the in vivo embryonic development of the thyroid gland and explain how emerging discoveries in developmental biology have been utilized as a roadmap for driving PSCs, which resemble cells of the early embryo, into mature functional thyroid follicles in vitro. Finally, we discuss the bioengineering, biological, and clinical hurdles that now need to be addressed if the goals of life-long cure of hypothyroidism through cell- and/or gene-based therapies are to be attained.

Summary: Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway. : In this article, Kotton and colleagues show that human pluripotent stem cell-derived airway epithelial spheres contain basal and secretory airway cells. Using reporter lines for the secretory airway lineage alongside transcriptomic and functional analyses, they demonstrate that these cells have characteristic features of this population but are susceptible to Wnt-dependent phenotypic drift toward an alveolar type II cell-like program. Keywords: pluripotent stem cells, lung epithelium, directed differentiation, single-cell RNA sequencing, airway, alveoli

The clinical importance of anterior foregut endoderm (AFE) derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs). Here, we identify transient overexpression of the transcription factor (TF) NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex) and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd) at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates.

Induced pluripotent stem cells (iPSCs) provide an inexhaustible source of cells for modeling disease and testing drugs. Here we develop a bioinformatic approach to detect differences between the genomic programs of iPSCs derived from diseased versus normal human cohorts as they emerge during in vitro directed differentiation. Using iPSCs generated from a cohort carrying mutations (PiZZ) in the gene responsible for alpha-1 antitrypsin (AAT) deficiency, we find that the global transcriptomes of PiZZ iPSCs diverge from normal controls upon differentiation to hepatic cells. Expression of 135 genes distinguishes PiZZ iPSC-hepatic cells, providing potential clues to liver disease pathogenesis. The disease-specific cells display intracellular accumulation of mutant AAT protein, resulting in increased autophagic flux. Furthermore, we detect beneficial responses to the drug carbamazepine, which further augments autophagic flux, but adverse responses to known hepatotoxic drugs. Our findings support the utility of iPSCs as tools for drug development or prediction of toxicity.

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

[This corrects the article DOI: 10.1371/journal.ppat.1010268.].

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human-induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (μTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage μT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

We previously reported that Mycobacterium tuberculosis triggers macrophage necrosis in vitro at a threshold intracellular load of ~25 bacilli. This suggests a model for tuberculosis where bacilli invading lung macrophages at low multiplicity of infection proliferate to burst size and spread to naïve phagocytes for repeated cycles of replication and cytolysis. The current study evaluated that model in vivo, an environment significantly more complex than in vitro culture. In the lungs of mice infected with M. tuberculosis by aerosol we observed three distinct mononuclear leukocyte populations (CD11b(-) CD11c(+/hi), CD11b(+/lo) CD11c(lo/-), CD11b(+/hi) CD11c(+/hi)) and neutrophils hosting bacilli. Four weeks after aerosol challenge, CD11b(+/hi) CD11c(+/hi) mononuclear cells and neutrophils were the predominant hosts for M. tuberculosis while CD11b(+/lo) CD11c(lo/-) cells assumed that role by ten weeks. Alveolar macrophages (CD11b(-) CD11c(+/hi)) were a minority infected cell type at both time points. The burst size model predicts that individual lung phagocytes would harbor a range of bacillary loads with most containing few bacilli, a smaller proportion containing many bacilli, and few or none exceeding a burst size load. Bacterial load per cell was enumerated in lung monocytic cells and neutrophils at time points after aerosol challenge of wild type and interferon-γ null mice. The resulting data fulfilled those predictions, suggesting a median in vivo burst size in the range of 20 to 40 bacilli for monocytic cells. Most heavily burdened monocytic cells were nonviable, with morphological features similar to those observed after high multiplicity challenge in vitro: nuclear condensation without fragmentation and disintegration of cell membranes without apoptotic vesicle formation. Neutrophils had a narrow range and lower peak bacillary burden than monocytic cells and some exhibited cell death with release of extracellular neutrophil traps. Our studies suggest that burst size cytolysis is a major cause of infection-induced mononuclear cell death in tuberculosis.

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Memory CD8(+) T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8(+) T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8(+) T-cell expansion.

Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to generate induced pluripotent stem cell (iPSC) lines from both normal and disease-specific somatic tissues, providing an invaluable resource for medical research and drug development. Residual reprogramming transgene expression from integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs) and that the expression profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON) vs Gtl2(OFF) iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and approximately 350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs.