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Severe lung injury causes airway basal stem cells to migrate and outcompete alveolar stem cells, resulting in dysplastic repair. We found that this "stem cell collision" generates an injury-induced tissue niche containing keratin 5 + epithelial cells and plastic Pdgfra + mesenchymal cells. Single-cell analysis revealed that the injury-induced niche is governed by mesenchymal proliferation and Notch signaling, which suppressed Wnt/Fgf signaling in the injured niche. Conversely, loss of Notch signaling rewired alveolar signaling patterns to promote functional regeneration and gas exchange. Signaling patterns in injury-induced niches can differentiate fibrotic from degenerative human lung diseases through altering the direction of Wnt/Fgf signaling. Thus, we have identified an injury-induced niche in the lung with the ability to discriminate human lung disease phenotypes.

Over the past decade, the use of human stem cell-derived β cells (SC-β cells) to model pancreatic β cell development, function and disease has become increasingly common. Though protocols are rapidly improving, current directed differentiation strategies do not yield a pure population of insulin-positive SC-β cells in vitro. Therefore, it is experimentally advantageous to have reporter lines that allow for live sorting of insulin-positive populations. To aid in these studies, we have knocked mNeonGreen fluorescent protein into the endogenous insulin locus of the commonly used H1 (WA01) human embryonic stem cell line., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Severe lung injury causes basal stem cells to migrate and outcompete alveolar stem cells resulting in dysplastic repair and a loss of gas exchange function. This "stem cell collision" is part of a multistep process that is now revealed to generate an injury-induced tissue niche (iTCH) containing Keratin 5+ epithelial cells and plastic Pdgfra+ mesenchymal cells. Temporal and spatial single cell analysis reveals that iTCHs are governed by mesenchymal proliferation and Notch signaling, which suppresses Wnt and Fgf signaling in iTCHs. Conversely, loss of Notch in iTCHs rewires alveolar signaling patterns to promote euplastic regeneration and gas exchange. The signaling patterns of iTCHs can differentially phenotype fibrotic from degenerative human lung diseases, through apposing flows of FGF and WNT signaling. These data reveal the emergence of an injury and disease associated iTCH in the lung and the ability of using iTCH specific signaling patterns to discriminate human lung disease phenotypes., Competing Interests: Declaration of interests The authors declare no competing interests.

AT2 cells harbor alveolar stem cell activity in the lung and can self-renew and differentiate into AT1 cells during homeostasis and after injury. To identify epigenetic pathways that control the AT2-AT1 regenerative response in the lung, we performed an organoid screen using a library of pharmacological epigenetic inhibitors. This screen identified DOT1L as a regulator of AT2 cell growth and differentiation. In vivo inactivation of Dot1l leads to precocious activation of both AT1 and AT2 gene expression during lung development and accelerated AT1 cell differentiation after acute lung injury. Single-cell transcriptome analysis reveals the presence of a new AT2 cell state upon loss of Dot1l, characterized by increased expression of oxidative phosphorylation genes and changes in expression of critical transcription and epigenetic factors. Taken together, these data demonstrate that Dot1l controls the rate of alveolar epithelial cell fate acquisition during development and regeneration after acute injury., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Alveolar epithelial type 2 (AT2) cells harbor the facultative progenitor capacity in the lung alveolus to drive regeneration after lung injury. Using single-cell transcriptomics, software-guided segmentation of tissue damage, and in vivo mouse lineage tracing, we identified the grainyhead transcription factor cellular promoter 2-like 1 (Tfcp2l1) as a regulator of this regenerative process. Tfcp2l1 loss in adult AT2 cells inhibits self-renewal and enhances AT2-AT1 differentiation during tissue regeneration. Conversely, Tfcp2l1 blunts the proliferative response to inflammatory signaling during the early acute injury phase. Tfcp2l1 temporally regulates AT2 self-renewal and differentiation in alveolar regions undergoing active regeneration. Single-cell transcriptomics and lineage tracing reveal that Tfcp2l1 regulates cell fate dynamics across the AT2-AT1 differentiation and restricts the inflammatory program in murine AT2 cells. Organoid modeling shows that Tfcp2l1 regulation of interleukin-1 (IL-1) receptor expression controlled these cell fate dynamics. These findings highlight the critical role Tfcp2l1 plays in balancing epithelial cell self-renewal and differentiation during alveolar regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses 1-3 . In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins 4-8 , particularly those containing post-translational modifications required for transcriptional regulation 9-11 . Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation 12-14 . However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear. Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation. ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction. Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number. These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Alveolar epithelial cell fate decisions drive lung development and regeneration. Using transcriptomic and epigenetic profiling coupled with genetic mouse and organoid models, we identified the transcription factor Klf5 as an essential determinant of alveolar epithelial cell fate across the lifespan. We show that although dispensable for both adult alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cell homeostasis, Klf5 enforces AT1 cell lineage fidelity during development. Using infectious and non-infectious models of acute respiratory distress syndrome, we demonstrate that Klf5 represses AT2 cell proliferation and enhances AT2-AT1 cell differentiation in a spatially restricted manner during lung regeneration. Moreover, ex vivo organoid assays identify that Klf5 reduces AT2 cell sensitivity to inflammatory signaling to drive AT2-AT1 cell differentiation. These data define the roll of a major transcriptional regulator of AT1 cell lineage commitment and of the AT2 cell response to inflammatory crosstalk during lung regeneration., Competing Interests: Declaration of interests E.E.M. is on the Developmental Cell advisory board. All other authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Epithelial cell organoids have increased opportunities to probe questions on tissue development and disease in vitro and for therapeutic cell transplantation. Despite their potential, current protocols to grow these organoids almost exclusively depend on culture within 3D Matrigel, which limits defined culture conditions, introduces animal components, and results in heterogenous organoids (i.e., shape, size, composition). Here, a method is described that relies on hyaluronic acid hydrogels for the generation and expansion of lung alveolar organoids (alveolospheres). Using synthetic hydrogels with defined chemical and physical properties, human-induced pluripotent stem cell (iPSC)-derived alveolar type 2 cells (iAT2s) self-assemble into alveolospheres and propagate in Matrigel-free conditions. By engineering predefined microcavities within these hydrogels, the heterogeneity of alveolosphere size and structure is reduced when compared to 3D culture, while maintaining the alveolar type 2 cell fate of human iAT2-derived progenitor cells. This hydrogel system is a facile and accessible system for the culture of iPSC-derived lung progenitors and the method can be expanded to the culture of primary mouse tissue derived AT2 and other epithelial progenitor and stem cell aggregates., (© 2022 Wiley-VCH GmbH.)

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549 ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549 ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549 ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2., Competing Interests: Competing interest statement: S.R.W. is on the scientific advisory board of Immunome, Inc. and Ocugen, Inc. R.H.S. is a consultant to Cutherna, Inc., (Copyright © 2021 the Author(s). Published by PNAS.)

There is an urgent need for antivirals to treat the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To identify new candidates, we screen a repurposing library of ∼3,000 drugs. Screening in Vero cells finds few antivirals, while screening in human Huh7.5 cells validates 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we find that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we find nine drugs are antiviral in respiratory cells, seven of which have been used in humans, and three are US Food and Drug Administration (FDA) approved, including cyclosporine. We find that the antiviral activity of cyclosporine is targeting Cyclophilin rather than calcineurin, revealing essential host targets that have the potential for rapid clinical implementation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims/hypothesis: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B., Methods: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry., Results: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca 2+ . Binding partners for both included secretory-granule-localised PTPRN2/phogrin., Conclusions/interpretation: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans., Data Availability: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.

Using chromatin conformation capture, we show that an enhancer cluster in the STARD10 type 2 diabetes (T2D) locus forms a defined 3-dimensional (3D) chromatin domain. A 4.1-kb region within this locus, carrying 5 T2D-associated variants, physically interacts with CTCF-binding regions and with an enhancer possessing strong transcriptional activity. Analysis of human islet 3D chromatin interaction maps identifies the FCHSD2 gene as an additional target of the enhancer cluster. CRISPR-Cas9-mediated deletion of the variant region, or of the associated enhancer, from human pancreas-derived EndoC-βH1 cells impairs glucose-stimulated insulin secretion. Expression of both STARD10 and FCHSD2 is reduced in cells harboring CRISPR deletions, and lower expression of STARD10 and FCHSD2 is associated, the latter nominally, with the possession of risk variant alleles in human islets. Finally, CRISPR-Cas9-mediated loss of STARD10 or FCHSD2, but not ARAP1, impairs regulated insulin secretion. Thus, multiple genes at the STARD10 locus influence β cell function., Competing Interests: Declaration of interests G.A.R. has received grant funding from Les Laboratoires Servier and is a consultant for Sun Pharmaceuticals., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Remarkable strides have been made over the past decade on the development of pancreatic β-cells from human stem cells through directed differentiation, allowing for modeling of β-cell development, function and disease. However, in vitro models and future therapeutic applications will require the use of stem cell-derived islets with multiple monohormonal endocrine cells types, including α, β, and δ cells. Using the previously reported Mel1 Ins GFP/w human embryonic stem cell (hESC) line, we have knocked-in Red Fluorescence Protein (RFP) under the control of the endogenous somatostatin promoter using CRISPR/Cas9, generating a dual insulin and somatostatin reporter hESC line., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549 ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549 ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549 ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2., Significance: SARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.

GATA6 is a critical regulator of pancreatic development, with heterozygous mutations in this transcription factor being the most common cause of pancreatic agenesis. To study the variability in disease phenotype among individuals harboring these mutations, a patient-induced pluripotent stem cell model was used. Interestingly, GATA6 protein expression remained depressed in pancreatic progenitor cells even after correction of the coding mutation. Screening the regulatory regions of the GATA6 gene in these patient cells and 32 additional agenesis patients revealed a higher minor allele frequency of a SNP 3' of the GATA6 coding sequence. Introduction of this minor allele SNP by genome editing confirmed its functionality in depressing GATA6 expression and the efficiency of pancreas differentiation. This work highlights a possible genetic modifier contributing to pancreatic agenesis and demonstrates the usefulness of using patient-induced pluripotent stem cells for targeted discovery and validation of non-coding gene variants affecting gene expression and disease penetrance., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Human in vitro model systems of diabetes are critical to both study disease pathophysiology and offer a platform for drug testing. We have generated a set of tools in the human β-cell line EndoC-βH1 that allows the efficient and inexpensive characterization of β-cell physiology and phenotypes driven by disruption of candidate genes. First, we generated a dual reporter line that expresses a preproinsulin-luciferase fusion protein along with GCaMP6s. This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy. Using these tools, we demonstrate that the generation of the reporter human β-cell line was highly efficient and validated that luciferase activity could accurately reflect insulin secretion. Second, we used a lentiviral vector carrying the CRISPR-Cas9 system to generate candidate gene disruptions in the reporter line. We also show that we can achieve gene disruption in ~90% of cells using a CRISPR-Cas9 lentiviral system. As a proof of principle, we disrupt the β-cell master regulator, PDX1, and show that mutant EndoC-βH1 cells display impaired calcium responses and fail to secrete insulin when stimulated with high glucose. Furthermore, we show that PDX1 mutant EndoC-βH1 cells exhibit decreased expression of the β-cell-specific genes MAFA and NKX6.1 and increased GCG expression. The system presented here provides a platform to quickly and easily test β-cell functionality in wildtype and cells lacking a gene of interest., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Islet transplantation is an effective therapy for achieving and maintaining normoglycemia in patients with type 1 diabetes mellitus. However, the supply of transplantable human islets is limited. Upon removal from the pancreas, islets rapidly disintegrate and lose function, resulting in a short interval for studies of islet biology and pretransplantation assessment. Here, we developed a biomimetic platform that can sustain human islet physiology for a prolonged period ex vivo. Our approach involved the creation of a multichannel perifusion system to monitor dynamic insulin secretion and intracellular calcium flux simultaneously, enabling the systematic evaluation of glucose-stimulated insulin secretion under multiple conditions. Using this tool, we developed a nanofibrillar cellulose hydrogel-based islet-preserving platform (iPreP) that can preserve islet viability, morphology, and function for nearly 12 weeks ex vivo, and with the ability to ameliorate glucose levels upon transplantation into diabetic hosts. Our platform has potential applications in the prolonged maintenance of human islets, providing an expanded time window for pretransplantation assessment and islet studies.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

The transcription factor GATA6 has been shown to be important for lung development and branching morphogenesis in mouse models, but its role in human lung development is largely unknown. Here, we studied the role of GATA6 during lung differentiation using human pluripotent stem cells. We found that the human stem cell lines most efficient at generating NKX2.1+ lung progenitors express lower endogenous levels of GATA6 during endoderm patterning and that knockdown of GATA6 during endoderm patterning increased the generation of these cells. Complete ablation of GATA6 resulted in the generation of lung progenitors displaying increased cell proliferation with up to a 15-fold expansion compared with control cells, whereas the null cell line displayed a defect in further development into mature lung cell types. Furthermore, transgenic expression of GATA6 at the endoderm anteriorization stage skewed development toward a liver fate at the expense of lung progenitors. Our results suggest a critical dosage effect of GATA6 during human endoderm patterning and a later requirement during terminal lung differentiation. These studies offer an approach of modulating GATA6 expression to enhance the production of lung progenitors from human stem cell sources.

Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into β-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient's disease. GATA6 mutant β-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and β-like cell functionality in humans., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Patient-derived induced pluripotent stem cells (iPSCs) are valuable tools for the study of developmental biology and disease modeling. In both applications, genetic correction of patient iPSCs is a powerful method to understand the specific contribution of a gene(s) in development or diseased state(s). Here, we describe a protocol for the targeted integration of a doxycycline-inducible transgene expression system in a safe harbor site in iPSCs. Our gene targeting strategy uses zinc finger nucleases (ZFNs) to enhance homologous recombination at the AAVS1 safe harbor locus, thus increasing the efficiency of the site-specific integration of the two targeting vectors that make up the doxycycline-inducible system. Importantly, the use of dual-drug selection in our system increases the efficiency of positive selection for double-targeted clones to >50 %, permitting a less laborious screening process. If desired, this protocol can also be adapted to allow the use of tissue-specific promoters to drive gene expression instead of the doxycycline-inducible promoter (TRE). Additionally, this protocol is also compatible with the use of Transcription-Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system in place of ZFNs.