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Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.

Organoids derived from human pluripotent stem cells (hPSCs) have emerged as important models for investigating human-specific aspects of development and disease. Here we discuss hPSC-derived organoids through the lens of development—highlighting how stages of human development align with the development of hPSC-derived organoids in the tissue culture dish. Using hPSC-derived lung and intestinal organoids as examples, we discuss the value and application of such systems for understanding human biology, as well as strategies for enhancing organoid complexity and maturity.

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. The current work aimed to develop an iPSC organoid model enriched with stable NKX2-1+ BTP-like cells. Building on prior work, we optimized a directed differentiation paradigm to generate spheroids with robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs can be purified and expanded as induced bud tip organoids (iBTO), which maintain an enriched population of bud tip progenitors. When iBTOs are directed to differentiate into airway or alveolar cell types using well-established methods, they give rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model to study human lung development and differentiation.SUMMARY STATEMENTiPSC-derived lung bud tip progenitors emerge in organoid culture, can be isolated and expanded, are transcriptionally similar to primary bud tip progenitors, and can differentiate into airway or alveolar organoids.

SUMMARYMammalian respiratory system development is regulated by complex reciprocal signaling events that take place between epithelial cells and the surrounding mesenchymal cells; however, mesenchymal heterogeneity and function in the developing human lung is poorly understood. We interrogated single cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonistR-SPONDIN2(RSPO2), and we found that adjacent epithelial bud tip progenitors are enriched for the RSPO2 receptorLGR5. By carrying out functional experiments using organoid models, lung explant cultures, and FACS-isolatedRSPO2+mesenchyme, we show that RSPO2 is a critical niche cue that potentiates WNT signaling in human lung progenitors to maintain their multipotency.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These ""bud tip progenitors'' are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of theWNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2(+) mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency., This project has been made possible in part by grant number CZF2019-002440 from the Chan Zuckerberg Initiative DAF, an advised fund from the Silicon Valley Community Foundation, and in part by the NIH-NHLBI (R01HL119215) funding to J.R.S.; R.F.C.H. was supported by a NIH Tissue En- gineering and Regenerative Medicine Training Grant (NIH-NIDCR T32DE 007057) and by a Ruth L. Kirschstein Predoctoral Individual National Research Service Award (NIH-NHLBI F31HL152531); A.J.M. was supported by a Ruth L. Kirschstein Predoctoral Individual National Research Service Award (NIH- NHLBI F31HL142197); E.M.H. was supported by a Ruth L. Kirschstein Predoc- toral Individual National Research Service Award (NIH-NHBLI F31HL146162); T.F. was supported by a NIH Tissue Engineering and Regenerative Medicine Training Grant (NIH-NIDCR T32DE007057); A.S.C. was supported by the T32 Michigan Medical Scientist Training Program (5T32GM007863-40); I.G. and the University of Washington Laboratory of Developmental Biology was supported by NIH award number 5R24HD000836 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). B.R. is a fellow of the Branco Weiss Foundation (Switzerland) and the National Research Foundation (Singapore), and an A*STAR and EMBO Young Investi- gator. This work was supported by an inaugural Use-Inspired Basic Research (UIBR) central fund from the Agency for Science, Technology and Research (A*STAR) and National Medical Research Council Open Fund– Individual Research Grants (OF-YIRG,#OFYIRG18May-0053; and OF-IRG, #OFIRG20- Nov-0057), to E.S.-R and B.R., respectively. We would like to thank Judy Opp and the University of Michigan Advanced Genomics core for the operation of the 103 chromium single-cell capture plat- form, the University of Michigan Microscopy core for providing access to confocal microscopes, the Flow Cytometry core for providing access to flow cytometers, and the Vector core for providing lentivirus production and adeno- virus purification and expansion services. We would also like to thank the Uni- versity of Washington Laboratory of Developmental Biology. Lastly, we would like to thank Michael Dame and the Translational Tissue Modeling Laboratory (TTML) for providing helpful suggestions regarding the use of WNT3a afamin conditioned media as well as Lindy K. Brastrom for her careful technical editing of the manuscript.

The mouse preimplantation embryo is a paradigm for discovery of the molecular principles governing formation of specific cell types during development. We show that conditions commonly used for ex vivo culture of preimplantation development are themselves antagonistic to a pathway that is critical for blastocyst lineage commitment.

An abundance of information about lung development in animal models exists; however, comparatively little is known about lung development in humans. Recent advances using primary human lung tissue combined with the use of human in vitro model systems, such as human pluripotent stem cell-derived tissue, have led to a growing understanding of the mechanisms governing human lung development. They have illuminated key differences between animal models and humans, underscoring the need for continued advancements in modeling human lung development and utilizing human tissue. This review discusses the use of human tissue and the use of human in vitro model systems that have been leveraged to better understand key regulators of human lung development and that have identified uniquely human features of development. This review also examines the implementation and challenges of human model systems and discusses how they can be applied to address knowledge gaps.

In mice, pluripotent cells are thought to derive from cells buried inside the embryo around the 16-cell stage. Sox2 is the only pluripotency gene known to be expressed specifically within inside cells at this stage. To understand how pluripotency is established, we therefore investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4 (Pou5f1), highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1 and Tead4. Interestingly, the repression of premature Sox2 expression was sensitive to LATS kinase activity, even though LATS proteins normally do not limit activity of TEAD4, YAP1 and WWTR1 during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1, WWTR1 and TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the four-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created., Highlighted Article: The pluripotency marker SOX2 is not initially regulated by OCT4 and NANOG, but by HIPPO pathway members during the first 2 days of mouse embryogenesis.

In the mouse embryo, pluripotent cells arise inside the embryo around the 16-cell stage. During these early stages, Sox2 is the only gene whose expression is known to be induced specifically within inside cells as they are established. To understand how pluripotent cells are created, we investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4, highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious, ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1, and Tead4. Interestingly, the repression of premature Sox2 expression was sensitive to LATS1/2 activity, even though it normally does not limit TEAD4/YAP1/WWTR1 activity during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1/WWTR1/TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the 4-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created.

During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway in controlling cell positioning and expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation., eLife digest As an embryo develops, its cells divide, grow and migrate in specific patterns to build an organized collection of cells that go on to form our tissues and organs. One of the first steps – well before the embryo has implanted into the womb – is to allocate cells to make part of the placenta. Once this process is complete, the remaining cells continue building the organism. These cells are pluripotent, meaning they can develop into any part of the body. Scientists think that the embryo manages to sort ‘placenta cells’ from pluripotent ones with the help of certain proteins, which the mother has packaged into her eggs. To investigate this further, Frum et al. used genetic tools to track a specific gene called Sox2 that identifies pluripotent cells as soon as they are formed in mouse embryos. The experiments revealed that the mother places two closely related proteins known as YAP1 and WWTR1 within each egg, which help to make placenta cells different from pluripotent cells. Moreover, both proteins enable the embryo to segregate these two cell types to two different locations: placenta cells are moved to the outer layer of the embryo, while pluripotent cells are moved to the inside. Current technologies allow researchers to create pluripotent cells in the laboratory. But these approaches often result in error, failing to replicate the embryo’s natural ability. By studying how embryos form and arrange pluripotent cells, scientists hope to advance stem cell technology (which emerge from pluripotent cells). This may help to find new ways to heal damaged tissues and organs, or to treat or even prevent many diseases.

A new study by Zenker et al. uses time-lapse imaging to discover how dynamic actin movements contribute to epithelialization of living mouse embryos. Together with work from other labs, this study presents exciting new ways to think about the emergence of cell fates during mammalian development.

The first cell fate decisions during mammalian development establish tissues essential for healthy pregnancy. The mouse has served as a valuable model for discovering pathways regulating the first cell fate decisions because of the ease with which early embryos can be recovered and the availability of an arsenal of classical and emerging methods for manipulating gene expression. We summarize the major pathways that govern the first cell fate decisions in mouse development. This knowledge serves as a paradigm for exploring how emergent properties of a self-organizing system can dynamically regulate gene expression and cell fate plasticity. Moreover, it brings to light the processes that establish healthy pregnancy and ES cells. We also describe unsolved mysteries and new technologies that could help to overcome experimental challenges in the field.

SummaryIn embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here, we show that neither maternal nor zygotic Oct4 is required for the formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for the expression of multiple EPI and PE genes as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo.

In many invertebrate and vertebrate species, cell fates are assigned through the cellular inheritance of differentially localized maternal determinants. Whether mammalian embryogenesis is also regulated by deterministic mechanisms is highly controversial. The caudal domain transcription factor CDX2 has been reported to act as a maternal determinant regulating cell fate decisions in mouse development. However, this finding is contentious because of reports that maternal Cdx2 is not essential for development. Notably, all of the previously published studies of maternal Cdx2 relied on injected RNA interference constructs, which could introduce experimental variation. Only deletion of the maternal gene can unambiguously resolve its requirement in mouse development. Here, we genetically ablated maternal Cdx2 using a Cre/lox strategy, and we definitively establish that maternal Cdx2 is not essential for mouse development.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation. An extensive map of human DNase I hypersensitive sites, markers of regulatory DNA, in 125 diverse cell and tissue types is described; integration of this information with other ENCODE-generated data sets identifies new relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. This paper describes the first extensive map of human DNaseI hypersensitive sites — markers of regulatory DNA — in 125 diverse cell and tissue types. Integration of this information with other data sets generated by ENCODE (Encyclopedia of DNA Elements) identified new relationships between chromatin accessibility, transcription, DNA methylation and regulatory-factor occupancy patterns. Evolutionary-conservation analysis revealed signatures of recent functional constraint within DNaseI hypersensitive sites.

Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog., Author Summary Pluripotent stem cells can give rise to any cell type in the body, making them an attractive tool for regenerative medicine. Pluripotent stem cells can be derived from the mammalian embryo at the blastocyst stage or they can be created from mature adult cells by reprogramming. During reprogramming, SOX2 helps establish pluripotency, but it is not clear how SOX2 establishes pluripotency in the blastocyst. We evaluated where SOX2 is present, how SOX2 is regulated, and where SOX2 is active during blastocyst formation. Our data show that the roles and the regulation of SOX2 are unique compared to other pluripotency/reprogramming factors, such as OCT4 and NANOG. SOX2 marks pluripotent cells earlier than do other factors, but does not regulate pluripotency until several days later. Rather, the earlier role of SOX2 is to help establish the yolk sac lineage, which is essential for gestation.

The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. The Consortium is funded by grants from the NHGRI as follows: production grants: U54HG004570 (B. E. Bernstein); U01HG004695 (E. Birney); U54HG004563 (G. E. Crawford); U54HG004557 (T. R. Gingeras); U54HG004555 (T. J. Hubbard); U41HG004568 /n(W. J. Kent); U54HG004576 (R. M. Myers); U54HG004558 (M. Snyder);/nU54HG004592 (J. A. Stamatoyannopoulos). Pilot grants: R01HG003143 (J. Dekker); RC2HG005591 and R01HG003700 (M. C. Giddings); R01HG004456-03 (Y. Ruan); U01HG004571 (S. A. Tenenbaum); U01HG004561 (Z. Weng); RC2HG005679 (K. P. White). This project was supported in part by American Recovery and/nReinvestment Act (ARRA) funds from the NHGRI through grants U54HG004570, U54HG004563, U41HG004568, U54HG004592, R01HG003143, RC2HG005591,R01HG003541, U01HG004561, RC2HG005679andR01HG003988(L. Pennacchio). In addition, work from NHGRI Groups was supported by the Intramural Research/nProgram of the NHGRI (L. Elnitski, ZIAHG200323; E. H. Margulies, ZIAHG200341). Research in the Pennachio laboratory was performed at Lawrence Berkeley National Laboratory and at the United States Department of Energy Joint Genome Institute, Department of Energy Contract DE-AC02-05CH11231, University of California.

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome., National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.)

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

Significance Since its emergence in China in December 2019, SARS-CoV-2 has caused a global pandemic. Repurposing of FDA-approved drugs is a promising strategy for identifying rapidly deployable treatments for COVID-19. Herein, we developed a pipeline for quantitative, high-throughput, image-based screening of SARS-CoV-2 infection in human cells that led to the identification of several FDA-approved drugs and clinical candidates with in vitro antiviral activity., The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.