Jak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3-/- bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium.

Adult stem cells maintain regenerative tissue structure and function by producing tissue-specific progeny, but the factors that preserve their tissue identities are not well understood. The small and large intestines differ markedly in cell composition and function, reflecting their distinct stem cell populations. Here we show that SATB2, a colon-restricted chromatin factor, singularly preserves LGR5+ adult colonic stem cell and epithelial identity in mice and humans. Satb2 loss in adult mice leads to stable conversion of colonic stem cells into small intestine ileal-like stem cells and replacement of the colonic mucosa with one that resembles the ileum. Conversely, SATB2 confers colonic properties on the mouse ileum. Human colonic organoids also adopt ileal characteristics upon SATB2 loss. SATB2 regulates colonic identity in part by modulating enhancer binding of the intestinal transcription factors CDX2 and HNF4A. Our study uncovers a conserved core regulator of colonic stem cells able to mediate cross-tissue plasticity in mature intestines.

Severe acute lung injury has few treatment options and a high mortality rate. Upon injury, neutrophils infiltrate the lungs and form neutrophil extracellular traps (NETs), damaging the lungs and driving an exacerbated immune response. Unfortunately, no drug preventing NET formation has completed clinical development. Here, we report that disulfiram - an FDA-approved drug for alcohol use disorder - dramatically reduced NETs, increased survival, improved blood oxygenation, and reduced lung edema in a transfusion-related acute lung injury (TRALI) mouse model. We then tested whether disulfiram could confer protection in the context of SARS-CoV-2 infection, as NETs are elevated in patients with severe COVID-19. In SARS-CoV-2-infected golden hamsters, disulfiram reduced NETs and perivascular fibrosis in the lungs, and it downregulated innate immune and complement/coagulation pathways, suggesting that it could be beneficial for patients with COVID-19. In conclusion, an existing FDA-approved drug can block NET formation and improve disease course in 2 rodent models of lung injury for which treatment options are limited.

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19.

The effect of SARS-CoV-2 infection on placental function is not well understood. Analysis of placentas from women who tested positive at delivery showed SARS-CoV-2 genomic and subgenomic RNA in 22 out of 52 placentas. Placentas from two mothers with symptomatic COVID-19 whose pregnancies resulted in adverse outcomes for the fetuses contained high levels of viral Alpha variant RNA. The RNA was localized to the trophoblasts that cover the fetal chorionic villi in direct contact with maternal blood. The intervillous spaces and villi were infiltrated with maternal macrophages and T cells. Transcriptome analysis showed an increased expression of chemokines and pathways associated with viral infection and inflammation. Infection of placental cultures with live SARS-CoV-2 and spike protein-pseudotyped lentivirus showed infection of syncytiotrophoblast and, in rare cases, endothelial cells mediated by ACE2 and Neuropilin-1. Viruses with Alpha, Beta, and Delta variant spikes infected the placental cultures at significantly greater levels.

KRAS is one of the driver oncogenes in non-small-cell lung cancer (NSCLC) but remains refractory to current modalities of targeted pathway inhibition, which include inhibiting downstream kinase MEK to circumvent KRAS activation. Here, we show that pulsatile, rather than continuous, treatment with MEK inhibitors (MEKis) maintains T cell activation and enables their proliferation. Two MEKis, selumetinib and trametinib, induce T cell activation with increased CTLA-4 expression and, to a lesser extent, PD-1 expression on T cells in vivo after cyclical pulsatile MEKi treatment. In addition, the pulsatile dosing schedule alone shows superior anti-tumor effects and delays the emergence of drug resistance. Furthermore, pulsatile MEKi treatment combined with CTLA-4 blockade prolongs survival in mice bearing tumors with mutant Kras. Our results set the foundation and show the importance of a combinatorial therapeutic strategy using pulsatile targeted therapy together with immunotherapy to optimally enhance tumor delay and promote long-term anti-tumor immunity.

There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.

Chronic obstructive pulmonary disease (COPD) is marked by airway inflammation and airspace enlargement (emphysema) leading to airflow obstruction and eventual respiratory failure. Microvasculature dysfunction is associated with COPD/emphysema. However, it is not known if abnormal endothelium drives COPD/emphysema pathology and/or if correcting endothelial dysfunction has therapeutic potential. Here, we show the centrality of endothelial cells to the pathogenesis of COPD/emphysema in human tissue and using an elastase-induced murine model of emphysema. Airspace disease showed significant endothelial cell loss, and transcriptional profiling suggested an apoptotic, angiogenic, and inflammatory state. This alveolar destruction was rescued by intravenous delivery of healthy lung endothelial cells. Leucine-rich α-2-glycoprotein-1 (LRG1) was a driver of emphysema, and deletion of Lrg1 from endothelial cells rescued vascular rarefaction and alveolar regression. Hence, targeting endothelial cell biology through regenerative methods and/or inhibition of the LRG1 pathway may represent strategies of immense potential for the treatment of COPD/emphysema.

The lymphatic vasculature regulates lung homeostasis through drainage of fluid and trafficking of immune cells and plays a key role in the response to lung injury in several disease states. We have previously shown that lymphatic dysfunction occurs early in the pathogenesis of chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) and that this is associated with increased thrombin and fibrin clots in lung lymph. However, the direct effects of CS and thrombin on lymphatic endothelial cells (LECs) in COPD are not entirely clear. Studies of the blood vasculature have shown that COPD is associated with increased thrombin after CS exposure that causes endothelial dysfunction characterized by changes in the expression of coagulation factors and leukocyte adhesion proteins. Here, we determined whether similar changes occur in LECs. We used an in vitro cell culture system and treated human lung microvascular lymphatic endothelial cells with cigarette smoke extract (CSE) and/or thrombin. We found that CSE treatment led to decreased fibrinolytic activity in LECs, which was associated with increased expression of plasminogen activator inhibitor 1 (PAI-1). LECs treated with both CSE and thrombin together had a decreased expression of tissue factor pathway inhibitor (TFPI) and increased expression of adhesion molecules. RNA sequencing of lung LECs isolated from mice exposed to CS also showed upregulation of prothrombotic and inflammatory pathways at both acute and chronic exposure time points. Analysis of publicly available single-cell RNA sequencing of LECs as well as immunohistochemical staining of lung tissue from COPD patients supported these data and showed increased expression of inflammatory markers in LECs from COPD patients compared to those from controls. These studies suggest that in parallel with blood vessels, the lymphatic endothelium undergoes inflammatory changes associated with CS exposure and increased thrombin in COPD. Further research is needed to unravel the mechanisms by which these changes affect lymphatic function and drive tissue injury in COPD.