A Comparative Study of Human Pluripotent Stem Cell-Derived Macrophages in Modeling Viral Infections.

Macrophages play multiple roles in innate immunity including phagocytosing pathogens, modulating the inflammatory response, presenting antigens, and recruiting other immune cells. Tissue-resident macrophages (TRMs) adapt to the local microenvironment and can exhibit different immune responses upon encountering distinct pathogens. In this study, we generated induced macrophages (iMACs) derived from human pluripotent stem cells (hPSCs) to investigate the interactions between the macrophages and various human pathogens, including the hepatitis C virus (HCV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Streptococcus pneumoniae. iMACs can engulf all three pathogens. A comparison of the RNA-seq data of the iMACs encountering these pathogens revealed that the pathogens activated distinct gene networks related to viral response and inflammation in iMACs. Interestingly, in the presence of both HCV and host cells, iMACs upregulated different sets of genes involved in immune cell migration and chemotaxis. Finally, we constructed an image-based high-content analysis system consisting of iMACs, recombinant GFP-HCV, and hepatic cells to evaluate the effect of a chemical inhibitor on HCV infection. In summary, we developed a human cell-based in vitro model to study the macrophage response to human viral and bacterial infections; the results of the transcriptome analysis indicated that the iMACs were a useful resource for modeling pathogen-macrophage-tissue microenvironment interactions.

Bradykinin-pretreated Human cardiac-specific c-kit+ Cells Enhance Exosomal miR-3059-5p and Promote Angiogenesis Against Hindlimb Ischemia in mice.

BACKGROUND: Protection of cardiac function following myocardial infarction was largely enhanced by bradykinin-pretreated cardiac-specific c-kit+ (BK-c-kit+) cells, even without significant engraftment, indicating that paracrine actions of BK-c-kit+ cells play a pivotal role in angiogenesis. Nevertheless, the active components of the paracrine actions of BK-c-kit+ cells and the underlying mechanisms remain unknown. This study aimed to define the active components of exosomes from BK-c-kit+ cells and elucidate their underlying protective mechanisms. METHODS: Matrigel tube formation assay, cell cycle, and mobility in human umbilical vein endothelial cells (HUVECs) and hindlimb ischemia (HLI) in mice were applied to determine the angiogenic effect of condition medium (CM) and exosomes. Proteome profiler, microRNA sponge, Due-luciferase assay, microRNA-sequencing, qRT-PCR, and Western blot were used to determine the underlying mechanism of the angiogenic effect of exosomes from BK-c-kit+. RESULTS: As a result, BK-c-kit+ CM and exosomes promoted tube formation in HUVECs and the repair of HLI in mice. Angiogenesis-related proteomic profiling and microRNA sequencing revealed highly enriched miR-3059-5p as a key angiogenic component of BK-c-kit+ exosomes. Meanwhile, loss- and gain-of-function experiments revealed that the promotion of angiogenesis by miR-3059-5p was mainly through suppression of TNFSF15-inhibited effects on vascular tube formation, cell proliferation and cell migration. Moreover, enhanced angiogenesis of miR-3059-5p-inhibited TNFSF15 has been associated with Akt/Erk1/2/Smad2/3-modulated signaling pathway. CONCLUSION: Our results demonstrated a novel finding that BK-c-kit+ cells enrich exosomal miR-3059-5p to suppress TNFSF15 and promote angiogenesis against hindlimb ischemia in mice.

FGF21-Sirtuin 3 Axis Confers the Protective Effects of Exercise Against Diabetic Cardiomyopathy by Governing Mitochondrial Integrity.

BACKGROUND: Exercise is an effective nonpharmacological strategy to alleviate diabetic cardiomyopathy (DCM) through poorly defined mechanisms. FGF21 (fibroblast growth factor 21), a peptide hormone with pleiotropic benefits on cardiometabolic homeostasis, has been identified as an exercise responsive factor. This study aims to investigate whether FGF21 signaling mediates the benefits of exercise on DCM, and if so, to elucidate the underlying mechanisms. METHODS: The global or hepatocyte-specific FGF21 knockout mice, cardiomyocyte-selective ß-klotho (the obligatory co-receptor for FGF21) knockout mice, and their wild-type littermates were subjected to high-fat diet feeding and injection of streptozotocin to induce DCM, followed by a 6-week exercise intervention and assessment of cardiac functions. Cardiac mitochondrial structure and function were assessed by electron microscopy, enzymatic assays, and measurements of fatty acid oxidation and ATP production. Human induced pluripotent stem cell-derived cardiomyocytes were used to investigate the receptor and postreceptor signaling pathways conferring the protective effects of FGF21 against toxic lipids-induced mitochondrial dysfunction. RESULTS: Treadmill exercise markedly induced cardiac expression of ß-klotho and significantly attenuated diabetes-induced cardiac dysfunction in wild-type mice, accompanied by reduced mitochondrial damage and increased activities of mitochondrial enzymes in hearts. However, such cardioprotective benefits of exercise were largely abrogated in mice with global or hepatocyte-selective ablation of FGF21, or cardiomyocyte-specific deletion of ß-klotho. Mechanistically, exercise enhanced the cardiac actions of FGF21 to induce the expression of the mitochondrial deacetylase SIRT3 by AMPK-evoked phosphorylation of FOXO3, thereby reversing diabetes-induced hyperacetylation and functional impairments of a cluster of mitochondrial enzymes. FGF21 prevented toxic lipids-induced mitochondrial dysfunction and oxidative stress by induction of the AMPK/FOXO3/SIRT3 signaling axis in human induced pluripotent stem cell-derived cardiomyocytes. Adeno-associated virus-mediated restoration of cardiac SIRT3 expression was sufficient to restore the responsiveness of diabetic FGF21 knockout mice to exercise in amelioration of mitochondrial dysfunction and DCM. CONCLUSIONS: The FGF21-SIRT3 axis mediates the protective effects of exercise against DCM by preserving mitochondrial integrity and represents a potential therapeutic target for DCM. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03240978.

Integrative epigenomic and transcriptomic analysis reveals the requirement of JUNB for hematopoietic fate induction.

Human pluripotent stem cell differentiation towards hematopoietic progenitor cell can serve as an in vitro model for human embryonic hematopoiesis, but the dynamic change of epigenome and transcriptome remains elusive. Here, we systematically profile the chromatin accessibility, H3K4me3 and H3K27me3 modifications, and the transcriptome of intermediate progenitors during hematopoietic progenitor cell differentiation in vitro. The integrative analyses reveal sequential opening-up of regions for the binding of hematopoietic transcription factors and stepwise epigenetic reprogramming of bivalent genes. Single-cell analysis of cells undergoing the endothelial-to-hematopoietic transition and comparison with in vivo hemogenic endothelial cells reveal important features of in vitro and in vivo hematopoiesis. We find that JUNB is an essential regulator for hemogenic endothelium specialization and endothelial-to-hematopoietic transition. These studies depict an epigenomic roadmap from human pluripotent stem cells to hematopoietic progenitor cells, which may pave the way to generate hematopoietic progenitor cells with improved developmental potentials.

Differential effects of macrophage subtypes on SARS-CoV-2 infection in a human pluripotent stem cell-derived model.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.

Clinical analysis and pluripotent stem cells-based model reveal possible impacts of ACE2 and lung progenitor cells on infants vulnerable to COVID-19.

Introduction: An increasing number of children with severe coronavirus disease 2019 (COVID-19) is being reported, yet the spectrum of disease severity and expression patterns of angiotensin-converting enzyme 2 (ACE2) in children at different developmental stages are largely unknow. Methods: We analysed clinical features in a cohort of 173 children with COVID-19 (0-15 yrs.-old) between January 22, 2020 and March 15, 2020. We systematically examined the expression and distribution of ACE2 in different developmental stages of children by using a combination of children's lung biopsies, pluripotent stem cell-derived lung cells, RNA-sequencing profiles, and ex vivo SARS-CoV-2 pseudoviral infections. Results: It revealed that infants (< 1yrs.-old), with a weaker potency of immune response, are more vulnerable to develop pneumonia whereas older children (> 1 yrs.-old) are more resistant to lung injury. The expression levels of ACE2 however do not vary by age in children's lung. ACE2 is notably expressed not only in Alveolar Type II (AT II) cells, but also in SOX9 positive lung progenitor cells detected in both pluripotent stem cell derivatives and infants' lungs. The ACE2+SOX9+ cells are readily infected by SARS-CoV-2 pseudovirus and the numbers of the double positive cells are significantly decreased in older children. Conclusions: Infants (< 1 yrs.-old) with SARS-CoV-2 infection are more vulnerable to lung injuries. ACE2 expression in multiple types of lung cells including SOX9 positive progenitor cells, in cooperation with an unestablished immune system, could be risk factors contributing to vulnerability of infants with COVID-19. There is a need to continue monitoring lung development in young children who have recovered from SARS-CoV-2 infection.

Homozygous MESP1 knock-in reporter hESCs facilitated cardiovascular cell differentiation and myocardial infarction repair.

Different populations of cardiovascular progenitor cells have been shown to possess varying differentiation potentials. They have also been used to facilitate heart repair. However, sensitive reporter cell lines that mark the human cardiovascular progenitors are in short supply. Methods: MESP1 marks the earliest population of cardiovascular progenitor cells during embryo development. Here, we generated a homozygous MESP1 knock-in reporter hESC line where mTomato gene joined to the MESP1 coding region via a 2A peptide, in which both MESP1 alleles were preserved. We performed transcriptome and functional analysis of human MESP1+ cardiovascular progenitor cells and tested their therapeutic potential using a rat model of myocardial infarction. Results: MESP1-mTomato knock-in reporter faithfully recapitulated the endogenous level of MESP1. Transcriptome analysis revealed that MESP1+ cells highly expressed early cardiovascular genes and heart development genes. The activation of MESP1 relied on the strength of canonical Wnt signaling, peak MESP1-mTomato fluorescence correlated with the window of canonical Wnt inhibition during in vitro differentiation. We further showed that MESP1 bound to the promoter of the WNT5A gene and the up-regulation of WNT5A expression suppressed canonical Wnt/ß-CATENIN signaling. Moreover, induced MESP1 expression could substitute the canonical Wnt inhibition step and promote robust cardiomyocyte formation. We used a configurable, chemically defined, tri-lineage differentiation system to obtain cardiomyocytes, endothelial cells, and smooth muscle cells from MESP1+ cells at high efficiency. Finally, we showed that the engraftment of MESP1+ cells repaired rat myocardial infarction model. Conclusions: MESP1-mTomato reporter cells offered a useful platform to study cardiovascular differentiation from human pluripotent stem cells and explore their therapeutic potential in regenerative medicine.

Anti-apoptotic Mutations Desensitize Human Pluripotent Stem Cells to Mitotic Stress and Enable Aneuploid Cell Survival.

Human pluripotent stem cells (hPSCs) are susceptible to numerical and structural chromosomal alterations during long-term culture. We show that mitotic errors occur frequently in hPSCs and that prometaphase arrest leads to very rapid apoptosis in undifferentiated but not in differentiated cells. hPSCs express high levels of proapoptotic protein NOXA in undifferentiated state. Knocking out NOXA by CRISPR or upregulation of the anti-apoptosis gene BCL-XL significantly reduced mitotic cell death, allowing the survival of aneuploid cells and the formation of teratomas significantly larger than their wild-type parental hPSCs. These results indicate that the normally low threshold of apoptosis in hPSCs can safeguard their genome integrity by clearing cells undergoing abnormal division. The amplification of BCL2L1 on chromosome 20q11.21, a frequent mutation in hPSCs, although not directly oncogenic, reduces the sensitivity of hPSCs to damage caused by erroneous mitosis and increases the risk of gaining aneuploidy.

Biphasic modulation of insulin signaling enables highly efficient hematopoietic differentiation from human pluripotent stem cells.

BACKGROUND: Hematopoietic lineage cells derived from human pluripotent stem cells (hPSCs) hold great promise for the treatment of hematological diseases and providing sufficient cells for immune therapy. However, a simple, cost-effective method to generate large quantities of hematopoietic stem/progenitor cells (HSPCs) is not yet available. METHODS: We established a monolayer, chemically defined culture system to induce hematopoietic differentiation from hPSCs in 8 days. RESULTS: We found that insulin-free medium allowed hPSCs to leave pluripotency promptly and preferably enter the vascular lineage. Addition of insulin during the later stage of differentiation was essential for the efficient induction of hemogenic endothelium and the emergence of large numbers of CD34+CD43+ HSPCs, while no insulin condition preferably permits endothelial differentiation. Global transcriptome profiling revealed that HSPCs differentiated using our protocol were similar to embryoid body-derived HSPCs. HSPCs obtained from our differentiation system formed robust erythroid, granulocyte and monocyte/macrophage colonies in CFU assay, and can be induced to generate functional macrophages with robust phagocytic ability. CONCLUSION: Our results demonstrated that proper manipulation of insulin-mTOR signaling can greatly facilitate HSPC formation. This finding can be further exploited to formulate cost-effective differentiation medium to generate large quantities of cells of desired blood lineages for regenerative medicine.

Optimizing mesoderm progenitor selection and three-dimensional microniche culture allows highly efficient endothelial differentiation and ischemic tissue repair from human pluripotent stem cells.

BACKGROUND: Generation of large quantities of endothelial cells is highly desirable for vascular research, for the treatment of ischemia diseases, and for tissue regeneration. To achieve this goal, we developed a simple, chemically defined culture system to efficiently and rapidly differentiate endothelial cells from human pluripotent stem cells by going through an MESP1 mesoderm progenitor stage. METHODS: Mesp1 is a key transcription factor that regulates the development of early cardiovascular tissue. Using an MESP1-mTomato knock-in reporter human embryonic stem cell line, we compared the gene expression profiles of MESP1+ and MESP1- cells and identified new signaling pathways that may promote endothelial differentiation. We also used a 3D scaffold to mimic the in vivo microenvironment to further improve the efficiency of endothelial cell generation. Finally, we performed cell transplantation into a critical limb ischemia mouse model to test the repairing potential of endothelial-primed MESP1+ cells. RESULTS: MESP1+ mesoderm progenitors, but not MESP1- cells, have strong endothelial differentiation potential. Global gene expression analysis revealed that transcription factors essential for early endothelial differentiation were enriched in MESP1+ cells. Interestingly, MESP1 cells highly expressed Sphingosine-1-phosphate (S1P) receptor and the addition of S1P significantly increased the endothelial differentiation efficiency. Upon seeding in a novel 3D microniche and priming with VEGF and bFGF, MESP1+ cells markedly upregulated genes related to vessel development and regeneration. 3D microniches also enabled long-term endothelial differentiation and proliferation from MESP1+ cells with minimal medium supplements. Finally, we showed that transplanting a small number of endothelial-primed MESP1+ cells in 3D microniches was sufficient to mediate rapid repair of a mouse model of critical limb ischemia. CONCLUSIONS: Our study demonstrates that combining MESP1+ mesoderm progenitor cells with tissue-engineered 3D microniche and a chemically defined endothelial induction medium is a promising route to maximizing the production of endothelial cells in vitro and augment their regenerative power in vivo.