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Summary A major limitation in anti-tuberculosis drug screening is the lack of reliable and scalable models for homogeneous human primary macrophage cells of non-cancer origin. Here we report a modified protocol for generating homogeneous populations of macrophage-like cells from human embryonic stem cells. The induced macrophages, referred to as iMACs, presented similar transcriptomic profiles and characteristic immunological features of classical macrophages and were permissive to viral and bacterial infection, in particular Mycobacterium tuberculosis (Mtb). More importantly, iMAC production was amenable to scale up. To evaluate iMAC efficiency in high-throughput anti-tuberculosis drug screening, we performed a phenotypic screening against intracellular Mtb, involving a library of 3,716 compounds that included FDA-approved drugs and other bioactive compounds. Our primary screen identified 120 hits, which were validated in a secondary screen by dose-intracellular and -extracellular Mtb assays. Our confirmatory studies identified a novel anti-Mtb compound, 10-DEBC, also showing activity against drug-resistant strains., Graphical Abstract, Highlights • Methods for large-scale production of hPSC-derived macrophage-like cells (iMACs) • iMACs recapitulate immune response of human monocyte in M. tuberculosis infection • High-throughput screening of 3,716 compounds using iMACs identified novel anti-tuberculosis compounds • 10-DEBC inhibited intra- and extracellular growth of drug-resistant M. tuberculosis, In this article, Jung-Hyun Kim and colleagues show a protocol for generating macrophage-like cells from PSCs that exhibit features of classical phagocytes, are amenable to scale up, permissive to Mycobacterium tuberculosis infection, and suitable for intracellular high-throughput screening. By performing screening of 3,716 compounds, they found a new, potent anti-tuberculosis drug against drug-resistant strains of M. tuberculosis.

Somatic cell nuclear transfer (SCNT) or the forced expression of transcription factors can be used to generate autologous pluripotent stem cells (PSCs). Although transcriptomic and epigenomic comparisons of isogenic human NT-embryonic stem cells (NT-ESCs) and induced PSCs (iPSCs) in the undifferentiated state have been reported, their functional similarities and differentiation potentials have not been fully elucidated. Our study showed that NT-ESCs and iPSCs derived from the same donors generally displayed similar in vitro commitment capacity toward three germ layer lineages as well as proliferative activity and clonogenic capacity. However, the maturation capacity of NT-ESC-derived hematopoietic progenitors was significantly greater than the corresponding capacity of isogenic iPSC-derived progenitors. Additionally, donor-dependent variations in hematopoietic specification and commitment capacity were observed. Transcriptome and methylome analyses in undifferentiated NT-ESCs and iPSCs revealed a set of genes that may influence variations in hematopoietic commitment and maturation between PSC lines derived using different reprogramming methods. Here, we suggest that genetically identical iPSCs and NT-ESCs could be functionally unequal due to differential transcription and methylation levels acquired during reprogramming. Our proof-of-concept study indicates that reprogramming mechanisms and genetic background could contribute to diverse functionalities between PSCs.

Background: Inflammation is an important priming event in the pathogenesis of pulmonary diseases, including chronic obstructive pulmonary disease (COPD). Increasing evidence indicates that microRNAs (miRNAs) contribute to the pathogenesis of COPD by regulating inflammatory response. Therefore, it is necessary to investigate novel molecular targets in COPD without any validation in COPD samples in airway inflammation. The aim of our study is to reveal novel miRNAs that can influence molecular targets for COPD and to examine the underlying mechanism in airway inflammation. Methods: We identified the downregulation of miR-181a-2-3p in the serum of COPD patients and further investigated the role of miR-181a-2-3p in cadmium (Cd)-induced inflammation of a human bronchial epithelial cell line (BEAS-2B) and normal human bronchial epithelial (NHBE) cells. Results: Our results showed that expression of miR-181a-2-3p was significantly decreased in Cd-treated cells and silencing of miR-181a-2-3p enhanced Cd-induced inflammatory responses and inflammasome activation. This negative regulatory effect of miR-181a-2-3p on inflammation is partly mediated by the calcium signaling pathway. Furthermore, global gene expression profiling revealed that Toll-like receptor 4 or sequestosome 1 genes were identified as potential targets of miR-181a-2-3p, which were significantly upregulated by knockdown of miR-181a-2-3p in Cd-treated cells. Conclusions: Our results strongly suggest that miR-181a-2-3p has a critical role in Cd-induced inflammation of airway by regulating its potential target genes, which could be molecular targets for COPD.

Several epidemiological studies concluded that inhalation of diesel exhaust particles (DEP) is associated with an increase in the relative risk of lung cancer. In vitro research evaluating the genetic damage and/or changes in gene expression have been attempted to explain the relationship between DEP exposure and carcinogenicity. However, to date, investigations have been largely confined to studies in immortalized or tumorigenic epithelial cell models. Few studies have investigated damage at the chromosomal level to DEP exposure in normal cell lines. Here, we present the genotoxic effects of DEP in normal cells (embryonic human lung fibroblasts) by conventional genotoxicity testing (micronuclei (MN) and comet assay). We show the differentially expressed genes and enriched pathways in DEP-exposed WI-38 cells using RNA sequencing data. We observed a significant increase in single-strand DNA breaks and the frequency of MN in DEP-exposed cells in a dose-dependent manner. The differentially expressed genes following DEP exposure were significantly enriched in the pathway for responding to xenobiotics and DNA damage. Taken together, these results show that DEP exposure induced DNA damage at the chromosomal level in normal human lung cells and provide information on the expression of genes associated with genotoxic stress.

The stem cell factor (SCF)/c-KIT axis plays an important role in the hematopoietic differentiation of human pluripotent stem cells (hPSCs), but its regulatory mechanisms involving microRNAs (miRs) are not fully elucidated. Here, we demonstrated that supplementation with SCF increases the hematopoietic differentiation of hPSCs via the interaction with its receptor tyrosine kinase c-KIT, which is modulated by miR-221 and miR-222. c-KIT is comparably expressed in undifferentiated human embryonic and induced pluripotent stem cells. The inhibition of SCF signaling via treatment with a c-KIT antagonist (imatinib) during hPSC-derived hematopoiesis resulted in reductions in the yield and multi-lineage potential of hematopoietic progenitors. We found that the transcript levels of miR-221 and miR-222 targeting c-KIT were significantly lower in the pluripotent state than they were in terminally differentiated somatic cells. Furthermore, suppression of miR-221 and miR-222 in undifferentiated hPSC cultures induced more hematopoiesis by increasing c-KIT expression. Collectively, our data implied that the modulation of c-KIT by miRs may provide further potential strategies to expedite the generation of functional blood cells for therapeutic approaches and the study of the cellular machinery related to hematologic malignant diseases such as leukemia.

Human pluripotent stem cell (hPSC)-derived alveolar epithelial cells (AECs) provide new opportunities for understanding lung development and the treatment of pulmonary diseases. However, toxicity assessments using hPSC-AECs have not been undertaken. In this study, we generated functional AECs from hPSCs and evaluated their inflammatory and apoptotic responses to cadmium (Cd) exposure (1, 5, and 10 μM) for 24 h compared with the human bronchial epithelial cell line (BEAS-2B) and primary AECs as controls. Our data showed that Cd (10 μM) treatment induced substantial inflammatory responses and apoptosis in BEAS-2B cells, but not in both hPSC-AECs and primary AECs. Interestingly, conditioned medium from AEC cultures significantly alleviated apoptotic and inflammatory responses to Cd exposure in BEAS-2B cells. Using cytokine arrays, several potential factors secreted from hPSC-AECs and primary AECs were detected and may be involved in reducing Cd-induced cytotoxicity. We also observed higher expression of surfactant proteins B and C in both hPSC-AECs and primary AECs, which may contribute to protection against Cd-induced cytotoxicity. These results suggested that hPSC-AECs phenotypically and functionally resemble primary AECs and could be more biologically relevant alternatives for evaluating the pathological contribution of confirmed or potential pulmotoxic materials included in smoking and microdust.

In recent years, perivascular stem cells (PVCs) have gained increasing attention as a promising source for regenerative medicine due to their greater differentiation potential compared to multipotent mesenchymal stem cells (MSCs). It has been reported that growth differentiation factor-5 (GDF-5) is involved in regulating proliferation and chondrogenic differentiation of MSCs. In this study, we investigated the effect of GDF-5 on the proliferation and chondrogenic differentiation of PVCs isolated from human umbilical cords. The supplementation of PVC cultures with GDF-5 (100 ng/mL) significantly enhanced their proliferative rate and augmented the size of pellets in micromass pellet cultures for chondrogenic induction. Although similar expression levels of chondrogenic-related genes were observed in chondrogenic pellets treated with GDF-5 compared to the pellets without GDF-5 treatment, these results indicate that supplementation of GDF-5 is able to acquire more chondrocytes when starting with equal amount of PVCs. Our study suggests that GDF-5 is an effective agent for the enhancement of PVC proliferation, thereby achieving a higher number of chondrocytes that are applicable in therapeutic doses for cartilage regeneration.

Self-renewal and differentiation are hallmarks of stem cells and controlled by various intrinsic and extrinsic factors. Increasing evidence indicates that estrogen (E2), the primary female sex hormone, is involved in regulating the proliferation and lineage commitment of adult and pluripotent stem cells as well as modulating the stem cell niche. Thus, a detailed understanding of the role of E2 in behavior of stem cells may help to improve their therapeutic potential. Recently, it has been reported that E2 promotes cell cycle activity of hematopoietic stem and progenitor cells and induces them to megakaryocyte-erythroid progenitors during pregnancy. This study paves the way towards a previously unexplored endocrine mechanism that controls stem cell behavior. In this review, we will focus on the scientific findings regarding the regulatory effects of E2 on the hematopoietic system including its microenvironment.

Introduction: A receptor for advanced glycation end products (RAGE) has been identified as a susceptibility gene for emphysema/chronic obstructive pulmonary disease (COPD) in genome-wide association studies (GWAS). However, less is known how RAGE is involved in the pathogenesis of COPD. Aim: In this study, we aimed to determine the molecular mechanism by which RAGE influences COPD and to assess the efficacy of RAGE-specific antagonist FPS-ZM1 administration in in vivo and in vitro COPD model. Methods: We injected intratracheally porcine pancreatic elastase (PPE) and RAGE antagonist FPS-ZM1 in mice. Inflammatory cells and cytokine were assessed by ELISA. The expression of RAGE was determined in protein, serum and bronchoalveolar (BAL) fluid of mice. Downstream DAMP pathway activation in vivo, in vitro and patients with COPD was assessed using immunofluorescence, western blotting, and ELISA. Results: we investigated the efficacy of RAGE-specific antagonist FPS-ZM1 administration in in vivo and in vitro COPD model. FPS-ZM1 reversed airspace enlargement and inflammation in emphysema mice. Moreover, we found that expression of membrane RAGE and soluble RAGE was associated with up-regulation of damage-associated molecular patterns (DAMP)-related signaling pathway via NF-E2 related factor 2 (Nrf2). FPS-ZM1 treatment significantly reduced lung inflammation in Nrf2+/+, but not in Nrf2-/- mice. Conclusions: Our data indicated for the first time that RAGE inhibition has an essential protective role in COPD. Our observation provided new insight into the therapeutic target in emphysema/COPD. Acknowledgments: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2014R1A2A2A01003737)

Introduction: Perivascular stem cells (PVCs) have been considered as a promising source of mesenchymal progenitors for cell-based therapy due to their greater regenerative potential. However, the therapeutic effects of PVCs in pulmonary diseases have not been investigated. Aims: We investigated the therapeutic effects of PVCs in an emphysema mouse model and understand the molecular mechanisms underlying alveolar epithelial repair. Methods: We isolated PVCs from human umbilical cords of pregnant women with IRB-approved written consent at KNUH. We collected the conditioned medium (CM) from PVC cultures and intratracheally injected it into an emphysema mouse model induced by elastase. We performed histological and biochemical analysis to determine the therapeutic effects of PVCs in lung injury. Results: Emphysema mouse lung injected with PVC-CM were recovered by a significant reduction in the MLI (84.6±2.1 mm) compared with the MLI of control mice (100.1±3.6 mm). In addition, expression levels of keratinocyte growth factor, as known for regulating pulmonary epithelial cell proliferation and surfactant protein, were increased. Conclusions: We demonstrated that lung injury was ameliorated by PVC-CM, which could be an alternative source for cell-based therapy in pulmonary diseases (This research was supported by Global PH.D Fellowship Program through the National Research Foundation of Korea funded by the Ministry of Education (2016R1A2B4014890 and 2016H1A2A1909769)).

The metastatic spread of tumor cells via lymphatic vessels affects the relapse of tumor patients. New lymphatic vessel formation, including lymphangiogenesis, is promoted in the tumor environment. The lymphangiogenic factor VEGF-C can mediate lymphatic vessel formation and induce tumor metastasis by binding with FLT4. In melanoma, metastasis via lymphatics such as lymph nodes is one of the main predictors of poor outcome. Thus, we investigated whether blockade of FLT4 can reduce metastasis via the suppression of lymphatic capillaries. Proliferative lymphatic capillaries in melanoma were estimated by immunohistochemistry using FLT4 antibody after the injection of the FLT4 antagonist MAZ51. The numbers of tumor modules in metastasised lungs were calculated by gross examination and lymphatic related factors were examined by qRT-PCR. MAZ51 injection resulted in the suppression of tumor size and module number and the inhibition of proliferative lymphatic vessels in the intratumoral region in the lung and proliferating melanoma cells in the lung compared to those of untreated groups. Additionally, high FLT4 and TNF-alpha were detected in melanoma-induced tissue, while lymphatic markers such as VEGF-C, FLT4 and Prox-1 were significantly decreased in MAZ51 treated groups, implying that anti-lymphangiogenesis by MAZ51 may provide a potential strategy to prevent tumor metastasis in melanoma and high number of lymphatic capillaries could be used diagnosis for severe metastasis.

Additional file 2: Table S1. Temporal changes (%) of ER-Îą and hematopoietic phenotypes during hiPSC-derived hematopoietic differentiation.

Cadmium (Cd), a major component of cigarette smoke, disrupts the normal functions of airway cells and can lead to the development of various pulmonary diseases such as chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms involved in Cd-induced pulmonary diseases are poorly understood. Here, we identified a cluster of genes that are altered in response to Cd exposure in human bronchial epithelial cells (BEAS-2B) and demonstrated that Cd-induced ER stress and inflammation are mediated via CCAAT-enhancer-binding proteins (C/EBP)-DNA-damaged-inducible transcript 3 (DDIT3) signaling in BEAS-2B cells. Cd treatment led to marked upregulation and downregulation of genes associated with the cell cycle, apoptosis, oxidative stress and inflammation as well as various signal transduction pathways. Gene set enrichment analysis revealed that Cd treatment stimulated the C/EBP signaling pathway and induced transcriptional activation of its downstream target genes, including DDIT3. Suppression of DDIT3 expression using specific small interfering RNA effectively alleviated Cd-induced ER stress and inflammatory responses in both BEAS-2B and normal primary normal human bronchial epithelial cells. Taken together, these data suggest that C/EBP signaling may have a pivotal role in the early induction of ER stress and inflammatory responses by Cd exposure and could be a molecular target for Cd-induced pulmonary disease.

Background Airway epithelial cells are the first line of defense, against pathogens and environmental pollutants, in the lungs. Cellular stress by cadmium (Cd), resulting in airway inflammation, is assumed to be directly involved in tissue injury, linked to the development of lung cancer, and chronic obstructive pulmonary disease (COPD). We had earlier shown that ACN9 (chromosome 7q21), is a potential candidate gene for COPD, and identified significant interaction with smoking, based on genetic studies. However, the role of ACN9 in the inflammatory response, in the airway cells, has not yet been reported. Methods We first checked the anatomical distribution of ACN9 in lung tissues, using mRNA in situ hybridization, and immunohistochemistry. Gene expression profiling in bronchial epithelial cells (BEAS-2B), was performed, after silencing ACN9. We further tested the roles of ACN9, in the intracellular mechanism, leading to Cd-induced production, of proinflammatory cytokines in BEAS-2B. Results ACN9 was localized in lymphoid, and epithelial cells, of human lung tissues. ACN9 silencing, led to differential expression of 216 genes. Pathways of sensory perception to chemical stimuli, and cell surface receptor-linked signal transduction, were significantly enriched. ACN9 silencing, further increased the expression of proinflammatory cytokines, in BEAS-2B after Cd exposure. Conclusion Our findings suggest, that ACN9 may have a role, in the inflammatory response in the airway.

Background Aside from its importance in reproduction, estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However, the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs). Results E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-α)-dependent pathway. During hematopoietic differentiation of hPSCs, ER-α is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-α positive cells. Interestingly, continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-α selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors, and subsequent erythropoiesis, whereas ER-β selective agonist did not. Furthermore, ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods, does E2 signaling potentiate hematopoietic development, suggesting universal function of E2 on hematopoiesis. Conclusions Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions. Electronic supplementary material The online version of this article (doi:10.1186/s13578-016-0111-9) contains supplementary material, which is available to authorized users.