Suspension-Induced Stem Cell Transition: A Non-Transgenic Method to Generate Adult Stem Cells from Mouse and Human Somatic Cells.

Adult stem cells (ASCs) can be cultured with difficulty from most tissues, often requiring chemical or transgenic modification to achieve adequate quantities. We show here that mouse primary fibroblasts, grown in suspension, change from the elongated and flattened morphology observed under standard adherent culture conditions of generating rounded cells with large nuclei and scant cytoplasm and expressing the mesenchymal stem cell (MSC) marker (Sca1; Ly6A) within 24 h. Based on this initial observation, we describe here a suspension culture method that, irrespective of the lineage used, mouse fibroblast or primary human somatic cells (fibroblasts, hepatocytes and keratinocytes), is capable of generating a high yield of cells in spheroid form which display the expression of ASC surface markers, circumventing the anoikis which often occurs at this stage. Moreover, mouse fibroblast-derived spheroids can be differentiated into adipogenic and osteogenic lineages. An analysis of single-cell RNA sequence data in mouse fibroblasts identified eight distinct cell clusters with one in particular comprising approximately 10% of the cells showing high levels of proliferative capacity expressing high levels of genes related to MSCs and self-renewal as well as the extracellular matrix (ECM). We believe the rapid, high-yield generation of proliferative, multi-potent ASC-like cells via the process we term suspension-induced stem cell transition (SIST) could have significant implications for regenerative medicine.

Role of Ceramides and Lysosomes in Extracellular Vesicle Biogenesis, Cargo Sorting and Release.

Cells have the ability to communicate with their immediate and distant neighbors through the release of extracellular vesicles (EVs). EVs facilitate intercellular signaling through the packaging of specific cargo in all type of cells, and perturbations of EV biogenesis, sorting, release and uptake is the basis of a number of disorders. In this review, we summarize recent advances of the complex roles of the sphingolipid ceramide and lysosomes in the journey of EV biogenesis to uptake.

Thrombospondin-1 Plays a Major Pathogenic Role in Experimental and Human Bronchopulmonary Dysplasia.

Rationale: Extremely preterm infants develop bronchopulmonary dysplasia (BPD), a chronic lung injury that lacks effective treatment. TSP-1 (thrombospondin-1) is an antiangiogenic protein that activates TGF-ß1 (transforming growth factor-ß1), a cytokine strongly linked to both experimental and human BPD. Objectives:1) To examine effects of inhibiting TSP-1-mediated TGF-ß1 activation (LSKL [leucine-serine-lysine-leucine]) in neonatal rats with bleomycin-induced lung injury; 2) to examine effects of a TSP-1 mimic (ABT-510) on lung morphology; and 3) to determine whether TSP-1 and related signaling peptides are increased in lungs of human preterm infants at risk for BPD. Methods: From Postnatal Days 1 to 14, rat pups received daily intraperitoneal bleomycin (1 mg/kg) or vehicle and were treated with daily subcutaneous LSKL (20 mg/kg) or vehicle alone. Separate animals were treated with vehicle or ABT-510 (30 mg/kg/d). Paraffin-embedded lung tissues from 47 autopsies (controls; death <28 d, n = 30 and BPD at risk; death ⩾28 d, n = 17) performed on infants born <29 completed weeks' gestation were semiquantified for injury markers (collagen, macrophages, and 3-nitrotyrosine), TSP-1, and TGF-ß1. Measurements and Main Results: Bleomycin or ABT-510 increased lung TGF-ß1 activity and macrophage influx, caused pulmonary hypertension, and led to alveolar and microvascular hypoplasia. Treatment with LSKL partially prevented abnormal lung morphology secondary to bleomycin. Lungs from human infants at risk for BPD had increased contents of TSP-1 and TGF-ß1 when compared with controls. TGF-ß1 content correlated with markers of lung injury. Conclusions: TSP-1 inhibits alveologenesis in neonatal rats, in part via the upregulated activity of TGF-ß1. Observations in human lungs suggest a similar pathogenic role for TSP-1 in infants at risk for BPD.

Autophagy in Tumor Immunity and Viral-Based Immunotherapeutic Approaches in Cancer.

Autophagy is a fundamental catabolic process essential for the maintenance of cellular and tissue homeostasis, as well as directly contributing to the control of invading pathogens. Unsurprisingly, this process becomes critical in supporting cellular dysregulation that occurs in cancer, particularly the tumor microenvironments and their immune cell infiltration, ultimately playing a role in responses to cancer therapies. Therefore, understanding "cancer autophagy" could help turn this cellular waste-management service into a powerful ally for specific therapeutics. For instance, numerous regulatory mechanisms of the autophagic machinery can contribute to the anti-tumor properties of oncolytic viruses (OVs), which comprise a diverse class of replication-competent viruses with potential as cancer immunotherapeutics. In that context, autophagy can either: promote OV anti-tumor effects by enhancing infectivity and replication, mediating oncolysis, and inducing autophagic and immunogenic cell death; or reduce OV cytotoxicity by providing survival cues to tumor cells. These properties make the catabolic process of autophagy an attractive target for therapeutic combinations looking to enhance the efficacy of OVs. In this article, we review the complicated role of autophagy in cancer initiation and development, its effect on modulating OVs and immunity, and we discuss recent progress and opportunities/challenges in targeting autophagy to enhance oncolytic viral immunotherapy.

Autophagy is required for lung development and morphogenesis.

Bronchopulmonary dysplasia (BPD) remains a major respiratory illness in extremely premature infants. The biological mechanisms leading to BPD are not fully understood, although an arrest in lung development has been implicated. The current study aimed to investigate the occurrence of autophagy in the developing mouse lung and its regulatory role in airway branching and terminal sacculi formation. We found 2 windows of epithelial autophagy activation in the developing mouse lung, both resulting from AMPK activation. Inhibition of AMPK-mediated autophagy led to reduced lung branching in vitro. Conditional deletion of beclin 1 (Becn1) in mouse lung epithelial cells (Becn1Epi-KO), either at early (E10.5) or late (E16.5) gestation, resulted in lethal respiratory distress at birth or shortly after. E10.5 Becn1Epi-KO lungs displayed reduced airway branching and sacculi formation accompanied by impaired vascularization, excessive epithelial cell death, reduced mesenchymal thinning of the interstitial walls, and delayed epithelial maturation. E16.5 Becn1Epi-KO lungs had reduced terminal air sac formation and vascularization and delayed distal epithelial differentiation, a pathology similar to that seen in infants with BPD. Taken together, our findings demonstrate that intrinsic autophagy is an important regulator of lung development and morphogenesis and may contribute to the BPD phenotype when impaired.

Acid Sphingomyelinase Inhibition Attenuates Cell Death in Mechanically Ventilated Newborn Rat Lung.

RATIONALE: Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. OBJECTIVES: To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. METHODS: Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. MEASUREMENTS AND MAIN RESULTS: Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. CONCLUSIONS: Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

Explant Culture for Studying Lung Development.

Lung development is a complex process that requires the input of various signaling pathways to coordinate the specification and differentiation of multiple cell types. Ex vivo culture of the lung is a very useful technique that represents an attractive model for investigating many different processes critical to lung development, function, and disease pathology. Ex vivo cultured lungs remain comparable to the in vivo lung both in structure and function, which makes them more suitable than cell cultures for physiological studies. Lung explant cultures offer several significant advantages for studies of morphogenetic events that guide lung development including budding, branching, and fusion. It also maintains the native physiological interactions between cells in the developing lung, enabling investigations of the direct and indirect signaling taking place between tissues and cells throughout the developmental process. Studying temporal and spatial control of gene expression by transcriptional factors using different reporters to understand their regulatory function at different moments of development is another valuable advantage of lung explants culture.

Autophagy and the unfolded protein response promote profibrotic effects of TGF-ß1 in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-ß1 (TGF-ß1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3ßII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3ßII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-ß1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-ß1 induced the accumulation of LC3ßII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-ß1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-ß1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-ß1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-ß1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia.

Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women.

mTOR-Notch3 signaling mediates pulmonary hypertension in hypoxia-exposed neonatal rats independent of changes in autophagy.

BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) is a pivotal regulator of cell proliferation, survival, and autophagy. Autophagy is increased in adult experimental chronic pulmonary hypertension (PHT), but its contributory role to pulmonary vascular disease remains uncertain and has yet to be explored in the neonatal animal. Notch is a major pro-proliferative pathway activated by mTOR. A direct relationship between autophagy and Notch signaling has not been previously explored. Our aim was to examine changes in mTOR-, Notch-, and autophagy-related pathways and the therapeutic effects of autophagy modulators in experimental chronic neonatal PHT secondary to chronic hypoxia. METHODS: Rat pups were exposed to normoxia or hypoxia (13% O2 ) from postnatal days 1-21, while receiving treatment with temsirolimus (mTOR inhibitor), DAPT (Notch inhibitor), or chloroquine (inhibitor of autophagic flux). RESULTS: Exposure to hypoxia up-regulated autophagy and Notch3 signaling markers in lung, pulmonary artery (PA), and PA-derived smooth muscle cells (SMCs). Temsirolimus prevented chronic PHT and attenuated PA and SMC signaling secondary to hypoxia. These effects were replicated by DAPT. mTOR or Notch inhibition also down-regulated smooth muscle content of platelet-derived growth factor ß-receptor, a known contributor to vascular remodeling. In contrast, chloroquine had no modifying effects on markers of chronic PHT. Knockdown of Beclin-1 in SMCs had no effect on hypoxia-stimulated Notch3 signaling. CONCLUSIONS: mTOR-Notch3 signaling plays a critical role in experimental chronic neonatal PHT. Inhibition of autophagy did not suppress Notch signaling and had no effect on markers of chronic PHT.

Imaging mass spectrometry identifies prognostic ganglioside species in rodent intracranial transplants of glioma and medulloblastoma.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (MALDI-MSI) allows us to investigate the distribution of lipid molecules within tissues. We used MALDI-MSI to identify prognostic gangliosides in tissue sections of rat intracranial allografts of rat glioma and mouse intracranial xenografts of human medulloblastoma. In the healthy adult rodent brain, GM1 and GD1 were the main types of glycolipids. Both gangliosides were absent in both intracranial transplants. The ganglioside GM3 was not present in the healthy adult brain but was highly expressed in rat glioma allografts. In combination with tandem mass spectrometry GM3 (d18:1/C24:0) was identified as the most abundant ganglioside species in the glioma allotransplant. By contrast, mouse xenografts of human medulloblastoma were characterized by prominent expression of the ganglioside GM2 (d18:0/C18:0). Together, these data demonstrate that tissue-based MALDI-MSI of gangliosides is able to discriminate between different brain tumors and may be a useful clinical tool for their classification and grading.

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2.

Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.

Effect of chloroquine on some clinical and biochemical parameters in non-response chronic hepatitis C virus infection patients: pilot clinical trial.

BACKGROUND: Hepatitis C virus infection frequently leads to chronic hepatitis C which may progress to cirrhosis and can be ended to hepatocellular carcinoma. This study aimed to investigate the effect of Anthropometric Parameters, Vit D3, Thyroid Function, Ferritin and Biochemistry Parameters in patients chronically infected with HCV with non-response criteria which was treated by chloroquine. METHODS: This study was the continuation of our previous investigation with a triple-blind method in a randomized controlled pilot study. After understanding the study procedures, patients signed an informed consent form and were randomized into the treatment (chloroquine 150 mg once daily, for 8 weeks) and control (placebo once daily, for 8 weeks) groups. The inclusion criteria were male, between 18 and 60 years of age, confirmed chronic hepatitis C with non-response criteria, and Genotype 1.  Data were analyzed with an intention to treat perspective at the end follow up (12 weeks) considering to variables such as anthropometric parameters, Vit D3, thyroid function, ferritin and biochemistry parameters evaluated. RESULTS: Although there were decreases in total weight (P-value=0.4), mid-arm circumference (P-value=0.05), and body mass index (P-value=0.04) there were increases in total body fat (P-value=0.8) and triceps skin fold thickness  (P-value=0.7) in the intervention group compared to the control group. Also, a reduction of AST (P-value=0.30), ALT (P-value=0.10), cholesterol (P-value=0.005), triglyceride (P-value=0.40) and ferritin (P-value=0.030) levels was seen in the intervention group during the follow up period. Our results also showed that serum TSH levels (P-value=0.5) were slightly higher in the chloroquine group than in the placebo group, though the trend was reversed for T3 (P-value=0.05) and T4 (P-value=0.04) levels. however, median of T3 and T4 were similar in both groups. A significant increase in vitamin D levels from 15 to 34 ng/ml was observed in the chloroquine group (P-value=0.04). CONCLUSIONS: The results suggest that chloroquine therapy may be very useful for HCV treatment in patients with non-response criteria, and helps to normalize some anthropometric parameters, biochemical, ferritin, and vitamin D status.

New use of an old drug: chloroquine reduces viral and ALT levels in HCV non-responders (a randomized, triple-blind, placebo-controlled pilot trial).

Hepatitis C virus (HCV) infection induces autophagy, but the virus assimilates the autophagic response into its own life cycle. Chloroquine (CQ) is an autophagy inhibitor that is clinically used to treat malaria. The aims of this pilot clinical trial were to evaluate the therapeutic potential and short-term safety of CQ in patients with chronic HCV genotype 1, who were unresponsive to a combination of pegylated interferon alpha and ribavirin. Ten non-responders to previous antiviral treatment(s) were randomized to receive either CQ (150 mg daily for 8 weeks) or placebo, and were followed for 4 weeks after CQ therapy. HCV RNA load and plasma alanine transaminase (ALT) levels were measured at baseline, week 4 (initial response), week 8 (end-of-treatment response), and at the end of 12 weeks. A significant decrease in HCV RNA after the treatments (week 8) was observed in all patients in the CQ group (P = 0.04). However, HCV RNA levels increased within 4 weeks after discontinuation of CQ treatment although they were still lower than baseline. In addition, the ALT normalized during treatment in the CQ group. However, this response was also lost after treatment cessation. This study provides preliminary evidence that CQ is possibly a safe treatment option for HCV non-responders.

Hepatitis B and C virus-induced hepatitis: Apoptosis, autophagy, and unfolded protein response.

AIM: To investigate the co-incidence of apoptosis, autophagy, and unfolded protein response (UPR) in hepatitis B (HBV) and C (HCV) infected hepatocytes. METHODS: We performed immunofluorescence confocal microscopy on 10 liver biopsies from HBV and HCV patients and tissue microarrays of HBV positive liver samples. We used specific antibodies for LC3ß, cleaved caspase-3, BIP (GRP78), and XBP1 to detect autophagy, apoptosis and UPR, respectively. Anti-HCV NS3 and anti-HBs antibodies were also used to confirm infection. We performed triple blind counting of events to determine the co-incidence of autophagy (LC3ß punctuate), apoptosis (cleaved caspase-3), and unfolded protein response (GRP78) with HBV and HCV infection in hepatocytes. All statistical analyses were performed using SPSS software for Windows (Version 16 SPSS Inc, Chicago, IL, United States). P-values < 0.05 were considered statistically significant. Statistical analyses were performed with Mann-Whitney test to compare incidence rates for autophagy, apoptosis, and UPR in HBV- and HCV-infected cells and adjacent non-infected cells. RESULTS: Our results showed that infection of hepatocytes with either HBV and HCV induces significant increase (P < 0.001) in apoptosis (cleavage of caspase-3), autophagy (LC3ß punctate), and UPR (increase in GRP78 expression) in the HCV- and HBV-infected cells, as compared to non-infected cells of the same biopsy sections. Our tissue microarray immunohistochemical expression analysis of LC3ß in HBV(Neg) and HBV(Pos) revealed that majority of HBV-infected hepatocytes display strong positive staining for LC3ß. Interestingly, although XBP splicing in HBV-infected cells was significantly higher (P < 0.05), our analyses show a slight increase of XBP splicing was in HCV-infected cells (P > 0.05). Furthermore, our evaluation of patients with HBV and HCV infection based on stage and grade of the liver diseases revealed no correlation between these pathological findings and induction of apoptosis, autophagy, and UPR. CONCLUSION: The results of this study indicate that HCV and HBV infection activates apoptosis, autophagy and UPR, but slightly differently by each virus. Further studies are warranted to elucidate the interconnections between these pathways in relation to pathology of HCV and HBV in the liver tissue.

Embryonic stages in cerebellar afferent development.

The cerebellum is important for motor control, cognition, and language processing. Afferent and efferent fibers are major components of cerebellar circuitry and impairment of these circuits causes severe cerebellar malfunction, such as ataxia. The cerebellum receives information from two major afferent types - climbing fibers and mossy fibers. In addition, a third set of afferents project to the cerebellum as neuromodulatory fibers. The spatiotemporal pattern of early cerebellar afferents that enter the developing embryonic cerebellum is not fully understood. In this review, we will discuss the cerebellar architecture and connectivity specifically related to afferents during development in different species. We will also consider the order of afferent fiber arrival into the developing cerebellum to establish neural connectivity.

Sphingolipids as cell fate regulators in lung development and disease.

Sphingolipids are a diverse class of signaling molecules implicated in many important aspects of cellular biology, including growth, differentiation, apoptosis, and autophagy. Autophagy and apoptosis are fundamental physiological processes essential for the maintenance of cellular and tissue homeostasis. There is great interest into the investigation of sphingolipids and their roles in regulating these key physiological processes as well as the manifestation of several disease states. With what is known to date, the entire scope of sphingolipid signaling is too broad, and a single review would hardly scratch the surface. Therefore, this review attempts to highlight the significance of sphingolipids in determining cell fate (e.g. apoptosis, autophagy, cell survival) in the context of the healthy lung, as well as various respiratory diseases including acute lung injury, acute respiratory distress syndrome, bronchopulmonary dysplasia, asthma, chronic obstructive pulmonary disease, emphysema, and cystic fibrosis. We present an overview of the latest findings related to sphingolipids and their metabolites, provide a short introduction to autophagy and apoptosis, and then briefly highlight the regulatory roles of sphingolipid metabolites in switching between cell survival and cell death. Finally, we describe functions of sphingolipids in autophagy and apoptosis in lung homeostasis, especially in the context of the aforementioned diseases.

Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans.

Efficient differentiation of pluripotent cells to proximal and distal lung epithelial cell populations remains a challenging task. The 3D extracellular matrix (ECM) scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone. Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.