Generation of 6 lines of human pluripotent stem cells from hypertensive patients and 3 lines of human pluripotent stem cell from normotensive patients.

Hypertension is a complex multifactorial disease characterized by a chronic increase of arterial pressure. Ninety percent of the cases are idiopathic and thus classified as essential hypertension. Uncontrolled arterial pressure has devasting consequences including cardiac insufficiency, stroke, dementia, chronic renal disease, ischemic heart disease and death. The hiPSC lines described here from six hypertensive patients and three controls were characterized according to established criteria and were shown to maintain pluripotency, differentiation into the three germ layers and genomic integrity. These cell lines can contribute to the understanding of the molecular mechanisms involved in hypertension in different cell types.

Generation of genetically modified human induced pluripotent stem cell lines harboring haploin sufficient or dominant negative variants in the FBN1 gene.

Marfan Syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the FBN1 gene. To investigate the molecular mechanisms of pathogenesis for the syndrome, we genetically modified the FBN1 gene in a line of induced pluripotent stem cells (hiPSCs) derived from a healthy donor using the CRISPR/Cas9 gene editing technology. The sublines described here were characterized according to established criteria and were shown to maintain pluripotency, three germ layer differentiation potential and genomic integrity. These clones can now be used to better understand the pathogenesis of MFS in different cell types.

Generation of two human induced pluripotent stem cell (hiPSC) lines derived from unrelated Marfan Syndrome patients.

Marfan Syndrome (MFS) is a pleiotropic and autosomal dominant condition caused by pathogenic variants in FBN1. Although fully penetrant, clinical variability is frequently observed among patients and there are only few genotype-phenotype correlations described so far. Here, we describe the generation and characterization of hiPSC lines derived from two unrelated MFS patients harboring heterozygous variants in FBN1. Human iPSCs were obtained from erythroblasts reprogrammed with episomal vectors carrying the reprogramming factors OCT4, SOX2, KLF4, c-MYC and LIN-28, and characterized according to established criteria. Differentiated cells demonstrated different patterns of fibrillin-1 expression suggesting different molecular mechanisms between the two patients.

Monitoring cell line identity in collections of human induced pluripotent stem cells.

The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs) has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations.

Induced Pluripotent Stem Cell for the Study and Treatment of Sickle Cell Anemia.

Sickle cell anemia (SCA) is a monogenic disease of high mortality, affecting millions of people worldwide. There is no broad, effective, and safe definitive treatment for SCA, so the palliative treatments are the most used. The establishment of an in vitro model allows better understanding of how the disease occurs, besides allowing the development of more effective tests and treatments. In this context, iPSC technology is a powerful tool for basic research and disease modeling, and a promise for finding and screening more effective and safe drugs, besides the possibility of use in regenerative medicine. This work obtained a model for study and treatment of SCA using iPSC. Then, episomal vectors were used for reprogramming peripheral blood mononuclear cells to obtain integration-free iPSC. Cells were collected from patients treated with hydroxyurea and without treatment. The iPSCP Bscd lines were characterized for pluripotent and differentiation potential. The iPSC lines were differentiated into HSC, so that we obtained a dynamic and efficient protocol of CD34+CD45+ cells production. We offer a valuable tool for a better understanding of how SCA occurs, in addition to making possible the development of more effective drugs and treatments and providing better understanding of widely used treatments, such as hydroxyurea.

Increasing The Genetic Admixture of Available Lines of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) may significantly improve drug development pipeline, serving as an in vitro system for the identification of novel leads, and for testing drug toxicity. Furthermore, these cells may be used to address the issue of differential drug response, a phenomenon greatly influenced by genetic factors. This application depends on the availability of hPSC lines from populations with diverse ancestries. So far, it has been reported that most lines of hPSCs derived worldwide are of European or East Asian ancestries. We have established 23 lines of hPSCs from Brazilian individuals, and we report the analysis of their genomic ancestry. We show that embryo-derived PSCs are mostly of European descent, while induced PSCs derived from participants of a national-wide Brazilian cohort study present high levels of admixed European, African and Native American genomic ancestry. Additionally, we use high density SNP data and estimate local ancestries, particularly those of CYP genes loci. Such information will be of key importance when interpreting variation among cell lines with respect to cellular phenotypes of interest. The availability of genetically admixed lines of hPSCs will be of relevance when setting up future in vitro studies of drug response.

A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo.

Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1-2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo's missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage.

TNF-alpha and melphalan modulate a specific group of early expressed genes in a murine melanoma model.

BACKGROUND: Cutaneous melanoma displays high morbidity and mortality rates. Isolated limb perfusion with melphalan (Mel) is used for the treatment of non-resectable, locally advanced extremity melanomas. When combined with tumor necrosis factor alpha (TNF-alpha) treatment, the complete response varies between 70% and 90%. The mechanisms underlying the effects of Mel and TNF-alpha are not completely understood. We evaluated the impact of systemic Mel and TNF-alpha administration on tumor growth, analyzed the morphological changes promoted by each treatment, and identified early expressed genes in response to Mel and TNF-alpha treatment, either alone or in combination, in a murine melanoma model. METHODS: Six- to eight-week-old male mice were subcutaneously inoculated with B16F10 melanoma cells and then intravenously injected with TNF-alpha, melphalan or a combination of both drugs when the tumors reached 1.0 cm(2). Tumor growth was monitored every other day, and histological analysis was performed when the tumors reached 3.0 cm(2). Total RNA was extracted from the resected tumors and submitted to amplification, labeling and hybridization on an oligonucleotide microarray (Fox Chase Cancer Center). Tumor growth and histological parameters were compared using ANOVA. Survival curves were calculated using the Kaplan-Meier method. Two-way ANOVA was used to identify differentially expressed genes among the various treatments, and Dunn's test was used for pair-wise comparisons. RESULTS: Systemic administration of Mel impaired tumor growth (p<0.001), improved animal survival (p<0.001), and decreased mitotic rate (p=0.049). Treatment with TNF-alpha alone had no impact, neither on tumor growth, nor on survival, but it increased necrosis (p<0.024) and decreased mitotic rates (p=0.001) in the tumors. Combined treatment with Mel and TNF-alpha had similar effects in tumor growth, survival, necrosis and mitotic rate as observed with individual treatments. Moreover, 118 genes were found differentially expressed by microarray analysis and 10% of them were validated by RT- real time PCR. In our model we found that the treatments regulate genes that play important roles in tumorigenesis such as cell adhesion (Pard3, Pecam1, Ilk, and Dlg5), proliferation (Tcfe3 and Polr1e), cell motility (Kifap3, Palld, and Arhgef6), apoptosis (Bcl2l11), and angiogenesis (Flt1 and Ptprj). CONCLUSIONS: Our data reproduces, in mice, some of the features observed in melanoma patients treated with the combination of Mel and TNF-alpha. The identification of genes with altered expression by these drugs both individually and in combination might help in the understanding of their mechanism of action and, as a consequence, improved strategies that could impact their clinical application.

Macrophage dectin-1 expression is controlled by leukotriene B4 via a GM-CSF/PU.1 axis.

Pattern recognition receptors for fungi include dectin-1 and mannose receptor, and these mediate phagocytosis, as well as production of cytokines, reactive oxygen species, and the lipid mediator leukotriene B(4) (LTB(4)). The influence of G protein-coupled receptor ligands such as LTB(4) on fungal pattern recognition receptor expression is unknown. In this study, we investigated the role of LTB(4) signaling in dectin-1 expression and responsiveness in macrophages. Genetic and pharmacologic approaches showed that LTB(4) production and signaling through its high-affinity G protein-coupled receptor leukotriene B(4) receptor 1 (BLT1) direct dectin-1-dependent binding, ingestion, and cytokine production both in vitro and in vivo. Impaired responses to fungal glucans correlated with lower dectin-1 expression in macrophages from leukotriene (LT)- and BLT1-deficent mice than their wild-type counterparts. LTB(4) increased the expression of the transcription factor responsible for dectin-1 expression, PU.1, and PU.1 small interfering RNA abolished LTB(4)-enhanced dectin-1 expression. GM-CSF controls PU.1 expression, and this cytokine was decreased in LT-deficient macrophages. Addition of GM-CSF to LT-deficient cells restored expression of dectin-1 and PU.1, as well as dectin-1 responsiveness. In addition, LTB(4) effects on dectin-1, PU.1, and cytokine production were blunted in GM-CSF(-/-) macrophages. Our results identify LTB(4)-BLT1 signaling as an unrecognized controller of dectin-1 transcription via GM-CSF and PU.1 that is required for fungi-protective host responses.

Pentraxin 3 accelerates lung injury in high tidal volume ventilation in mice.

Mechanical ventilation is the major cause of iatrogenic lung damage in intensive care units. Although inflammation is known to be involved in ventilator-induced lung injury (VILI), several aspects of this process are still unknown. Pentraxin 3 (PTX3) is an acute phase protein with important regulatory functions in inflammation which has been found elevated in patients with acute respiratory distress syndrome. This study aimed at investigating the direct effect of PTX3 production in the pathogenesis of VILI. Genetically modified mice deficient and that over express murine Ptx3 gene were subjected to high tidal volume ventilation (V(T)=45 mL/kg, PEEP(zero)). Morphological changes and time required for 50% increase in respiratory system elastance were evaluated. Gene expression profile in the lungs was also investigated in earlier times in Ptx3-overexpressing mice. Ptx3 knockout and wild-type mice developed same lung injury degree in similar times (156±42 min and 148±41 min, respectively; p=0.8173). However, Ptx3 over-expression led to a faster development of VILI in Ptx3-overexpressing mice (77±29 min vs 118±41 min, p=0.0225) which also displayed a faster kinetics of Il1b expression and elevated Ptx3, Cxcl1 and Ccl2 transcripts levels in comparison with wild-type mice assessed by quantitative real-time polymerase chain reaction. Ptx3 deficiency did not impacted the time for VILI induced by high tidal volume ventilation but Ptx3-overexpression increased inflammatory response and reflected in a faster VILI development.

Leukotrienes target F-actin/cofilin-1 to enhance alveolar macrophage anti-fungal activity.

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.