Turning skin into brain: Using patient‐derived cells to model X‐linked adrenoleukodystrophy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87162/1/22546_ftp.pd

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models. Copyright (c) 2012 John Wiley & Sons, Ltd

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

Background & Aims: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Methods: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes

White Blood Cells and Blood Pressure: A Mendelian Randomization Study

Background: High blood pressure (BP) is a risk factor for cardiovascular morbidity and mortality. While BP is regulated by the function of kidney, vasculature, and sympathetic nervous system, recent experimental data suggest that immune cells may play a role in hypertension. Methods: We studied the relationship between major white blood cell types and blood pressure in the UK Biobank population and used Mendelian randomization (MR) analyses using the ≈750 000 UK-Biobank/International Consortium of Blood Pressure-Genome-Wide Association Studies to examine which leukocyte populations may be causally linked to BP. Results: A positive association between quintiles of lymphocyte, monocyte, and neutrophil counts, and increased systolic BP, diastolic BP, and pulse pressure was observed (eg, adjusted systolic BP mean±SE for 1st versus 5th quintile respectively: 140.13±0.08 versus 141.62±0.07 mm Hg for lymphocyte, 139.51±0.08 versus 141.84±0.07 mm Hg for monocyte, and 137.96±0.08 versus 142.71±0.07 mm Hg for neutrophil counts; all P<10-50). Using 121 single nucleotide polymorphisms in MR, implemented through the inverse-variance weighted approach, we identified a potential causal relationship of lymphocyte count with systolic BP and diastolic BP (causal estimates: 0.69 [95% CI, 0.19-1.20] and 0.56 [95% CI, 0.23-0.90] of mm Hg per 1 SD genetically elevated lymphocyte count, respectively), which was directionally concordant to the observational findings. These inverse-variance weighted estimates were consistent with other robust MR methods. The exclusion of rs3184504 SNP in the SH2B3 locus attenuated the magnitude of the signal in some of the MR analyses. MR in the reverse direction found evidence of positive effects of BP indices on counts of monocytes, neutrophils, and eosinophils but not lymphocytes or basophils. Subsequent MR testing of lymphocyte count in the context of genetic correlation with renal function or resting and postexercise heart rate demonstrated a positive association of lymphocyte count with urine albumin-to-creatinine ratio. Conclusions: Observational and genetic analyses demonstrate a concordant, positive and potentially causal relationship of lymphocyte count with systolic BP and diastolic BP

New horizons for stem cell therapy in liver disease

SummaryThere is an increasing range of potential applications of stem cells in liver diseases, with many clinical studies already undertaken. We identify four of the main areas which we propose stem cell therapy could be a realistic aim for in the future: (1) to improve regeneration and reduce scarring in liver cirrhosis by modulating the liver’s own regenerative processes, (2) to down-regulate immune mediated liver damage, (3) supplying hepatocyte-like cells (HLCs) derived from stem cells for use in extracorporeal bio-artificial liver machines, and (4) to use stem cell derived HLCs for cell transplantation to supplement or replace hepatocyte function

White blood cells and blood pressure: a Mendelian randomization study

Background: High blood pressure (BP) is a risk factor for cardiovascular morbidity and mortality. While BP is regulated by the function of kidney, vasculature and sympathetic nervous system, recent experimental data suggest that immune cells may play a role in hypertension. Methods: We studied the relationship between major white blood cell types and blood pressure in the UK Biobank population and employed Mendelian randomization (MR) analyses using the ∼750,000 UK-Biobank/International Consortium of Blood Pressure-Genome-Wide Association Studies to examine which leukocyte populations may be causally linked to BP. Results: A positive association between quintiles of lymphocyte, monocyte, neutrophil counts and increased systolic (SBP), diastolic (DBP) and pulse pressure (PP) was observed (e.g. adjusted SBP mean±SE for 1st vs 5th quintile respectively: 140.13±0.08 vs. 141.62±0.07 mmHg for lymphocyte, 139.51±0.08 vs. 141.84±0.07 mmHg for monocyte, and 137.96±0.08 vs. 142.71±0.07 mmHg for neutrophil counts, all p&lt;10-50). Using 121 SNPs in MR implemented through the inverse-variance weighted (IVW) approach, we identified a potential causal relationship of lymphocyte count with SBP and DBP (causal estimates: 0.69 (95%CI: 0.19-1.20) and 0.56 (95%CI: 0.23-0.90) of mmHg per 1 SD genetically elevated lymphocyte count, respectively), which was directionally concordant to the observational findings. These IVW estimates were consistent with other, robust MR methods. Interestingly, the exclusion of rs3184504 SNP in the SH2B3 locus attenuated the magnitude of the signal in some of the MR analyses. MR in the reverse direction found evidence of positive effects of BP indices on counts of monocytes, neutrophils and eosinophils, but not lymphocytes or basophils. Subsequent MR testing of lymphocyte count in the context of genetic correlation with renal function or resting and post-exercise heart rate demonstrated a positive association of lymphocyte count with urinary albumin to creatinine ratio. Conclusions: Observational and genetic analyses demonstrate a concordant, positive and potentially causal relationship of lymphocyte count with SBP and DBP

Metabolomic and transcriptomic stress response of Escherichia coli

GC-MS-based analysis of the metabolic response of Escherichia coli exposed to four different stress conditions reveals reduction of energy expensive pathways.Time-resolved response of E. coli to changing environmental conditions is more specific on the metabolite as compared with the transcript level.Cease of growth during stress response as compared with stationary phase response invokes similar transcript but dissimilar metabolite responses.Condition-dependent associations between metabolites and transcripts are revealed applying co-clustering and canonical correlation analysis

Systemic and local vascular inflammation and arterial reactive oxygen species generation in patients with advanced cardiovascular diseases

BackgroundSystemic inflammation may cause endothelial activation, mediate local inflammation, and accelerate progression of atherosclerosis. We examined whether the levels of circulating inflammatory cytokines reflect local vascular inflammation and oxidative stress in two types of human arteries.MethodsHuman internal mammary artery (IMA) was obtained in 69 patients undergoing coronary artery bypass graft (CABG) surgery and left anterior descending (LAD) artery was obtained in 17 patients undergoing heart transplantation (HTx). Plasma levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were measured using ELISA, high-sensitivity C-reactive protein (hs-CRP) was measured using Luminex, and mRNA expression of proinflammatory cytokines in the vascular tissues was assessed. Furthermore, formation of superoxide anion was measured in segments of IMA using 5 uM lucigenin-dependent chemiluminescence. Vascular reactivity was measured using tissue organ bath system.ResultsTNF-α, IL-6 and IL-1β mRNAs were expressed in all studied IMA and LAD segments. Plasma levels of inflammatory cytokines did not correlate with vascular cytokine mRNA expression neither in IMA nor in LAD. Plasma TNF-α and IL-6 correlated with hs-CRP level in CABG group. Hs-CRP also correlated with TNF-α in HTx group. Neither vascular TNF-α, IL-6 and IL-1β mRNA expression, nor systemic levels of either TNF-α, IL-6 and IL-1β were correlated with superoxide generation in IMAs. Interestingly, circulating IL-1β negatively correlated with maximal relaxation of the internal mammary artery (r = −0.37, p = 0.004). At the same time the mRNA expression of studied inflammatory cytokines were positively associated with each other in both IMA and LAD. The positive correlations were observed between circulating levels of IL-6 and TNF-α in CABG cohort and IL-6 and IL-1β in HTx cohort.ConclusionsThis study shows that peripheral inflammatory cytokine measurements may not reflect local vascular inflammation or oxidative stress in patients with advanced cardiovascular disease (CVD). Circulating pro-inflammatory cytokines generally correlated positively with each other, similarly their mRNA correlated in the arterial wall, however, these levels were not correlated between the studied compartments

Detection of Perturbation Phases and Developmental Stages in Organisms from DNA Microarray Time Series Data

Available DNA microarray time series that record gene expression along the developmental stages of multicellular eukaryotes, or in unicellular organisms subject to external perturbations such as stress and diauxie, are analyzed. By pairwise comparison of the gene expression profiles on the basis of a translation-invariant and scale-invariant distance measure corresponding to least-rectangle regression, it is shown that peaks in the average distance values are noticeable and are localized around specific time points. These points systematically coincide with the transition points between developmental phases or just follow the external perturbations. This approach can thus be used to identify automatically, from microarray time series alone, the presence of external perturbations or the succession of developmental stages in arbitrary cell systems. Moreover, our results show that there is a striking similarity between the gene expression responses to these a priori very different phenomena. In contrast, the cell cycle does not involve a perturbation-like phase, but rather continuous gene expression remodeling. Similar analyses were conducted using three other standard distance measures, showing that the one we introduced was superior. Based on these findings, we set up an adapted clustering method that uses this distance measure and classifies the genes on the basis of their expression profiles within each developmental stage or between perturbation phases

Corrigendum: whole-transcriptome analysis of Verocytotoxigenic Escherichia coli O157:H7 (Sakai) Suggests Plant-Species-Specific Metabolic Responses on Exposure to Spinach and Lettuce Extracts.

Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonisation. As such, we compared the ability of the VTEC strain, E. coli O157:H7 ‘Sakai’, to colonise the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum) and prickly lettuce (L. serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analysed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonisation. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 hour) transcriptional response to extracts as well as longer-term (10 days) plant colonisation or persistence. We show that propagation temperature (37 versus 18 oC) has a major impact on the expression profile and therefore preadaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilisation. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress or translational stalling. A large proportion of differentially regulated genes are uncharacterised (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai