An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

BACKGROUND: The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. RESULTS: Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity. CONCLUSION: hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells

Public Deposits In Biological Resource Centres Is An Essential Part Of Fair Science

F A I R s c i e n c e a i m s a t s h a r i n g s c i e n t i f i c o u t p u t i n s u c h a w a y a s t o m a x i m i z e t h e a c c e s s , r e u s e a n d i m p a c t o f r e s e a r c h . T h i s a l l o w s t r a n s p a r e n c y o f r e s u l t s , r e p r o d u c i b i l i t y o f e x p e r i m e n t s , c u m u l a t i v e r e s e a r c h , a n d a v o i d s a w a s t e o f r e s s o u r c e s ( 1 ) . A l t h o u g h t h e F A I R d a t a p r i n c i p l e i s b e c o m i n g a w e l l - k n o w n c o n c e p t , l e s s a t t e n t i o n i s g i v e n t o i t s a p p l i c a t i o n t o b i o l o g i c a l r e s o u r c e s . I n l i f e s c i e n c e s , p u b l i c m i c r o b i a l - a n d p l a s m i d c o l l e c t i o n s r e p r e s e n t a h i s t o r i c a l e x a m p l e o f F A I R s c i e n c e , t h a n k s t o t h e i r l o n g s t a n d i n g e x p e r i e n c e i n t h e p r e s e r v a t i o n a n d d i s t r i b u t i o n o f l i v i n g m i c r o b i a l s t r a i n s a n d ( g ) D N A f o r f u r t h e r s c i e n t i f i c i n v e s t i g a t i o n s o r d e v e l o p m e n t , w h i l e r e s p e c t i n g ( i n t e r ) n a t i o n a l l e g i s l a t i o n s a n d c a s e - s p e c i f i c r e s t r i c t i o n s a s d e f i n e d b y t h e c l i e n t s . T h e s e b i o l o g i c a l r e s o u r c e c e n t r e s ( B R C ) p r o v i d e w e l l - c h a r a c t e r i z e d , q u a l i t y - c o n t r o l l e d a n d a u t h e n t i c a t e d s t r a i n s , p l a s m i d s a n d a s s o c i a t e d d a t a ( 2 ) . T h e y a l s o s u p p o r t t h e b i o - i n d u s t r y , f o r w h i c h t h e d i v e r s i t y o f n a t u r a l l y o c c u r r i n g o r g e n e t i c a l l y e n g i n e e r e d m i c r o o r g a n i s m s a r e a n i n v a l u a b l e s o u r c e o f a p p l i c a t i o n s . T h e r e s p o n s i b i l i t y t o d e p o s i t t h e m i c r o o r g a n i s m s a n d g e n e t i c r e s o u r c e s i n p u b l i c B R C s i s s h a r e d b y r e s e a r c h e r s , f u n d i n g a g e n c i e s a n d p u b l i s h e r s ( 2 ) . L i f e s c i e n t i s t s n e e d t o b e c o m e m o r e a w a r e o f t h e i m p o r t a n c e o f s t r a i n a n d p l a s m i d c o n s e r v a t i o n a n d g r o w a c c u s t o m e d t o d e p o s i t t h e m d u r i n g t h e p u b l i c a t i o n p r o c e s s o r a t t h e e n d o f p r o j e c t s . G o v e r n m e n t a l f u n d i n g p o l i c i e s s h o u l d r e q u e s t i n t h e i r c o n t r a c t s t h e d e p o s i t o f b i o l o g i c a l m a t e r i a l s i s o l a t e d o r c o n s t r u c t e d d u r i n g f i n a n c e d p r o j e c t s . R e g a r d i n g p u b l i s h e r s , m o s t j o u r n a l s e n c o u r a g e a u t h o r s t o d e p o s i t t h e i r d a t a s e t s ( c o d e s , s e q u e n c e s , e t c ) i n p u b l i c r e p o s i t o r i e s b u t v e r y f e w s p e c i f i c a l l y r e q u i r e d e p o s i t o f b i o l o g i c a l m a t e r i a l a n d c u l t i v a t e d s t r a i n s i n p u b l i c c o l l e c t i o n s . E d i t o r s s h o u l d t h e r e f o r e i m p l e m e n t m e c h a n i s m s f o r a c t i v e a g r e e m e n t b y a u t h o r s t o d e p o s i t s t r a i n s a n d o t h e r g e n e t i c r e s o u r c e s w h e n s u b m i t t i n g a n a r t i c l e , o r t o j u s t i f y w h y i t w o u l d n o t b e p o s s i b l e . S u c h m e c h a n i s m s c o u l d f o l l o w T r a n s p a r e n c y a n d O p e n n e s s P r o m o t i o n g u i d e l i n e s ( 3 ) f o r j o u r n a l s t h a t i n c l u d e s t a n d a r d s f o r research materials.Belgian Consortium of Collections of Microorganism

Image acquisition and quality assurance in the Boston Adolescent Neuroimaging of Depression and Anxiety study

The Connectomes Related to Human Diseases (CRHD) initiative was developed with the Human Connectome Project (HCP) to provide high-resolution, open-access, multi-modal MRI data to better understand the neural correlates of human disease. Here, we present an introduction to a CRHD project, the Boston Adolescent Neuroimaging of Depression and Anxiety (BANDA) study, which is collecting multimodal neuroimaging, clinical, and neuropsychological data from 225 adolescents (ages 14–17), 150 of whom are expected to have a diagnosis of depression and/or anxiety. Our transdiagnostic recruitment approach samples the full spectrum of depressed/anxious symptoms and their comorbidity, consistent with NIMH Research Domain Criteria (RDoC). We focused on an age range that is critical for brain development and for the onset of mental illness. This project sought to harmonize imaging sequences, hardware, and functional tasks with other HCP studies, although some changes were made to canonical HCP methods to accommodate our study population and questions. We present a thorough overview of our imaging sequences, hardware, and scanning protocol. We detail similarities and dif-ferences between this study and other HCP studies. We evaluate structural-, diffusion-, and functional-image-quality measures that may be influenced by clinical factors (e.g., disorder, symptomatology). Signal-to-noise and motion estimates from the first 140 adolescents suggest minimal influence of clinical factors on image quality. We anticipate enrollment of an additional 85 participants, most of whom are expected to have a diagnosis of anxiety and/or depression. Clinical and neuropsychological data from the first 140 participants are currently freely available through the National Institute of Mental Health Data Archive (NDA)

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

The future of medical diagnostics: Review paper

While histopathology of excised tissue remains the gold standard for diagnosis, several new, non-invasive diagnostic techniques are being developed. They rely on physical and biochemical changes that precede and mirror malignant change within tissue. The basic principle involves simple optical techniques of tissue interrogation. Their accuracy, expressed as sensitivity and specificity, are reported in a number of studies suggests that they have a potential for cost effective, real-time, in situ diagnosis. We review the Third Scientific Meeting of the Head and Neck Optical Diagnostics Society held in Congress Innsbruck, Innsbruck, Austria on the 11th May 2011. For the first time the HNODS Annual Scientific Meeting was held in association with the International Photodynamic Association (IPA) and the European Platform for Photodynamic Medicine (EPPM). The aim was to enhance the interdisciplinary aspects of optical diagnostics and other photodynamic applications. The meeting included 2 sections: oral communication sessions running in parallel to the IPA programme and poster presentation sessions combined with the IPA and EPPM posters sessions. © 2011 Jerjes et al; licensee BioMed Central Ltd

Genome-wide association analysis of genetic generalized epilepsies implicates susceptibility loci at 1q43, 2p16.1, 2q22.3 and 17q21.32

Genetic generalized epilepsies (GGEs) have a lifetime prevalence of 0.3% and account for 20-30% of all epilepsies. Despite their high heritability of 80%, the genetic factors predisposing to GGEs remain elusive. To identify susceptibility variants shared across common GGE syndromes, we carried out a two-stage genome-wide association study (GWAS) including 3020 patients with GGEs and 3954 controls of European ancestry. To dissect out syndrome-related variants, we also explored two distinct GGE subgroups comprising 1434 patients with genetic absence epilepsies (GAEs) and 1134 patients with juvenile myoclonic epilepsy (JME). Joint Stage-1 and 2 analyses revealed genome-wide significant associations for GGEs at 2p16.1 (rs13026414, Pmeta = 2.5 × 10−9, OR[T] = 0.81) and 17q21.32 (rs72823592, Pmeta = 9.3 × 10−9, OR[A] = 0.77). The search for syndrome-related susceptibility alleles identified significant associations for GAEs at 2q22.3 (rs10496964, Pmeta = 9.1 × 10−9, OR[T] = 0.68) and at 1q43 for JME (rs12059546, Pmeta = 4.1 × 10−8, OR[G] = 1.42). Suggestive evidence for an association with GGEs was found in the region 2q24.3 (rs11890028, Pmeta = 4.0 × 10−6) nearby the SCN1A gene, which is currently the gene with the largest number of known epilepsy-related mutations. The associated regions harbor high-ranking candidate genes: CHRM3 at 1q43, VRK2 at 2p16.1, ZEB2 at 2q22.3, SCN1A at 2q24.3 and PNPO at 17q21.32. Further replication efforts are necessary to elucidate whether these positional candidate genes contribute to the heritability of the common GGE syndrome

Soft windowing application to improve analysis of high-throughput phenotyping data.

MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce \u27soft windowing\u27, a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease