1,720,980 research outputs found
Development of three-dimensional models for the study of the hematopoietic stem cell niche
No full textLa maggior parte delle informazioni sulle principali proprietà delle cellule staminali ematopoietiche (HSCs), quali la capacità di auto-mantentenimento e di differenziazione multilineare, sono state acquisite mediante l’impiego dei tradizionali sistemi di coltura cellulare bidimensionale in vitro o dei modelli animali. Attraverso tali approcci sono stati raggiunti importanti avanzamenti nella conoscenza dei meccanismi che regolano lo sviluppo ed il mantenimento dell’emopoiesi. Nonostante ciò, nessuno dei sistemi di coltura a tutt’oggi descritti è in grado di mantenere in vitro l’emopoiesi a lungo termine, mentre i modelli animali non sempre riproducono adeguatamente le caratteristiche delle patologie onco-ematologiche umane. Recentemente sono stati sperimentati diversi sistemi artificiali tridimensionali (3D) per espandere i progenitori ematopoietici e per ottenere la differenziazione multilineare.
Lo scopo principale del nostro studio ha consistito nello sviluppo di un nuovo modello 3D che, mediante l’impiego di materiali naturali, permettesse di riprodurre in vitro il più fedelmente possibile il microambiente midollare e la struttura delle nicchie che regolano il mantenimento delle HSCs. A tal fine sono stati impiegati come supporto la matrice ossea inorganica e le cellule staminali mesenchimali (MSCs) di derivazione umana per ricostituire in 3D il microambiente stromale su cui effettuare la cultura delle CD34+ HSCs. Sono state analizzate tre diverse condizioni di differenziamento volte ad ottenere i principali elementi cellulari del midollo osseo e della nicchia staminale: osteoblasti, adipociti e cellule endoteliali. Dopo due settimane di coltura delle CD34+ HSCs nei sistemi 3D ottenuti con le tre diverse condizioni di differenziamento, sono stati effettuati saggi immunoistochimici volti ad identificare l’attecchimento e lo sviluppo/differenziazione di cloni di cellule ematopoietiche e saggi clonogenici (CFC- ed LTC-IC) per la valutazione del mantenimento di precursori immaturi. Tra i diversi stimoli differenziativi considerati, l’esposizione delle MSCs cresciute sui supporti ossei alle condizioni angiogeniche in presenza di placental derived growth factor (PlGF) ha reso possibile la riproduzione di alcune delle proprietà che rendono il microambiente stromale capace di sostenere l’emopoiesi. In base ai risultati dell’analisi immunoistochimica, le CD34+ HSCs cresciute in questi sistemi sono in grado di differenziarsi in elementi mieloidi CD45+/CD33+ e di mantenere una parte di esse nello stato non differenziato, come ulteriormente
mostrato dai saggi CFC e LTC-IC. L’analisi di espressione genica, condotta sugli mRNA estratti dai campioni ottenuti con lo stimolo angiogenico (PlGF) e bloccati al momento della semina delle CD34+ HSCs, ha escluso nelle MSCs il differenziamento in senso endoteliale e miogenico. Piuttosto, l’incremento nell’espressione di alcuni dei marcatori delle fasi precoci di differenziamento osteoblastico e dell’angiopoietina 1 (ANGPT-1), una delle molecole chiave coinvolte nel mantenimento della nicchia staminale midollare, suggerisce l’acquisizione di un fenotipo e funzionalità propri di quei progenitori osteoblastici che supportano le HSCs.
In conclusione, il modello 3D proposto in questo lavoro, basato sull’impiego delle MSCs e di un supporto naturale quale l’osso trabecolare, è in grado di sostenere la sopravvivenza e differenziazione delle HSCs attraverso una probabile creazione della nicchia osteoblastica. Sebbene siano necessari un ulteriore perfezionamento e standardizzazione e una caratterizzazione più ampia dei meccanismi che sostengono il mantenimento, l’espansione e la differenziazione delle HSCs, questo nuovo strumento può rappresentare un nuovo approccio per la riproduzione ex-vivo della nicchia midollare e per lo studio dell’ematopoiesi midollare in condizioni fisiologiche e patologiche.Most information about hematopoietic stem cell (HSC) self-renewal and multilineage commitment derives from standard bi-dimensional (2D) cell cultures or animal model systems. Through these approaches many important improvements in the understanding of the haematopoietic development have been achieved. However, none of the in vitro systems is capable of sustaining the haematopoiesis for a long time, whereas animal models may not adequately reproduce the features of human pathological conditions. Recently, several experiments using three-dimensional (3D) artificial systems have been carried out to expand haematopoietic progenitors and to achieve multilineage haematopoietic differentiation.
The main aim of our study was to develop a new in vitro three-dimensional (3D) model of BM by employing natural components, to reproduce as close as possible BM microenvironment and the structure of HSC niches. To this aim, we employed human bone mineral matrix as scaffold and mesenchymal stem cells (MSCs) to reproduce the stromal microenvironment. Three different culture conditions were compared, aimed to achieve osteoblast, adipocyte and endothelial differentiation, respectively. After 2 weeks of CD34+ cell culture in the 3D system, immunohistochemistry as well as CFC and LTC-IC assays were carried out to investigate the system ability to maintain and differentiate HSCs.
The exposure of MSCs to angiogenic stimuli in the presence of placental derived growth factor (PlGF) could reproduce, at least in part, the properties that make the microenvironment capable of supporting haematopoiesis. CD34+ cells grown in this 3D system differentiated into myeloid cells (CD45+/CD33+), but some of them remained in an undifferentiated status, as shown by the presence of CFC and LTC-IC after the culture. Immunohistochemical and gene expression analysis, carried out on the 3D system upon the angiogenic conditions before CD34+ cell culture, excluded the acquisition by MSCs of the endothelial-like phenotype, as well as the vascular smooth muscle cell differentiation. Instead, quite unexpectedly, the up-regulation of some of the early osteoblastic markers, together with angiopoietin 1 (ANGPT-1), suggested that the angiogenic conditions could have induced the differentiation of MSCs into HSC-supporting osteoblastic progenitors.
In conclusion, our findings suggest that this new 3D model, based on the employment of MSCs and a natural scaffold of trabecular bone, is capable of inducing survival and differentiation of HSCs, probably through the creation of a osteoblastic-like HSC niche. Although further improvements, standardization processes, and mechanism evaluation are needed, this new tool may be useful to reproduce in vitro the conditions for HSC maintenance, expansion and differentiation, thus permitting to study the mechanisms of BM haematopoiesis in normal conditions and in haematological diseases
Linkage analysis for prenatal diagnosis in a familial case of Stickler syndrome
No full textThe Stickler syndrome is among the most common heritable disorders of connective tissue. The syndrome fully expressed clinical phenotype includes the degeneration of the vitreous gel and retina, frequently associated with myopia, accompanied by non-ocular features, such as craniofacial dysmorphisms or malformations, hearing impairment, skeletal dysplasia and progressive arthropathy. So far, mutations at three collagen loci, COL2A1, COL11A1 and COL11A2, have been found in Stickler syndrome patients, with about two thirds of investigated familial cases found to be associated to COL2A1 gene mutations. We report on a three generation family in which a diagnosis of Stickler syndrome was made and linkage analysis suggested COL2A1 to be the causing gene. These data permitted us to perform two prenatal diagnosis analysing the 3'VNTR polymorphism of the involved gene on amniocytes' DNA and to provide the family with genetic counselling and paediatric support at the delivery
Four new cases of lethal Osteogenesis Imperfecta due to glycine substitutions in the COL1A1 and COL1A2 genes
No full textFour new cases of lethal osteogenesis imperfecta due to glycine substitutions in COL1A1 and genes. Mutations in brief no. 152.
No full textGoing Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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