Studi di knockout genico usando modelli 3D di cellule staminali in vitro

[Genever PG. The generation of three-dimensional tissue structures with mesenchymal stem cells. Altern Lab Anim. 2010 Dec;38 Suppl 1:31-4.]

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Mesenchymal stem cells (MSCs) are multipotent stem cells, found in the bone-marrow and other adult tissues, which give rise to various cell lineages. Although MSCs are biologically important, and may have widespread therapeutic potential, they are not well-characterised, particularly in terms of their cell surface receptors and in vivo phenotype. We aimed to develop a three-dimensional (3-D) MSC in vitro model, in order to understand the factors involved in the regulation of lineage specification routes. A suitable model, which replicates the MSC microenvironment as accurately as possible, will allow more detailed investigations into the phenotype of the cells. Our MSC spheroids appear to have an enhanced mesenchymal differentiation compared to two-dimensional MSC monolayers. With this in vitro system, it is possible to perform real-time analysis of cellular differentiation status. MSC spheroids may also be amenable for use in high-throughput assays. A more-recent research project aims to generate knockout micro-tissues, based on human 3-D MSCs, as an alternative to animal studies.


Da NC3Rs ( 

The tissues in our bodies are three-dimensional (3D) arrays of different cell types, whose behaviour is largely determined by the genes they express. Understanding the function of individual genes of cells within tissues is essential if we are to know more about human health and disease, and the development of new and better treatments. A common experiment to determine gene function is to “knockout” specific genes in animal models, usually mice, and examine the effects. However, producing knockout mice is difficult, inefficient, requires large numbers of animals and results may not translate satisfactorily to human physiology. Adult human stem cells can now be grown in the laboratory and induced to generate different tissue types. We have isolated so-called mesenchymal stem cells (MSCs) from human bone marrow and generated an immortal MSC “line” that appears to live forever when grown in a culture dish. This provides us with an opportunity to perform long-term genetic manipulations, which would not normally be possible in standard short-lived cells. We have also developed new techniques where MSCs in specialised Petri dishes can be coaxed into forming 3D tissues containing both bone and cartilage, in a manner similar to how our skeletons form. In this project, we will use sophisticated gene targeting methods to knockout particular genes in the MSCs that are normally involved in the development of healthy and diseased skeletons. We will then find out how the gene knockouts affect the ability of the MSCs to form 3D microskeletons in the laboratory. This system will using human stem cells, capable of complex tissue formation, in a system that is highly controllable and recognises the 3D nature of true tissues. If successful, this work will have far-reaching implications for cost and efficiency of biological research, our understanding of gene function and will contribute significantly to the replacement of mouse knockout experiments.



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