3D gene knockout tissue models using adult human stem cells

Aims

This project will model the development of skeletal tissue by using state of the art gene targeting techniques to study the ability of mesenchymal stem cells to form 3D micro-skeletons in culture.

Background

A common approach to determining gene function is to knockout specific genes in animal models, usually mice, and to examine the effects. However, producing knockout mice is inefficient and requires large numbers of animals. Results do not always translate satisfactorily to human physiology. Developing in vitro alternatives is challenging and this research will test a new approach which combines stem cell biology, complex 3D tissue models and gene targeting to provide a system for investigating gene function that avoids the use of mice. 

Research details and methods

The research will use immortalised mesenchymal stem cells from human bone marrow. The cells can form 3D micro-skeletons composed of bone and cartilage. Sophisticated gene targeting methods will be used to knockout genes in the stem cells and the impact on the ability to develop micro-skeletons will be investigated.  

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 use 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.

Carstairs A and Genever P (2014). Stem cell treatment for musculoskeletal disease.Curr Opin Pharmacol. 16: 1-6 doi:10.1016/j.coph.2014.01.005

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PhD Studentship

Status:

Closed

Principal investigator

Professor Paul Genever

Institution

University of York

Grant reference number

NC/K001671/1

Award date:

Oct 2013 - Sep 2016

Grant amount

£90,000