Reprogramming nuclei from adult cells

Lead Research Organisation: University of Oxford
Department Name: Zoology

Abstract

Reprogramming nuclei from adult cells.The challenge is to convert a patient’s own cells for use in transplantation therapy so that the graft and the host are compatible. In effect, the patient donates cells whose path of development can be changed to repair the patient’s own damaged tissues. At present, transplantation of solid tissues and organs between humans is limited by genetic mismatch between the donor tissue and the patient. In most cases, the patient has to be treated continuously with drugs that suppress the natural mechanism for rejecting foreign tissue. This immune suppression has side effects, and the patients tend to be susceptible to infection. The challenge can be met by refining a range of new cell microsurgery techniques. In the first instance, we hope to develop a general method for reprogramming nuclei from adult mice so that they code for the growth of cells that can be used to repair damaged muscle. Mice carrying particular transgenic markers have been bred to obtain suitable cells for the work because these cells express colours when reprogramming has worked.

Technical Summary

The challenge is to convert a patient?s own cells for use in transplantation therapy so that the graft and the host are histocompatible. This challenge can be met by refining a range of new cell microsurgery techniques and testing their efficacy in mice. We propose to make heterokaryons between non-muscle cells from adult mice and multinucleate myoblasts cells and myotubes: it is known that in such heterokaryons a range of ?muscle? genes are expressed from the non-muscle nucleus. Our plan is to isolate this reprogrammed nucleus in a cell with all the characteristics of a myoblast.

The 3 year deliverable is a general method for reprogramming nuclei from adult mice so that they code for the growth of cells that can be used to repair damaged muscle. We also plan to extend these techniques to neural stem cells during the second and third year of the grant. This programme of work combines the microsurgery expertise in the Zoology department with the current programmes of muscle and nerve stem cell research in the department of Anatomy & Genetics.

The novel cell manipulation techniques that must be refined during the course of the project are:
Electrofusion of selected and identified cells to make heterokaryons between single adult cells and multinucleate myoblasts/myotubes or multinucleate neural stem cells, and
Microsurgery to isolate the reprogrammed nucleus.

In principle these technique could be extended to any stem cell type. However this optimistic view should be balanced against the known problems of maintaining the other stem cell states in culture: in other systems, these states must be sufficiently robust to survive cell abuse by fusion and microsurgery. Our preliminary work and the results of others have demonstrated that both C2C12 myoblasts and their derived myotubes have these properties. Similar heterokaryons with multinucleate neural stem cells will begin the assessment of the general applicability of these techniques.
 
Title Micromanipulation equipment and methods 
Description Protocols for making giant myoblasts and subsequently fusing each homokaryon with single fibroblast. The main outcome was the development of containers for time-lapse video recording. This was required for the protocols above but it was not specific to those protocols. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2007 
Provided To Others? Yes  
Impact One of the methods has been used to keep mouse zygotes in place during video recording. There has been no interest by other groups in using the methods. 
 
Description Fabrication of microchambers 
Organisation University of Oxford
Department Department of Engineering Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Design of microchambers
Collaborator Contribution Microchambers were fabricated in collaboration with Professor Zheng Cui. In these chambers the giant homokaryons and the donor cells should have been kept apart but nuclei from each partner were able to either negotiate the narrow passage or to emerge from climb out from their compartment. Modification of these chambers might lead to the success of the main experiment. Prof Zheng Cui put me in touch with a microfabrication company on the Harwell campus and they made the most recent templates that I used. I continue in contact with Prof Cui becasue we are both memebers of the Oxford Stem Cell Institute ( a virtual Institute that sponsors reqular seminars).
Impact Multidisciplinary, involving engineering expertise. Resulted in microchambers tested in experiments.
Start Year 2007