Skip to main content

Generation of human disease models by homologous recombination in human embryonic stem cells

Final Activity Report Summary - HOMOL. RECOMB. HESC (Generation of human disease models by homologous recombination in human embryonic stem cells)

A new laboratory specific for stem cell culture has been set up in the research unit of the Hospital de Cruces. This cell culture facility is physically separated from the general tissue culture room where untested biopsy material is manipulated. The stem cell culture room is cleaned daily and disinfected once a week. To work in this "clean" room, lab coats and shoe covers are worn.

Using this facility we have implemented the thawing, culture and propagation of human embryonic stem cells (hESCs), using mouse embryonic fibroblasts as feeders. The production of a stock of hESC vials at low passages has been achieved; these vials provide a sufficient source of hESC for this project. Optimisation of the differentiation into hESC-cardiomyocytes has been completed. And genetic manipulation has been successfully optimised and will be used to introduce a point mutation associated with dilated cardiomyopathy into the genomic sequence of the LMNA gene.

These goals focus on the generation of an experimental model of a human disorder associated with mutations in the LMNA gene, specifically dilated cardiomiopathy. hESCs are regarded as the most promising tool for cellular/tissue repair because their indefinite replicative capacity makes them a renewable cell source and highly suitable for culturing in large quantities. Moreover the possibility of modelling human disease where the animal model has proven inadequate would open several areas of research to study etiologies and pathologies of these difficult disorders. hESCs are also an excellent tool for discovering and testing new drugs that could cure or delay the development of these diseases.

We also intend to study the effects of protease inhibitor treatments similar to those given to HIV patients on stem cells and which alter the maturation process of LMNA gene. Preliminary results suggest that human stem cells subjected to these protease inhibitor treatments show alterations in proliferation rates, morphology, mortality and chromatin organisation. Given the similar effects of lypodystrophic LMNA mutations on these cell characteristics, these stem cell models could provide an important tool in analysing the effects of protease inhibitor treatments, as well as the effects of potential ameliorative drug treatments for HIV patients.