Stem cell genetics

Background

Embryonic stem cells (ES) are a useful research tool because they can multiply indefinitely while maintaining their ability to develop into any type of cell found in the body. Furthermore, their genomes can be modified in a targeted way using homologous recombination. For the past three decades mouse ES cells have been successfully used to study loss-of-function effects in vivo as well as in vitro. However, the diploid nature of their genome means that both sets of each gene must be inactivated for successful phenotype-driven forward genetic screens, which has hampered research efforts.

Human ES cells are a relatively newer cell type with great research potential, but technical difficulties have hindered their use. These cells were first reported in 1998 and have considerably different properties from mouse ES cells, although both cell types are established from pre-implantation embryos. Compared with mouse ES cells, human ES cells are morphologically different, more prone to cell death upon single cell dissociation, and use a different signalling pathway to maintain their pluripotency. Moreover, homologous recombination is less efficient in human ES cells than in mouse ES cells.

However, the new technique of directly reprogramming of human somatic cells into human induced pluripotent stem (iPS) cells overcomes many the problems encountered with human ES cells. This approach is opening the door to cell-based genetic therapies, highlighting the need to develop robust and efficient genetic engineering techniques in these iPS cells.

Research

Our aims

The group's main focus is to develop novel genetic engineering technologies in human and mouse pluripotent stem cells and to apply them to genetic screening and new healthcare therapies.

Recessive genetic screening in mouse ES cells

We developed an efficient method to conduct recessive genetic screens in mouse ES cells by using the hyper-recombination phenotype of Bloom helicase-deficiency to produce genome-wide homozygous mutations. Using this technique, we identified novel factors involved in the toxification of Aeromonas toxin, aerolysin.

We are now expanding our recessive genetic screens by using newly isolated mouse haploid ES cells. These cells contain only one set of the genome and therefore can be altered by a 'single hit'. Because of this, these ES cells are a genetic tool of unprecedented potential for gene function studies of mammalian biological systems.

High-throughput sequencing will enable us able to identify mutations introduced into mouse haploid ES cells, allowing us to conduct a wide range of genetic screens: from finding the genes required for ES cell differentiation to looking for those involved in viral replication.

The piggyBac transposon system

piggyBac is a moth-derived DNA transposon (jumping gene) and is one of the most active transposon systems in mammalian cells. Transposons have been used in a wide range of organisms for genetic studies - especially random mutagenesis. The transposon sequence can be used as a molecular tag in the DNA and, therefore, we can easily identify mutated genes by searching for transposon integration sites.

In addition, the piggyBac transposon has a unique property that offers us a powerful way to reprogramme iPS cells in a manner that could be used in treatments. piggyBac has traceless excision, which enables removal of the transposon from the genome without altering the cells' DNA sequences. Using this important property, we have established a transposon-based, factor-free, method for iPS cell reprogramming. The iPS cells established by this means are clean from potentially mutagenic changes.

We are also developing more active transposon systems, to expand the usefulness of this approach in mammalian genetic research and gene therapy. In collaboration with Professor Nancy Craig at Johns Hopkins University, we have developed a hyperactive piggyBac transposase, which can increase transposition efficiency by one order of magnitude.

Genetic correction of disease-causing mutations in patient-derived iPS cells

Human iPS cells hold great promise for autologous cell-based therapy in a broad range of disorders. However, creating human iPS cells from a person with an inherited disease to treat their condition requires not only the correction of the disease-causing mutation, but also for it to be done cleanly to avoid other DNA changes or unwanted side-effects.

Until recently, cleanly editing the genome of human iPS cells has been particularly challenging because of the lack of necessary technologies. But now we have developed a technique that combines using zinc-finger nuclease (ZFN) with the hyperactive piggyBac transposon system to achieve efficient and precise genome editing. Using this approach, we have successfully performed genetic correction of a major inherited metabolic disease - alpha-1 antitrypsin deficiency - in human iPS cells.

To check that unwanted mutations were not introduced during the 'genetic surgery', we conducted high-resolution analysis of the corrected genome using array-based comparative genomic hybridization, single nucleotide polymorphism array and exome-sequencing. This revealed that genomic integrity of iPS cells is maintained throughout genetic correction.

This approach is now even more accessible with novel nuclease technologies such as TALE nuclease and the Cas9 systems. Patient-derived iPS cells and their disease-corrected counterparts offer the opportunity to carry out the best controlled in vitro modelling of diseases. We will investigate the potential of genome editing in human iPS cells to develop new ways to investigate the molecular mechanisms underlying disease as well as create new cell-based therapies.

Collaborations

On campus, we work closely with the following Sanger Institute faculty members:

• Ludovic Vallier
• Allan Bradley

Selected Publications

• Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

  Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordóñez A, Hannan NR, Rouhani FJ, Darche S, Alexander G, Marciniak SJ, Fusaki N, Hasegawa M, Holmes MC, Di Santo JP, Lomas DA, Bradley A and Vallier L

  Nature 2011;478;7369;391-4

  PUBMED: 21993621; PMC: 3198846; DOI: 10.1038/nature10424

• A hyperactive piggyBac transposase for mammalian applications.

  Yusa K, Zhou L, Li MA, Bradley A and Craig NL

  Proceedings of the National Academy of Sciences of the United States of America 2011;108;4;1531-6

  PUBMED: 21205896; PMC: 3029773; DOI: 10.1073/pnas.1008322108

• Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.

  Yusa K, Rad R, Takeda J and Bradley A

  Nature methods 2009;6;5;363-9

  PUBMED: 19337237; PMC: 2677165; DOI: 10.1038/nmeth.1323

• Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene.

  Yusa K, Horie K, Kondoh G, Kouno M, Maeda Y, Kinoshita T and Takeda J

  Nature 2004;429;6994;896-9

  PUBMED: 15215867; DOI: 10.1038/nature02646