Genomic integrity of human pluripotent stem cells
Nathalie Lefort : Research Engineer (INSERM)
Christine Varela : Qualified research technician (CECS)
Christine Varela : Qualified research technician (CECS)
Aims and background :
Human pluripotent stem cells have the ability to divide endlessly while maintaining their capacity to differentiate into all cell types of the organism. Due to these original properties human pluripotent stem cells and their progenies are highly valuable not only for regenerative medicine but also as tools to study development and pathologies.
Furthermore they are relevant candidate as cellular substrates to screen and test new drugs. However, ensuring their genomic integrity is one important prerequisite for both research and therapeutic applications. Indeed, in vivo the rate of spontaneous mutations in normal human cells is about 10-7 to 10-8 per nucleotide per cell division. In humans there are approximately 3x109 nucleotides per haploid genome which is about 100 mutations per cell at each cell cycle. As any other cell lines growing in culture, long-term culture of human pluripotent stem cells is inevitably subject to evolution, since any mutant that arises with a growth advantage will be selectively amplified. Selection of cells that have acquired proliferative advantage due to chromosomal alteration is a permanent worry when growing pluripotent stem cell lines. Since 2004, it has been well established that aneuploidies as well as more restricted abnormalities occur non-randomly in cultured human embryonic stem cells upon prolonged passaging in culture. The main objectives of the group are:
- The genomic integrity control of human pluripotent stem cells and their progenies for all the teams of the laboratory,
- The characterization of cells carrying recurrent chromosomal abnormalities. Strategy, means and methods :
The “Genomic Integrity” group uses various methods to assess karyotypic integrity of cells which differ both in their sensitivity and resolution. Conventional G-banding karyotypes and multiplex Fluorescent In Situ Hybridization (mFISH) methods allow the identification of abnormal chromosomes number (aneuploidy, polyploidy) and structural chromosomes changes such as translocations and gains or losses of large part of chromosomes. Consequently, conventional and virtual karyotyping technologies are complementary approaches which provide us meaningful information when combined.
These methods rely on the analysis of cells blocked in metaphase and then have an average resolution limited to 5 to 10 Mb depending on the location of the region of interest in the genome.
Virtual karyotyping such as array-based Comparative Genomic Hybridization (array-CGH) or Single Nucleotide Polymorphism (SNP) array relies on the analysis of genomic DNA. They have a much higher resolution ranging from less than 1Mb to less than 100kb but a lower sensitivity. 
(A) G-banding showing a normal female karyotype (B) mFISH (Metasystems’probes) showing a normal male karyotype
Results and future prospects :
Regular control of the genomic integrity of human embryonic stem cells has led us to the identification of a recurrent chromosomal amplification at 20q11.21. This region that contains about 23 genes and one microRNA is also amplified in numerous of cancers. We now wish to carry on this work by (1) the phenotypic characterization of cells carrying a duplication of the 20q11.21 region, (2) the high throughput DNA sequencing of the region near 20q11.21 in order to determine the nucleotide sequence of the breakpoint and identify sequence motifs localized around the breakpoint that could explain the instability, (3) cells behaviour when grafted into adult rat striatum.
(C) Array-CGH showing an amplification at 20q11.21 region (right panel) compared to array-CGH (IntegraChip genome wide BAC array) showing a normal chromosome 20 (left panel)
Publications :
Lefort N et al., Human embryonic stem cells and genomic instability. Regen Med. 2009 Nov;4(6):899-909. Review
Lefort N et al., A recurrent hotspot of genomic instability identified in human ES cells. Med Sci. 2009 Jan;25(1):99-101.
Lefort N et al., Human embryonic stem cells reveal recurrent genomic instability at 20q11.21 in the human. Nat Biotechnol. 2008 ; 26:1364-6.
Lefort N et al., A recurrent hotspot of genomic instability identified in human ES cells. Med Sci. 2009 Jan;25(1):99-101.
Lefort N et al., Human embryonic stem cells reveal recurrent genomic instability at 20q11.21 in the human. Nat Biotechnol. 2008 ; 26:1364-6.
Collaboration :
Dr. Gabor Gyapay and Dr. Eric Pelletier, Génoscope, Evry, France, (high throughput DNA sequencing)
