Exploiting natural and induced genetic variation to study hematopoiesis

PUZZLING WITH DNA Blood cell formation can be studied by making use of natural genetic variation across mouse strains. There are, for example, two mouse strains that do not only differ in fur color, but also in average life span and more specifically in the number of blood-forming stem cells in their bone marrow. The cause of these differences can be found in the DNA of these mice. This DNA differs slightly between the two mouse strains, making some genes in one strain just a bit more or less active compared to those same genes in the other strain. The aim of part I of this thesis was to study the influence of genetic variation on gene expression and how this might explain the specific characteristics of the mouse strains. One of the findings in this study was that the influence of genetic variation on gene expression is strongly cell-type-dependent. Additionally, blood cell formation can be studied by introducing genetic variation into the system. In part II of this thesis genetic variation was introduced into mouse blood-forming stem cells by letting random DNA sequences or “barcodes” integrate into the DNA of these cells. Thereby, these cells were provided with a unique and identifiable label that was heritable from mother- to daughter cell. In this manner the fate of blood-forming stem cells and their progeny could be tracked following transplantation in mice. This technique is very promising for monitoring blood cell formation in future clinical gene therapy studies in humans. PUZZELEN MET DNA Bloedvorming kan bestudeerd worden door gebruik te maken van natuurlijke genetische variatie tussen muizenstammen. Zo bestaan er bijvoorbeeld twee muizenstammen die niet alleen verschillen in vachtkleur, maar ook in gemiddelde levensduur en meer specifiek in het aantal bloedvormende stamcellen dat zich in hun beenmerg bevindt. De oorzaak van deze verschillen kan gevonden worden in het DNA van deze muizen. Dat DNA verschilt net iets tussen de twee muizenstammen, waardoor sommige genen in de ene stam actiever of juist minder actief zijn dan diezelfde genen in de andere stam. In deel I van dit proefschrift is onderzocht hoe genetische variatie de expressie van genen beïnvloedt en hoe dit de specifieke eigenschappen van de muizenstammen zou kunnen verklaren. Er is onder andere gevonden dat de invloed van genetische variatie op de expressie van genen sterk celtype-afhankelijk is. Daarnaast kan bloedvorming bestudeerd worden door genetische variatie te introduceren in het systeem. In deel II van dit proefschrift is genetische variatie in bloedvormende stamcellen van muizen geïntroduceerd door random DNA volgordes of “barcodes” te laten integreren in het DNA van deze cellen. Dit resulteert erin dat elke cel voorzien wordt van een uniek label dat overgegeven wordt van moeder- op dochtercel. De DNA volgorde van het label kan gelezen worden met behulp van een zogenaamde sequencing techniek. Op deze manier kan het lot van bloedvormende stamcellen en hun nakomelingen gevolgd worden na transplantatie in muizen. Deze techniek is zeer veelbelovend voor het monitoren van bloedvorming in toekomstige klinische gentherapie studies in de mens.

Expression quantitative trait loci are highly sensitive to cellular differentiation state

Blood cell development from multipotent hematopoietic stem cells to specialized blood cells is accompanied by drastic changes in gene expression for which the triggers remain mostly unknown. Genetical genomics is an approach linking natural genetic variation to gene expression variation, thereby allowing the identification of genomic loci containing gene expression modulators (eQTLs). In this paper, we used a genetical genomics approach to analyze gene expression across four developmentally close blood cell types collected from a large number of genetically different but related mouse strains. We found that, while a significant number of eQTLs (365) had a consistent “static” regulatory effect on gene expression, an even larger number were found to be very sensitive to cell stage. As many as 1,283 eQTLs exhibited a “dynamic” behavior across cell types. By looking more closely at these dynamic eQTLs, we show that the sensitivity of eQTLs to cell stage is largely associated with gene expression changes in target genes. These results stress the importance of studying gene expression variation in well-defined cell populations. Only such studies will be able to reveal the important differences in gene regulation between different ce

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Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a lowresolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important. (Blood. 2010;115(13):2610-2618

Genetical genomics: spotlight on QTL hotspots

No abstract available

Combining transcriptional profiling and genetic linkage analysis to uncover gene networks operating in hematopoietic stem cells and their progeny

Stem cells are unique in that they possess both the capacity to self-renew and thereby maintain their original pool as well as the capacity to differentiate into mature cells. In the past number of years, transcriptional profiling of enriched stem cell populations has been extensively performed in an attempt to identify a universal stem cell gene expression signature. While stem-cell-specific transcripts were identified in each case, this approach has thus far been insufficient to identify a universal group of core “stemness” genes ultimately responsible for self-renewal and multipotency. Similarly, in the hematopoietic system, comparisons of transcriptional profiles between different hematopoietic cell stages have had limited success in revealing core genes ultimately responsible for the initiation of differentiation and lineage specification. Here, we propose that the combined use of transcriptional profiling and genetic linkage analysis, an approach called “genetical genomics”, can be a valuable tool to assist in the identification of genes and gene networks that specify “stemness” and cell fate decisions. We review past studies of hematopoietic cells that utilized transcriptional profiling and/or genetic linkage analysis, and discuss several potential future applications of genetical genomics

Epigenetic control of hematopoietic stem cell aging - The case of Ezh2

Hematopoietic stem cells have potent, but not unlimited, selfrenewal potential. The mechanisms that restrict selfrenewal are likely to play a role during aging. Recent data suggest that the regulation of histone modifications by Polycomb group genes may be of crucial relevance to balance selfrenewal and aging. We provide evidence for the involvement of one of these Polycomb group genes, Ezh2, in aging of the hematopoietic stem cell system.</p

Modern genome-wide genetic approaches to reveal intrinsic properties of stem cells

Purpose of review The clinical use of hematopoietic stem cells, which produce all mature blood cell lineages in the circulation, is continuously increasing. Identification of genes and gene networks specifying either sternness or commitment will not only be of major relevance for a fundamental understanding of developmental biology, but also for the emerging fields of tissue engineering and regenerative medicine. Our appreciation of the transcriptional machinery that distinguishes stem cells from their nonstem cell progeny is, however, rudimentary. State-of-the art genome-wide tools are now becoming available to elucidate intrinsic properties of stem cells. Here, we review recent progress that has been made in this field. Recent findings Approaches to study stem cell-specific genes and gene networks include genetical genomics, mRNA and microRNA expression profiling of carefully selected cells, proteomics, chromatin studies using 'CHIP-on-chip' tools, genome-wide binding site analyses for transcription factors and chromatin-remodeling proteins, and tools to study the three-dimensional organization of gene loci. It is promising to see that the combined application of these tools has resulted in the identification of multiple novel genes that regulate stem cell self-renewal. Summary Exploitation of the available technology and integrating the data by translation into a dynamic model of networks, operating in all four dimensions, will be essential to fully comprehend the elusive concept of 'stemness'. It is time to harvest