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

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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

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

Cellular barcoding tool for clonal analysis in the hematopoietic system

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 low-resolution, 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

Genetic screen identifies microRNA cluster 99b/let-7e/125a as a regulator of primitive hematopoietic cells

Hematopoietic stem/progenitor cell (HSPC) traits differ between genetically distinct mouse strains. For example, DBA/2 mice have a higher HSPC frequency compared with C57BL/6 mice. We performed a genetic screen for microRNAs that are differentially expressed between LSK, LS(-)K(+), erythroid and myeloid cells isolated from C57BL/6 and DBA/2 mice. This analysis identified 131 micro-RNAs that were differentially expressed between cell types and 15 that were differentially expressed between mouse strains. Of special interest was an evolutionary conserved miR cluster located on chromosome 17 consisting of miR-99b, let-7e, and miR-125a. All cluster members were most highly expressed in LSKs and down-regulated upon differentiation. In addition, these microRNAs were higher expressed in DBA/2 cells compared with C57BL/6 cells, and thus correlated with HSPC frequency. To functionally characterize these microRNAs, we overexpressed the entire miR-cluster 99b/let-7e/125a and miR-125a alone in BM cells from C57BL/6 mice. Overexpression of the miR-cluster or miR-125a dramatically increased day-35 CAFC activity and caused severe hematopoietic phenotypes upon transplantation. We showed that a single member of the miR-cluster, namely miR-125a, is responsible for the majority of the observed miR-cluster overexpression effects. Finally, we performed genome-wide gene expression arrays and identified candidate target genes through which miR-125a may modulate HSPC fate. (Blood. 2012;119(2):377-387