For personal use only

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

Asymmetry in skeletal distribution of mouse hematopoietic stem cell clones and their equilibration by mobilizing cytokines

Hematopoietic stem cells (HSCs) are able to migrate through the blood stream and engraft bone marrow (BM) niches. These features are key factors for successful stem cell transplantations that are used in cancer patients and in gene therapy protocols. It is unknown to what extent transplanted HSCs distribute throughout different anatomical niches in the BM and whether this changes with age. Here we determine the degree of hematopoietic migration at a clonal level by transplanting individual young and aged mouse HSCs labeled with barcoded viral vector, followed by assessing the skeletal distribution of hundreds of HSC clones. We detected highly skewed representation of individual clones in different bones at least 11 mo after transplantation. Importantly, a single challenge with the clinically relevant mobilizing agent granulocyte colony-stimulating factor (G-CSF) caused rapid redistribution of HSCs across the skeletal compartments. Old and young HSC clones showed a similar level of migratory behavior. Clonal make- up of blood of secondary recipients recapitulates the barcode composition of HSCs in the bone of origin. These data demonstrate a previously unanticipated high skeletal disequilibrium of the clonal composition of HSC pool long- term after transplantation. Our findings have important implications for experimental and clinical and stem cell transplantation protocols

Clonal analysis of young and aged hematopoietic stem cells using cellular barcoding

De productie van bloed cellen gedurende het leven wordt bepaalt door een kleine popluatie hematopoëitsche stamcellen (HSC’s). Veranderingen in deze populatie veroorzaakt door stress, verwerving van mutaties of veroudering kunnen leiden tot de ontwikkeling van bloedziektes, zoals leukemie en beenmerg falen. Om dit te voorkomen en te genezen is moeten we de normale functie van HSC’s. Hiervoor moet er bekeken worden hoeveel HSC’s er nodig zijn voor productie van bloedcellen, of alle HSC’s gelijk zijn en hoe ze veranderen gedurende veroudering. Om deze vragen te beantwoorden is het belangrijk kwantitatief te kunnen meten wat de contributie is van meerder HSC’s. Wij hebben een techniek ontwikkeld en gevalideerd: cellulair barcoderen. Hierbij worden hematopoëitsche cellen gemarkeerd op basis van het ingereren van een DNA label van een virale vector in het genoom. Met deze techniek hebben we HSC’s van jonge en oude muizen gemarkeerd en getransplanteerd in muizen. Doordat elke HSC uniek is gelabeld met een barcode kunnen zijn nakomelingen worden gevolgd in de tijd. Ons onderzoek laat zien dat HSC klonen heterogeen zijn in hun bijdrage aan verschillende types uitgerijpte cellen. Door veroudering neemt het aantal uitgerijpte cellen dat een stamcel kan produceren af. Echter, jonge en oude HSC’s zitten bij voorkeur op bepaald plaatsen in het skelet, en reageren gelijk op een dosis cytokine die ook in de kliniek wordt gebruikt. Dit samenvattend hebben we een nieuwe techniek ontwikkeld dat het mogelijk maakt HSC’s in vivo te bestuderen en een antwoorden te geven op veelgestelde vragen in HSC biologie

Noncanonical Wnt Comes of Age in Hematopoietic Stem Cells

Understanding molecular mechanisms of aging is crucial in efforts to reverse it. In a recent issue of Nature, Florian et al. (2013) report that increasing levels of noncanonical Wnt signaling accompany hematopoietic stem cell (HSC) aging, which can be modulated to functionally rejuvenate HSCs

Aging of murine hematopoietic stem cells

Hematopoietic stem cells (HSCs) are unique in their ability to self-renew and differentiate into all mature blood lineages. The equilibrium between these processes is crucial for tissue maintenance during the lifetime of the organism. However, with age the functionality of HSCs declines, resulting indevelopment of anemias, deficiencies of immune response, and increased risk of hematopoietic malignancies. Aged HSCs are characterized by preferential differentiation toward myeloid lineage, impaired self-renewal, and engraftment. Recent evidence provides clues to the understanding of these processeson cellular and molecular levels. Key components contributing to stem cell aging are shifts in the transcriptome and epigenome, accompanied by dysfunction of DNA repair pathways. In this chapter we will focus on studies and conceptual models of murine HSC aging.</p

Barcoded vector libraries and retroviral or lentiviral barcoding of hematopoietic stem cells

Cellular barcoding is a relatively recent technique aimed at clonal analysis of a proliferating cell population of any kind. The method was shown to be particularly successful in monitoring clonal contributions of hematopoietic stem cells (HSCs). An essential step of the method is retroviral or lentiviral labeling of the hematopoietic cells. The unique feature of the method is the generation of a vector library containing specific artificial DNA tags, generally known as barcodes. The library must satisfy multiple essential requirements. Importantly, considering the number of possible variations within the barcode sequence, the actual size of the barcoded vector library, and the number of clonogenic (stem) cells in the given experiment should be in ratios far from saturation. Excessive bias in barcodes frequencies must be avoided, and the library size must be assessed prior to the sequencing analysis. The final sequencing results must undergo statistical filtering. If all requirements are met, the method ensures profound sensitivity and accuracy for monitoring of the clonal fluctuations in a wide range of biological experiments