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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

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development

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

Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding

<p>The number of hematopoietic stem cells (HSCs) that contributes to blood formation and the dynamics of their clonal contribution is a matter of ongoing discussion. Here, we use cellular barcoding combined with multiplex high-throughput sequencing to provide a quantitative and sensitive analysis of clonal behavior of hundreds of young and old HSCs. The majority of transplanted clones steadily contributes to hematopoiesis in the long-term, although clonal output in granulocytes, T cells, and B cells is substantially different. Contributions of individual clones to blood are dynamically changing; most of the clones either expand or decline with time. Finally, we demonstrate that the pool of old HSCs is composed of multiple small clones, whereas the young HSC pool is dominated by fewer, but larger, clones.</p>

Glucocorticoid-induced glucocorticoid-receptor expression and promoter usage is not linked to glucocorticoid resistance in childhood ALL

Glucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoplosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC expo- sure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure)

Glucocorticoid-induced glucocorticoid-receptor expression and promoter usage is not linked to glucocorticoid resistance in childhood ALL

Glucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoplosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC expo- sure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure)

Glucocorticoid-induced glucocorticoid-receptor expression and promoter usage is not linked to glucocorticoid resistance in childhood ALL

Glucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoplosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC expo- sure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure)

MicroRNA-125 family members exert a similar role in the regulation of murine hematopoiesis

MicroRNAs (miRNAs) are crucial for proper functioning of hematopoietic stem and progenitor cells (HSPCs). Members of the miRNA-125 family (consisting of miR-125a, miR-125b1, and miR-125b2) are known to confer a proliferative advantage on cells upon overexpression, to decrease the rate of apoptosis by targeting proapoptotic genes, and to promote differentiation toward the myeloid lineage in mice. However, many distinct biological effects of the three miR-125 species have been reported as well. In the current study, we set out to assess whether the three miRNA-125s that carry identical seed sequences could be functionally different. Our data show that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state in vitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. All miR-125 family members decreased the pool of phenotypically defined Lin(-)Sca(+)Kit(+)CD48(-)CD150(+) long-term hematopoietic stem cells, simultaneously increasing the self-renewal activity upon secondary transplantation. The downregulation of miR-125s in hematopoietic stem cells abolishes these effects and impairs long-term contribution to blood cell production. The introduction of a point mutation within the miRNA-125 seed sequence abolishes all abovementioned effects and leads to the restoration of normal hematopoiesis. Our results show that all miR-125 family members are similar in function, they likely operate in a seed-sequence-dependent manner, and they induce a highly comparable hematopoietic phenotype. (C) 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc