http://repub.eur.nl/res/aut/3289/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/34434/ 2011-11-01T00:00:00Z Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied 7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions. http://repub.eur.nl/res/pub/30754/ 2011-10-01T00:00:00Z Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg-/-mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required. http://repub.eur.nl/res/pub/26428/ 2011-05-01T00:00:00Z Extensive studies have demonstrated the potential applications of bone marrow-derived mesenchymal stem cells (BM-MSCs) as regenerative or immunosuppressive treatments in the setting of organ transplantation. The aims of the present study were to explore the presence and mobilization of mesenchymal stem cells (MSCs) in adult human liver grafts and to compare their functional capacities to those of BM-MSCs. The culturing of liver graft preservation fluids (perfusates) or end-stage liver disease tissues resulted in the expansion of MSCs. Liver-derived mesenchymal stem cells (L-MSCs) were equivalent to BM-MSCs in adipogenic and osteogenic differentiation and in wingless-type-stimulated proliferative responses. Moreover, the genome-wide gene expression was very similar, with a 2-fold or greater difference found in only 82 of the 32,321 genes (0.25%). L-MSC differentiation into a hepatocyte lineage was demonstrated in immunodeficient mice and in vitro by the ability to support a hepatitis C virus infection. Furthermore, a subset of engrafted MSCs survived over the long term in vivo and maintained stem cell characteristics. Like BM-MSCs, L-MSCs were found to be immunosuppressive; this was shown by significant inhibition of T cell proliferation. In conclusion, the adult human liver contains an MSC population with a regenerative and immunoregulatory capacity that can potentially contribute to tissue repair and immunomodulation after liver transplantation. http://repub.eur.nl/res/pub/34512/ 2011-04-12T00:00:00Z To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements. http://repub.eur.nl/res/pub/21129/ 2010-07-01T00:00:00Z Pompe disease (acid α-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid α-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid α-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid α-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue. http://repub.eur.nl/res/pub/17228/ 2009-10-02T00:00:00Z Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development. http://repub.eur.nl/res/pub/36301/ 2007-04-01T00:00:00Z The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rγ), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rγ had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rγ may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth. http://repub.eur.nl/res/pub/10012/ 2007-02-23T00:00:00Z Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. METHODS: Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs). RESULTS: Tendon-derived cells stained D7-FIB (fibroblast-marker) positive, but alpha-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor gamma). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. CONCLUSION: This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis. http://repub.eur.nl/res/pub/22858/ 2001-02-28T00:00:00Z Mature blood cells have a finite life span and are continuously replenished by the proliferation and differentiation of lineage-specific progenitor cells derived from rare pluripotent hematopoietic stem cells. l. 2 Stem cells are capable of reproducing them-selves (self-renewal) and of producing cells belonging to all lineages of blood (hematopoietic) cell differentiation (Figure 1.). Definitions of stem cells vary on the model of hematopoiesis used but. in general, these cells are referred to by the functional property of being capable of long-term reconstitution of the hematopoietic system of recipients.