Biomedical subjects

Ian D Dubé

Publications and source records attributed to Ian D Dubé.

3 recordsLinked to original sources

PubMed2004

Gene expression profiling in the inductive human hematopoietic microenvironment.

Human hematopoietic stem cells (HSCs) and their progenitors can be maintained in vitro in long-term bone marrow cultures (LTBMCs) in which constituent HSCs can persist within the adherent layers for up to 2 months. Media replenishment of LTBMCs has been shown to induce transition of HSCs from a quiescent state to an active cycling state. We hypothesize that the media replenishment of the LTBMCs leads to the activation of important regulatory genes uniquely involved in HSC proliferation and differentiation. To profile the gene expression changes associated with HSC activation, we performed suppression subtractive hybridization (SSH) on day 14 human LTBMCs following 1-h media replenishment and on unmanipulated controls. The generated SSH library contained 191 differentially up-regulated expressed sequence tags (ESTs), the majority corresponding to known genes related to various intracellular processes, including signal transduction pathways, protein synthesis, and cell cycle regulation. Nineteen ESTs represented previously undescribed sequences encoding proteins of unknown function. Differential up-regulation of representative genes, including IL-8, IL-1, putative cytokine 21/HC21, MAD3, and a novel EST was confirmed by semi-quantitative RT-PCR. Levels of fibronectin, G-CSF, and stem cell factor also increased in the conditioned media of LTBMCs as assessed by ELISA, indicating increased synthesis and secretion of these factors. Analysis of our library provides insights into some of the immediate early gene changes underlying the mechanisms by which the stromal elements within the LTBMCs contribute to the induction of HSC activation and provides the opportunity to identify as yet unrecognized factors regulating HSC activation in the LTBMC milieu.

Bone Marrow Cells↗

PubMed2002

In utero injection of alpha-L-iduronidase-carrying retrovirus in canine mucopolysaccharidosis type I: infection of multiple tissues and neonatal gene expression.

Canine alpha-L-iduronidase (alpha-ID) deficiency is caused by a single base pair mutation in the alpha-ID gene, resulting in no enzyme activity in homozygous affected pups. The disease clinically resembles human mucopolysaccharidosis type I (MPSI). We used the canine MPSI model system to address the efficacy of a new retroviral vector, MND-MFG, containing the human alpha-ID cDNA (MND-MFG-alpha-ID) for direct in utero gene delivery to MPSI cells. In vitro, the MND-MFG-alpha-ID vector showed high-level, long-term expression of the transgene in both canine and human alpha-ID-deficient fibroblasts. The effectiveness of this vector for in utero gene transfer and expression in multiple tissues was assessed by injecting viral supernatants into MPSI fetuses and evaluating transduction efficiency and enzyme expression at various times after birth. Transduction of a spectrum of cell types and tissues was observed in all seven live-born pups and in one stillborn pup. Although enzyme activity was not detected in adult tissues from the seven surviving pups, significant alpha-ID enzyme activity was detected in both the liver and kidney of the deceased pup. Our combined gene delivery vector and in utero transfer approach, while encouraging in terms of overall gene transfer efficiency to multiple tissues and successful short-term gene expression, was unable to meet the important requirement of sustained in vivo gene expression.

Animals↗

PubMed2002

Human hematopoietic progenitors engraft in fetal canine recipients and expand with neonatal injection of fibroblasts expressing human hematopoietic cytokines.

OBJECTIVE: The development of large-animal models for human hematopoiesis will facilitate the study of human hematopoietic stem cells and their progenitors in vivo. In previous studies, human hematopoietic progenitors engrafted in fetal dogs and contributed to hematopoiesis for one year. Despite initially high levels of human cells, the proportion declined to less than 0.1% at 6 months, possibly due to inability of the canine hematopoietic microenvironment to support ongoing human hematopoiesis. In the current experiments we examined the potential of co-transplanting fibroblasts expressing human hematopoietic cytokines with the hematopoietic graft to increase the contribution of human progenitors to chimeric hematopoiesis. METHODS: Mid-gestation canine fetuses were injected with 1-3 x 10(7) human cord blood cells and 1 x 10(7) murine fibroblasts engineered to express human cytokines. Neonatal pups were boosted with additional injections of cytokine-expressing fibroblasts. Human cell engraftment was monitored by PCR amplification of human-specific DNA sequences from recipient hematopoietic tissues. RESULTS: Human hematopoietic cells were detected in 13/15 fetal recipients for at least 7 months. At time points up to 30 weeks of age, human DNA was detected in stimulated lymphocyte cultures, approximately 0.1% of blood leukocytes and 1.5% (85/5757) of myeloid colonies. Eight months postinfusion, 1.7% of colony-forming units (CFUs) were of human origin. By one year 0.5% or less of myeloid colonies and less than 0.01% of blood leukocytes carried human DNA. Following an infusion of cytokine-expressing fibroblasts at one year, the proportion of human myeloid progenitors rose to 11.5% and remained detectable for 8 months. CONCLUSION: These studies confirm that human hematopoietic progenitors can engraft in fetal pups and contribute to multilineage hematopoiesis. Infusion of cells expressing human cytokines is one approach to stimulate human hematopoietic progenitors in vivo and thus increase their contributions to chimeric hematopoiesis.

Adoptive Transfer↗