Induction of antigen-specific tolerance through hematopoietic stem cell-mediated gene therapy: the future for therapy of autoimmune disease?

Based on the principle that immune ablation followed by HSC-mediated recovery purges disease-causing leukocytes to interrupt autoimmune disease progression, hematopoietic stem cell transplantation (HSCT) has been increasingly used as a treatment for severe autoimmune diseases. Despite clinically-relevant outcomes, HSCT is associated with serious iatrogenic risks and is suitable only for the most serious and intractable diseases. A further limitation of autologous HSCT is that relapse rates can be high, suggesting disease-causing leukocytes are incompletely purged or the environmental and genetic determinants that drive disease remain active. Incorporation of antigen-specific tolerance approaches that synergise with autologous HSCT could reduce or prevent relapse. Further, by reducing the requirement for highly toxic immune-ablation and instead relying on antigen-specific tolerance, the clinical utility of HSCT could be significantly diversified. Substantial progress has been made exploring HSCT-mediated induction of antigen-specific tolerance in animal models but studies have focussed on primarily on prevention of autoimmune diseases. However, as diagnosis of autoimmune disease is often not made until autoimmune disease is well developed and populations of autoantigen-specific pathogenic effector and memory T cells have become well established, immunotherapies must be developed to address effector and memory T-cell responses which have traditionally been considered the key impediment to immunotherapy. Here, focusing on T-cell mediated autoimmune diseases we review progress made in antigen-specific immunotherapy using HSCT-mediated approaches, induction of tolerance in effector and memory T cells and the challenges for progression and clinical application of antigen-specific ‘tolerogenic’ HSCT therapy

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described

Intrinsic potential of human hemopoietic stem cells to self-renewal during EX vivo cultures

Self-renewal of p I uri potential hemopoietic stem cells (HSCs) is a primary requirement for their ex vivo expansion as well as for the long-lasting genetic correction of the hemopoietic system. In our search for culture conditions that will facilitate HSC replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay based on the phenotypic properties of the primitive cells. The HSCs were defined as cells expressing high levels of CD34 but lack the CD38, CD33 and CD? I antigens. Cell replication in this study was determined by PKH26 membrane dye. Loading CD34+ cells before culture with PKH26 enables to monitor the replication history of cells meeting, at the lime of analysis, the characteristics of HSCs. In total 31 samples of bone marrow and umbilical cord blood were included in the study. Isolated CD34+ cells from each sample were submitted to parallel culture conditions including varying hemopoietic growth factor combinations, the presence or absence of pre-established allogeneic stroma and serum supplementation. This experimental set-up enabled us to compare the effects of various stimuli on the replication of the CD34+CD33,38,71- population of individual samples as well as the response pattern of cells from different samples. A most striking observation in this study was the large intra-sample variation in the proliferative response of the CD34+CD33.38.7S- cells. The replication potential of the CD34+CD33,38,71- population did not correspond to that of the other cellular components in the culture. The replication of the culture as a whole was determined clearly by culture stimuli. In general, the samples from the two sources could be divided according to the replication property of the CD34+CD33,38,71- sub-set into either good or poorly replicating. In comparison to this 'intrinsic' potential, the effect of growth stimuli on proliferation was négligeable. If the CD34+-CD33,38,71 sub set divided it was once and only occasionally twice, during a 6-day period. Other cell components replicated up to 8 times. In conclusion, ex vivo replication of CD34++CD33,38,71- HSCs is apparently intrinsically regulated and their replication potential remains limited under many culture conditions. As the determinants for the intrinsic capacity of HSCs to replicate in culture are unsolved, their ex vivo behavior remains unpredictable. This knowledge should be taken in consideration in the practice of ex vivo HSC expansion and gene therapy

Pressure Ulcers: Description of a New Model and Use of Mesenchymal Stem Cells for Repair

Gene regulation and cell differentiatio