Genetic Therapies and Xenotransplantation

Expert Opinion on Biological Therapy. Mar, 2002  |  Pubmed ID: 11890869

The number of patients in need of an organ transplant is increasing, while the number of satisfactory sources of organs has declined in many countries [101]. The resulting shortage of human organs has spurred an urgent effort to investigate alternative therapies, including the use of animal organs, tissues and cells (i.e., xenotransplantation). Advances in genetic engineering have provided essential tools for the development of practical solutions to human disease. The area of xenotransplantation is no exception. In fact, the use of genetic therapies is especially attractive in the transplant setting as it offers an opportunity to manipulate the donor tissue rather than the recipient. This review will describe the obstacles in the clinical application of xenotransplantation and how genetic engineering might be used to address them.

Manipulation of Remodeling Pathways to Enhance the Mechanical Properties of a Tissue Engineered Blood Vessel

Journal of Biomechanical Engineering. Dec, 2002  |  Pubmed ID: 12596641

There is a current need for a small diameter vascular graft due to the limited supply of autogenous grafts and the failure of synthetic grafts due to thrombosis and/or intimal hyperplasia. The use of living cells and tissues to fabricate a small diameter graft (i.e., tissue engineered blood vessel, TEBV) could be useful given the endothelialization potential and biocompatibility benefits of such a graft. However, while sufficient strength has been attained in a TEBV, coordinate compliance has yet to be fine-tuned. In this study we investigate the effects of biological response modifiers, retinoic acid (RA) and ascorbic acid (AA) on TEBV biomechanics as a function of time and subsequently correlate observed RA/AA induced changes in TEBV mechanics with alterations in smooth muscle cell (SMC) biochemistry. TEBVs were constructed using a fibrillar type I collagen network populated by human aortic smooth muscle cells (AoSMC). Following construction this TEBV was treated with 0.3 mM AA and 0.1 mM RA (concentrations found to induce changes in VSMC phenotype). Ultimate tensile stress (UTS), rate of relaxation (RR) and elastic efficiency (EE) of RA/AA treated and untreated TEBVs were measured following 1, 7, 15, 30, 45, and 60 days of treatment. At corresponding time points, the effect of these treatments on collagen and elastin protein synthesis and mRNA expression was examined. RA/AA treated TEBV strength increased and stiffness decreased compared to controls as a function of time. Relative collagen synthesis in treated TEBVs exceeded control levels by nearly two-fold at 15 and 30 days of incubation. RA/AA treated collagen gene expression followed a similar trend. Relative elastin synthesis was also greater in treated TEBVs as compared to untreated TEBVs at 15 and 30 days of incubation and correspondingly elastin mRNA expression was significantly elevated at 15 days of incubation. These data provide evidence that RA/AA treated TEBVs exhibit mechanical properties which more closely mimic those of a native vessel than their untreated counterparts and that changes in extracellular matrix composition and matrix gene expression in the presence of RA/AA treatment may play an important role in the development of said mechanical properties.

Direct Measurement of Lymphocyte Receptor Diversity

Nucleic Acids Research. Nov, 2003  |  Pubmed ID: 14602932

The ability to mount an immune defense against infectious microorganisms and their products, and against tumors is believed to be a direct function of lymphocyte diversity. Because the diversity of lymphocyte receptor genes is >1000-fold more diverse than the entire genome and varies between genetically identical individuals, measuring lymphocyte diversity has been a daunting challenge. We developed a novel technique for measuring lymphocyte diversity directly using gene chips. We reasoned and here demonstrate that the frequency of hybridization of nucleic acids coding for lymphocyte receptors to the oligonucleotides on a gene chip varies in direct proportion to diversity. We applied the technique to detect changes in lymphocyte diversity in mice with known B cell alterations and in persons with known T cell repertoire defects. This approach is the first to provide direct analysis of lymphocyte receptor diversity and should facilitate fundamental study of the adaptive immune system and clinical efforts to assess immunological diseases. In addition, this approach could be more broadly applied, for example to measure diversity of viral quasi-species.

Spontaneous Fusion of Cells Between Species Yields Transdifferentiation and Retroviral Transfer in Vivo

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Mar, 2004  |  Pubmed ID: 14715691

Human cells can fuse with damaged or diseased somatic cells in vivo. Whether human cells fuse in vivo in the absence of disease and with cells of disparate species is unknown. Such a question is of current interest because blood exchanges between species through direct physical contact, via insect vectors or parasitism, are thought to underlie the transmission of zoonotic agents. In a model of human-pig chimerism, we show that some human hematopoietic stem cells engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens. * differentiation * fusion * retrovirus

B Cell-dependent TCR Diversification

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2004  |  Pubmed ID: 15067046

T cell diversity was once thought to depend on the interaction of T cell precursors with thymic epithelial cells. Recent evidence suggests, however, that diversity might arise through the interaction of developing T cells with other cells, the identity of which is not known. In this study we show that T cell diversity is driven by B cells and Ig. The TCR V beta diversity of thymocytes in mice that lack B cells and Ig is reduced to 6 x 10(2) from wild-type values of 1.1 x 10(8); in mice with oligoclonal B cells, the TCR V beta diversity of thymocytes is 0.01% that in wild-type mice. Adoptive transfer of diverse B cells or administration of polyclonal Ig increases thymocyte diversity in mice that lack B cells 8- and 7-fold, respectively, whereas adoptive transfer of monoclonal B cells or monoclonal Ig does not. These findings reveal a heretofore unrecognized and vital function of B cells and Ig for generation of T cell diversity and suggest a potential approach to immune reconstitution.

Xenotransplantation and the Future of Renal Replacement

Journal of the American Society of Nephrology : JASN. May, 2004  |  Pubmed ID: 15100350

Biological Implications of Cell Fusion

Nature Reviews. Molecular Cell Biology. Jul, 2005  |  Pubmed ID: 15957005

Until recently, cells were thought to be integral and discrete components of tissues, and their state was determined by cell differentiation. However, under some conditions, stem cells or their progeny can fuse with cells of other types, mixing cytoplasmic and even genetic material of different (heterotypic) origins. The fusion of heterotypic cells could be of central importance for development, repair of tissues and the pathogenesis of disease.

Effacing of the T Cell Compartment by Cardiac Transplantation in Infancy

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2006  |  Pubmed ID: 16424228

For cardiac transplantation in infants, T cells are depleted and the thymus is removed. These manipulations should cause profound defects in the T cell compartment. To test this concept, 20 subjects who underwent cardiac transplantation in infancy and healthy age-matched subjects were studied. The number of T cells in the blood was nearly normal in all subjects 1-10 years after surgery. However, newly generated T cells were undetectable in 10 recipients and 10-fold less than controls in 10, suggesting absence of thymic function. TCRbeta chain diversity, measured by a novel technique, was approximately 100-fold lower than controls. T cell function, deduced from levels of human herpesvirus 7 and response to hepatitis B immunization, were notably impaired. Yet cardiac transplant recipients were generally free of opportunistic infections. Our findings demonstrate a novel approach to measuring lymphocyte diversity and suggest that understanding how these subjects resist infection could yield important insights into immune fitness.

Immunoglobulin Promotes the Diversity and the Function of T Cells

European Journal of Immunology. Jul, 2006  |  Pubmed ID: 16791877

It is generally accepted in immunology that while T and B cells collaborate for the production of antibodies in response to protein antigens, T cells develop and function in the absence of B cells. However, B cell-deficient subjects and mice have unexplained cellular immune defects. Here, we examined the contribution of B cells/Ig to the generation of diversity and function of T cells. Mice lacking B cells and Ig (JH(-/-)) or having oligoclonal B cells (QM) had a profoundly contracted T cell receptor (TCR) Vbeta repertoire: 0.08 and 1.3% of wild type, respectively. Rejection of H-Y-incompatible skin grafts in QM and JH(-/-) mice was significantly delayed (median, 43 and 22 days, respectively) compared to wild-type mice (median, 16 days). Furthermore, reduction of the TCR Vbeta diversity by thymectomy in wild-type mice significantly increased survival of H-Y-incompatible skin grafts, and reconstitution of the T cell diversity in QM mice with Ig Fab fragments significantly decreased survival of the skin grafts. These results indicate that B cells and/or Ig "help" T cells through the generation and maintenance of T cell diversity, improving T cell function. Our results may have important implications for therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post-myeloablative treatments.

The Future of Organ Transplantation

Annals of Transplantation : Quarterly of the Polish Transplantation Society. 2006  |  Pubmed ID: 17494288

No challenge in medicine can be more urgent than the devising of new strategies for replacing organs. The need for organ replacement not only exceeds by far the supply of organs available for transplantation, the need is likely to increase dramatically. The induction of tolerance to spare transplanted organs and the use of animal organs, i.e. xenotransplantation, could help address this problem but neither appears close to application. Here we discuss a strategy involving the sequential generation of pleuripotent stem cells, formation of human organs in an adoptive xenogeneic host, the harvesting of human cells, tissues or organs from that host and implantation into the individual from whom the stem cells were obtained as one potential way to generate histocompatible organs. We discuss as well the promise, limitations and uncertainties of these steps. This approach, while speculative and perhaps unlikely, may lead to development of further new technologies and insights, the pursuit of which could provide new approaches to replacing organ function.

Toward Development and Production of Human T Cells in Swine for Potential Use in Adoptive T Cell Immunotherapy

Tissue Engineering. Part A. May, 2009  |  Pubmed ID: 18826341

Immunotherapy and vaccination for cancer or infection are generally approached by administration of antigen or stimulation of antigen-presenting cells or both. These measures may fail if the treated individual lacks T cells specific for the immunogen(s). We tested another strategy-the generation of new T cells from hematopoietic stem cells that might be used for adoptive immunotherapy. To test this concept, we introduced T cell-depleted human bone marrow cells into fetal swine and tested the swine for human T cells at various times after birth. Human T cells were detected in the thymus and blood of the treated swine. These cells were generated de novo as they contained human T cell receptor excision circles not present in the T cell-depleted bone marrow. The human T cells were highly diverse and included novel specificities capable of responding to antigen presented by human antigen-presenting cells. Our findings constitute a first step in a new promising approach to immunotherapy in which tumor- or virus-specific T cell clones lacking in an individual might be generated in a surrogate host from hematopoietic stem cells of the individual to be treated.

Heterogeneous Differentiation of Human Mesenchymal Stem Cells in Response to Extended Culture in Extracellular Matrices

Tissue Engineering. Part A. Dec, 2009  |  Pubmed ID: 19911955

Extracellular matrix proteins (ECMs) guide differentiation of adult stem cells, but the temporal distribution of differentiation (i.e., heterogeneity) in a given population has not been investigated. We tested the effect of individual ECM proteins on lineage commitment of human bone marrow-derived mesenchymal stem cells (MSCs) over time. We exposed stem cell populations to ECM proteins representing the primary tissue structures of the body (i.e., collagens type I, III, IV; laminin; and fibronectin) and determined the lineage commitment of the stem cells at 1, 7, and 14 days. We found that collagens that can participate in the formation of fibrils guide differentiation of cardiomyocytes, adipocytes, and osteoblasts. ECMs of the basement membrane initiate differentiation of cardiomyocytes and osteoblasts but not adipocytes, and small facilitator ECMs (e.g., fibronectin) do not significantly affect stem cell differentiation. Differentiation was ECM-dependent because culture on tissue culture polystyrene, with consistent cell morphology, proliferation, and death, initiated differentiation of osteoblasts only. Thus, we show that ECMs independently trigger differentiation of human adult MSCs and that differentiation in this context can be guided down multiple lineages using the same ECM stimulus. This work highlights the importance of more clearly defining progenitor populations, especially those cultured in the presence of ECMs before transplantation.

Bimolecular Fluorescence Complementation Analysis of Eukaryotic Fusion Products

Biology of the Cell / Under the Auspices of the European Cell Biology Organization. Sep, 2010  |  Pubmed ID: 20590528

Cell fusion is known to underlie key developmental processes in humans and is postulated to contribute to tissue maintenance and even carcinogenesis. The mechanistic details of cell fusion, especially between different cell types, have been difficult to characterize because of the dynamic nature of the process and inadequate means to track fusion products over time. Here we introduce an inducible system for detecting and tracking live cell fusion products in vitro and potentially in vivo. This system is based on BiFC (bimolecular fluorescence complementation) analysis. In this approach, two proteins that can interact with each other are joined to fragments of a fluorescent protein and are expressed in separate cells. The interaction of said proteins after cell fusion produces a fluorescent signal, enabling the identification and tracking of fusion products over time.

Multiphoton Flow Cytometry to Assess Intrinsic and Extrinsic Fluorescence in Cellular Aggregates: Applications to Stem Cells

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada. Aug, 2011  |  Pubmed ID: 20684798

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

In Utero Cell Transfer Between Porcine Littermates

Reproduction, Fertility, and Development. 2011  |  Pubmed ID: 21211462

Trafficking of cells between mother and fetus during the course of normal pregnancy is well documented. Similarly, cells are known to travel between twins that share either a placenta (i.e. monozygotic) or associated chorion (i.e. monochorionic). Transferred cells are thought to be channelled via the vessels of the placenta or vascular connections established via the chorion and the long-term presence of these cells (i.e. microchimerism) can have important consequences for immune system function and reparative capacity of the host. Whether cells can be transferred between twins with separate placentas and separate chorions (i.e. no vascular connections between placentas) has not been investigated nor have the biological consequences of such a transfer. In the present study, we tested the possibility of this type of cell transfer by injecting human cord blood-derived cells into a portion of the littermates of swine and probing for human cells in the blood and tissues of unmanipulated littermates. Human cells were detected in the blood of 78% of unmanipulated littermates. Human cells were also detected in various tissues of the unmanipulated littermates, including kidney (56%), spleen (33%), thymus (11%) and heart (22%). Human cells were maintained in the blood until the piglets were sacrificed (8 months after birth), suggesting the establishment of long-term microchimerism. Our findings show that the transfer of cells between fetuses with separate placentas and separate chorions is significant and thus such twins may be subject to the same consequences of microchimerism as monozygotic or monochorionic counterparts.

Harnessing Endogenous Growth Factor Activity Modulates Stem Cell Behavior

Integrative Biology : Quantitative Biosciences from Nano to Macro. Aug, 2011  |  Pubmed ID: 21720642

The influence of specific serum-borne biomolecules (e.g. heparin) on growth factor-dependent cell behavior is often difficult to elucidate in traditional cell culture due to the random, non-specific nature of biomolecule adsorption from serum. We hypothesized that chemically well-defined cell culture substrates could be used to study the influence of sequestered heparin on human mesenchymal stem cell (hMSC) behavior. Specifically, we used bio-inert self-assembled monolayers (SAMs) chemically modified with a bioinspired heparin-binding peptide (termed "HEPpep") and an integrin-binding peptide (RGDSP) as stem cell culture substrates. Our results demonstrate that purified heparin binds to HEPpep SAMs in a dose-dependent manner, and serum-borne heparin binds specifically and in a dose-dependent manner to HEPpep SAMs. These heparin-sequestering SAMs enhance hMSC proliferation by amplifying endogenous fibroblast growth factor (FGF) signaling, and enhance hMSC osteogenic differentiation by amplifying endogenous bone morphogenetic protein (BMP) signaling. The effects of heparin-sequestering are similar to the effects of supraphysiologic concentrations of recombinant FGF-2. hMSC phenotype is maintained over multiple population doublings on heparin-sequestering substrates in growth medium, while hMSC osteogenic differentiation is enhanced in a bone morphogenetic protein-dependent manner on the same substrates during culture in osteogenic induction medium. Together, these observations demonstrate that the influence of the substrate on stem cell phenotype is sensitive to the culture medium formulation. Our results also demonstrate that enhanced hMSC proliferation can be spatially localized by patterning the location of HEPpep on the substrate. Importantly, the use of chemically well-defined SAMs in this study eliminated the confounding factor of random, non-specific biomolecule adsorption, and identified serum-borne heparin as a key mediator of hMSC response to endogenous growth factors.

A Nondenatured, Noncrosslinked Collagen Matrix to Deliver Stem Cells to the Heart

Regenerative Medicine. Sep, 2011  |  Pubmed ID: 21916593

Stem cell transplantation holds promise as a therapeutic approach for the repair of damaged myocardial tissue. One challenge of this approach is efficient delivery and long-term retention of the stem cells. Although several synthetic and natural biomaterials have been developed for this purpose, the ideal formulation has yet to be identified.

Imaging Cardiac Extracellular Matrices: a Blueprint for Regeneration

Trends in Biotechnology. Dec, 2011  |  Pubmed ID: 22209562

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated.

Environmental Parameters Influence Non-viral Transfection of Human Mesenchymal Stem Cells for Tissue Engineering Applications

Cell and Tissue Research. Jan, 2012  |  Pubmed ID: 22277991

Non-viral transfection is a promising technique that could be used to increase the therapeutic potential of stem cells. The purpose of this study was to explore practical culture parameters of relevance in potential human mesenchymal stem cell (hMSC) clinical and tissue engineering applications, including type of polycationic transfection reagent, N/P ratio and dose of polycation/pDNA polyplexes, cell passage number, cell density and cell proliferation. The non-viral transfection efficiency was significantly influenced by N/P ratio, polyplex dose, cell density and cell passage number. hMSC culture conditions that inhibited cell division also decreased transfection efficiency, suggesting that strategies to promote hMSC proliferation may be useful to enhance transfection efficiency in future tissue engineering studies. Non-viral transfection treatments influenced hMSC phenotype, including the expression level of the hMSC marker CD105 and the ability of hMSCs to differentiate down the osteogenic and adipogenic lineages. The parameters found here to promote hMSC transfection efficiency, minimize toxicity and influence hMSC phenotype may be instructive in future non-viral transfection studies and tissue engineering applications.