Morphological and neurochemical features of cultured primary skin fibroblasts of fischer 344 rats following striatal implantation

Authors

  • Dr. Michael D. Kawaja,

    1. Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0624
    Current affiliation:
    1. Division of Molecular Immunology and Neuroscience, Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5
    Search for more papers by this author
  • Dr. Fred H. Gage

    Corresponding author
    1. Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0624
    • Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0624
    Search for more papers by this author

Abstract

In order to assess the feasibility of using primary skin fibroblasts as a donor population for genetic modification and subsequent intracerebral grafting, the present study examines the structural and neurochemical characteristics of intrastriatal grafts of isogeneic primary fibroblasts over a period of 6 months. In culture, primary skin fibroblasts obtained from a female Fischer 344 rat display robust growth, but once confluent these cells exhibit contact inhibition. Following the implantation of cultured primary cells within the striatum of other adult rats from the same inbred strain, isologous grafts stain immunohistochemically for fibronectin at 1 week, and this immunostaining persists up to 6 months. Immunoreactivity for laminin is intense within the grafts from 1 to 8 weeks, but decreases by 6 months. Astrocytes within the striatum respond dramatically to the implantation of primary fibroblasts, such that immunohistochemical staining for glial fibrillary acidic protein increases markedly from 1 to 8 weeks after implantation. Although the intensity of immunostaining for glial fibrillary acidic protein diminishes among striatal astrocytes between 8 weeks and 6 months, the astrocytic border between the grafts and striatal neuropil remains intensely immunoreactive. Capillaries within the grafts stain immunohistochemically for glucose transporter (a facilitated glucose uptake carrier) as early as 3 weeks after implantation. Following intravenous infusions of peroxidase, capillaries within fibroblast grafts do not permit the extravasation of this macromolecule at 8 weeks and 6 months. Thus, capillaries formed within intracerebral grafts of primary skin fibroblasts exhibit a functional impermeable barrier to macromolecules similar to those capillaries of the host striatum. At the ultrastructural level, grafts possess numerous fibroblasts and have an extracellular matrix filled with collagen. Reactive astrocytic processes filled with intermediate filaments are found throughout the grafts. Hypertrophied astrocytes and their processes also appear to form a continuous border between the grafts and striatal neuropil. Grafts of primary fibroblasts also possess an extensive vasculature that is composed of capillaries with nonfenestrated endothelial cells; the occurrence of reactive astrocytic processes closely associated with or enveloping capillaries is variable. These results provide direct morphological and neurochemical evidence for the long-term survival of isologous fibroblasts after implantation within the rat striatum. From these data, we propose that isologous skin fibroblasts can be considered as donor candidates for successful intracerebral grafting following gene transfer.

Ancillary