Jennifer A. Cheeseman

Nestin-positive spheres derived from canine bone marrow stromal cells generate cells with early neuronal and glial phenotypic characteristics.

Bone marrow stromal cells (BMSCs) isolated from humans and rodents have been shown to generate neural cells under specific culture conditions and after transplantation in the central nervous system. The apparent plasticity of BMSCs has therefore been a target of intensive research in attempt to develop a novel therapy for neurological diseases. Canines sustain neurological disorders (e.g., traumatic spinal cord injury) that closely mirror pathology of those in humans. Therefore, we evaluated neural differentiation properties of canine BMSCs to provide insights into basic characterization of these cells for future neurotransplantation trials in canine patients with neurological disorders. We demonstrate that canine BMSCs form spherical cellular aggregates on anti-adhesive culture substrate in serum-free culture media, which morphologically and phenotypically resemble spherical aggregates of neural progenitor cells, so-called neurospheres. Upon dissociation and subculture on adhesive substrate, canine BMSCs express neuronal (ß Ш-tubulin) and glial (GFAP, A2B5, and CNPase) markers. Formation of spherical aggregates appears to be a critical preceding process for some of the glial marker expression (CNPase and A2B5). However, expression of more mature neuronal (MAP2) and glial (MBP) markers could not be induced with the protocol used in this study. We suggest that induction of canine BMSCs into cells with neural progenitor cell characteristics is possible and that these cells may have the potential for future cellular therapy for neurological disorders.

The frequency, growth kinetics, and osteogenic/adipogenic differentiation properties of canine bone marrow stromal cells

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38 × 104 bone marrow mononucleated cells (0.0042 ± 0.0019%, n = 5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.

The effects of canine bone marrow stromal cells on neuritogenesis from dorsal root ganglion neurons in vitro

The present in vitro study was designed to evaluate whether canine bone marrow stromal cells (BMSCs) promote neurite outgrowth from dorsal root ganglion (DRG) neurons. Bone marrow aspirates were collected from iliac crests of three young adult dogs. DRG neurons were cultured on BMSCs, fibroblasts, or laminin substrates. DRG neurons were also cultured in BMSC- or fibroblast-conditioned media. DRG neurons grown on BMSCs extended longer neurites and developed a much more elaborate conformation of branching neurites compared to those on fibroblasts or laminin. Quantitative analysis revealed that these effects were associated with the emergence of increased numbers of primary and branching neurites. The effect appears to be dependent upon cell-cell interactions rather than by elaboration of diffusible molecules. With more extensive investigations into the basic biology of canine BMSCs, their ability for promoting neurite outgrowth may be translated into a novel therapeutic strategy for dogs with a variety of neurological disorders.

Detection of oligoclonal bands in cerebrospinal fluid from German Shepherd dogs with degenerative myelopathy by isoelectric focusing and immunofixation

BACKGROUND Detection of intrathecal IgG synthesis is important in evaluating inflammatory diseases in the central nervous system. Isoelectric focusing (IEF) is currently the most sensitive method to demonstrate intrathecal IgG synthesis and may have diagnostic value for German Shepherd degenerative myelopathy (GSDM). OBJECTIVE The objective of this study was to adapt a modified IEF and immunofixation method for the detection of oligoclonal bands in cerebrospinal fluid (CSF) from dogs with GSDM. METHODS Serum and lumbar CSF samples were collected from 6 German Shepherd dogs clinically diagnosed with GSDM. Samples were also collected from 6 clinically healthy dogs for comparison. The concentration of IgG was measured by quantitative ELISA and the concentration of total protein was measured by the Bradford protein assay. The presence of oligoclonal bands was evaluated by use of modified IEF followed by immunofixation. RESULTS The concentrations of total protein and IgG, and the IgG/total protein ratio in CSF samples, were not significantly different between GSDM patients and control dogs. Four GSDM patients had oligoclonal bands in their CSF based on IEF-immunofixation. No oligoclonal bands were found in CSF from control dogs. CONCLUSION The results suggest that the detection of oligoclonal bands by IEF-immunofixation may have diagnostic value for GSDM, and support the idea that humoral immune responses may play a role in the pathogenesis of GSDM.