David Lipsitz

Generation of oligodendroglial progenitors from neural stem cells

To understand how the differentiation of stem cells to oligodendroglial progenitors is regulated, we established cultures of neural stem cells from neonatal rat striatum in the presence of epidermal growth factor (EGF) as free-floating neurospheres that were then exposed to an increasing amount of B104 cell-conditioned medium (B104CM). The resultant cells proliferated in response to B104CM but no longer to EGF. In vitro analysis and transplantation studies indicated that these cells were committed to the oligodendroglial lineage, and they were thus referred to as oligospheres. Further characterization of their expression of early markers, cell cycle, migration, and self-renewal suggests that they were pre-O2A progenitors. RT-PCR analysis indicated that the oligosphere cells expressed mRNAs of platelet-derived growth factor α receptor in addition to fibroblast growth factor receptor but not EGF receptor; the latter two receptor mRNAs were expressed by neurosphere cells. Thus, the progression of stem cells to oligodendroglial progenitors is likely induced by factors in B104CM.

Self‐renewing canine oligodendroglial progenitor expanded as oligospheres

We have previously shown that oligodendroglial progenitors (OP) can be generated from multipotent rat neural precursor cells. We now report the generation of a homogeneous culture of canine OP from neural precursor cells. In non‐adherent cultures, homogeneous OP cultures were obtained in 6–8 weeks of treatment with B104 cell conditioned medium (B104CM). In adherent cultures where astrocytes grew as a layer of substrate, colonies of OP invariably appeared at 10–14 days in vitro (DIV) and the colonies were expanded as free‐floating spheres (oligospheres), in the presence of B104CM, suggesting that astrocytes facilitate the generation of canine OP. The oligosphere cells were characterized by self‐renewal in the presence of B104CM and by terminal differentiation into oligodendrocytes after withdrawal of B104CM. Transplantation studies indicated that the extensively expanded oligosphere cells retained myelination capacity. The oligospheres thus provide a valuable source for experimental cell therapy studies. J. Neurosci. Res. 54:181–190, 1998.

Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain.

Glial cell transplantation into myelin‐deficient rodent models has resulted in myelination of axons and restoration of conduction velocity. The shaking (sh) pup canine myelin mutant is a useful model in which to test the ability to repair human myelin diseases, but as in humans, the canine donor supply for allografting is limited. A solution may be provided by self‐renewing epidermal growth factor (EGF)‐responsive multipotential neural progenitor cell populations (“neurospheres”). Nonadherent spherical clusters, similar in appearance to murine neurospheres, have been obtained from the brain of perinatal wildtype (wt) canine brain and expanded in vitro in the presence of EGF for at least 6 months. Most of the cells in these clusters express a nestin‐related protein. Within 1–2 weeks after removal of EGF, cells from the clusters generate neurons, astrocytes, and both oligodendroglial progenitors and oligodendrocytes. Transplantation of lacZ‐expressing wt neurospheres into the myelin‐deficient (md) rat showed that a proportion of the cells differentiated into oligodendrocytes and produced myelin. In addition, cells from the neurosphere populations survived at least 6 weeks after grafting into a 14‐day postnatal sh pup recipient and at least 2 weeks after grafting into an adult sh pup recipient. Thus, neurospheres provide a new source of allogeneic donor cells for transplantation studies in this mutant. J. Neurosci. Res. 50:862–871, 1997.

Cervical Myelopathy Associated with Extradural Synovial Cysts in 4 Dogs

Three Mastiffs and 1 Great Dane were presented to the University of Wisconsin Veterinary Medical Teaching Hospital for cervical myelopathy based on history and neurologic examination. All dogs were males and had progressive ataxia and tetraparesis. Degenerative arthritis of the articular facet joints was noted on survey spinal radiographs. Myelography disclosed lateral axial compression of the cervical spinal cord medial to the articular facets. Extradural compressive cystic structures adjacent to articular facets were identified on magnetic resonance imaging (1 dog). High protein concentration was the most important finding on cerebrospinal fluid analysis. Dorsal laminectomies were performed in all dogs for spinal cord decompression and cyst removal. Findings on cytologic examination of the cystic fluid were consistent with synovial fluid, and histopathologic results supported the diagnosis of synovial cysts. All dogs are ambulatory and 3 are asymptomatic after surgery with a follow-up time ranging from 1 to 8 months. This is the 1st report of extradural synovial cysts in dogs, and synovial cysts should be a differential diagnosis for young giant breed dogs with cervical myelopathy.

Apoptotic glial cell death and kinetics in the spinal cord of the myelin‐deficient rat

This study examined the glial cell kinetics and death in the thoracic spinal cord of normal and myelin‐deficient (md) rats between 1 and 21 days of age and determined whether the observed glial cell death primarily affected oligodendrocytes and had the morphologic and molecular features of apoptosis. In the md rat spinal cord there was an increase in cell division and death in a pattern that correlated with the onset of myelination. The dying cells were identified as oligodendrocytes ultrastructurally as many had the characteristic distention of the rough endoplasmic reticulum seen in the md rat glia. Double labeling using PLP in situ hybridization and a modified TUNEL method also suggested that the dying cells, in both mutant rats and control littermates, were oligodendrocytes. These findings were compared with previous studies on the md rat optic nerve and those in other PLP mutants. J. Neurosci. Res. 51:497–507, 1998.