Jeremy P. Brockes

Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve.

We have previously reported that in dissociated cultures of neonatal rat sciatic nerve, all of the cells could be identified by indirect immunofluorescence with two antisera to cell surface antigens. The Schwann cells, but not the fibroblasts, expressed the Ran-1 antigen, while the fibroblasts, but not the Schwann cells, expressed the Thy-1 antigen. We have exploited this difference to derive pure populations of Schwann cells. A combination of [3H]thymidine autoradiography and immunofluorescence marking showed that in Modified Eagles Medium with 10% foetal calf serum, the Schwann cells divided slowly while the fibroblasts divided rapidly. Accordingly, two day old cultures were exposed to cytosine arabinoside to select against the fibroblasts, followed by growth in medium containing an extract of bovine pituitary which stimulated division of the Schwann cells. After 7 days the confluent cultures, which contained 80-90% Schwann cells, were passaged after treatment in suspension with antiserum to Thy-1 and rabbit complement. After continued growth in medium with pituitary extract, the secondary cultures contained greater than 99.5% Schwann cells. These purified populations have been maintained in culture for as long as 150 days (6 passages) and retained the Ran-1 marker. The cultured Schwann cells expressed the S100 antigen, as shown by indirect immunofluorescence and complement fixation, and receptors for cholera toxin. They did not express the large external transformation sensitive protein, the glial fibrillary acidic protein, or receptors for tetanus toxin.

Appendage Regeneration in Adult Vertebrates and Implications for Regenerative Medicine

The regeneration of complex structures in adult salamanders depends on mechanisms that offer pointers for regenerative medicine. These include the plasticity of differentiated cells and the retention in regenerative cells of local cues such as positional identity. Limb regeneration proceeds by the local formation of a blastema, a growth zone of mesenchymal stem cells on the stump. The blastema can regenerate autonomously as a self-organizing system over variable linear dimensions. Here we consider the prospects for limb regeneration in mammals from this viewpoint.

Comparative Aspects of Animal Regeneration

Most but not all phyla include examples of species that are able to regenerate large sections of the body plan. The mechanisms underlying regeneration on this scale are currently being studied in a variety of contexts in both vertebrates and invertebrates. Regeneration generally involves the formation of a wound epithelium after transection or injury, followed by the generation of regenerative progenitor cells and morphogenesis to give the regenerate. Common mechanisms may exist in relation to each of these aspects. For example, the initial proliferation of progenitor cells often depends on the nerve supply, whereas morphogenesis reflects the generation of positional disparity between adjacent cells-the principle of intercalation. These mechanisms are reviewed here across a range of contexts. We also consider the evolutionary origins of regeneration and how regeneration may relate to both agametic reproduction and to ontogeny.

Schwann cell growth factors

Purified rat Schwann cells were found to proliferate very slowly in normal growth medium containing 10% fetal calf serum (FCS). Crude extracts of bovine pituitary or brain markedly enhanced Schwann cell growth, while similar extracts of nerve roots, liver and kidney did not. Pituitary extracts were more potent than brain extracts, and extracts from both anterior and posterior pituitary were active. The mitogenic activity of pituitary extracts was reduced by treatment with trypsin, and abolished by pronase and by boiling. A variety of known anterior and posterior pituitary hormones, as well as fibroblast, epidermal and nerve growth factors, were not mitogenic. FCS (greater than 1%) was required for Schwann cell proliferation, but even high concentrations of FCS did not substitute for pituitary or brain extracts, and serum from various other species did not support Schwann cell growth. Although various agents that increase cyclic AMP levels (such as cholera toxin) had been shown to be Schwann cell mitogens, extracts of pituitary or brain did not increase cyclic AMP levels. Extracts of various bovine tissues, including pituitary, brain, liver and kidney, acted synergistically with cholera toxin in stimulating Schwann cell proliferation, although the increase in cyclic AMP induced by the mixture was not greater than that seen with cholera toxin alone. We conclude that there are at least two separate pathways for stimulating Schwann cell division, only one of which involves an increase in intracellular cyclic AMP.

Recurrent turnover of senescent cells during regeneration of a complex structure

Cellular senescence has been recently linked to the promotion of age-related pathologies, including a decline in regenerative capacity. While such capacity deteriorates with age in mammals, it remains intact in species such as salamanders, which have an extensive repertoire of regeneration and can undergo multiple episodes through their lifespan. Here we show that, surprisingly, there is a significant induction of cellular senescence during salamander limb regeneration, but that rapid and effective mechanisms of senescent cell clearance operate in normal and regenerating tissues. Furthermore, the number of senescent cells does not increase upon repetitive amputation or ageing, in contrast to mammals. Finally, we identify the macrophage as a critical player in this efficient senescent cell clearance mechanism. We propose that effective immunosurveillance of senescent cells in salamanders supports their ability to undergo regeneration throughout their lifespan. DOI: http://dx.doi.org/10.7554/eLife.05505.001

Cell surface markers for distinguishing different types of rat dorsal root ganglion cells in culture

The distribution of three cell surface markers on dissociated cultures of the rat dorsal root ganglion has been investigated using indirect immunofluorescence. Tetanus toxin binds to a nondividing class of cells with neuronal morphology. Ran-1 is expressed by Schwann cells. Thy-1 is expressed both by fibroblasts and by all the neurones. These assignments have been confirmed by detection of any two of these markers in a single culture using antisera conjugated to different fluorochromes. Approximately 20% of the nonneuronal cells in these cultures were negative for all three markers.

Nerve dependence in tissue, organ, and appendage regeneration

Many regeneration contexts require the presence of regenerating nerves as a transient component of the progenitor cell niche. Here we review nerve involvement in regeneration of various structures in vertebrates and invertebrates. Nerves are also implicated as persistent determinants in the niche of certain stem cells in mammals, as well as in Drosophila. We consider our present understanding of the cellular and molecular mechanisms underlying nerve dependence, including evidence of critical interactions with glia and non-neural cell types. The example of the salamander aneurogenic limb illustrates that developmental interactions between the limb bud and its innervation can be determinative for adult regeneration. These phenomena provide a different perspective on nerve cells to that based on chemical and electrical excitability.

The Newt Ortholog of CD59 Is Implicated in Proximodistal Identity during Amphibian Limb Regeneration

The proximodistal identity of a newt limb regeneration blastema is respecified by exposure to retinoic acid, but its molecular basis is unclear. We identified from a differential screen the cDNA for Prod 1, a gene whose expression in normal and regenerating limbs is regulated by proximodistal location and retinoic acid: Prod 1 is the newt ortholog of CD59. Prod 1/CD59 was found to be located at the cell surface with a GPI anchor which is cleaved by PIPLC. A proximal newt limb blastema engulfs a distal blastema after juxtaposition in culture, and engulfment is specifically blocked by PIPLC, and by affinity-purified antibodies to two distinct Prod 1/CD59 peptides. Prod 1 is therefore a cell surface protein implicated in the local cell-cell interactions mediating positional identity.

Transfer of scrapie prion infectivity by cell contact in culture.

BACKGROUND When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. RESULTS We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. CONCLUSIONS Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.

Regeneration as an evolutionary variable

Regeneration poses a distinctive set of problems for evolutionary biologists, but there has been little substantive progress since these issues were clearly outlined in the monograph of T. H. Morgan (1901). The champions at regeneration among vertebrates are the urodele amphibians such as the newt, and we tend to regard urodele regeneration as an exceptional attribute. The ability to regenerate large sections of the body plan is widespread in metazoan phylogeny, although it is not universal. It is striking that in phylogenetic contexts where regeneration occurs, closely related species are observed which do not possess this ability. It is a challenge to reconcile such variation between species with a conventional selective interpretation of regeneration. The critical hypothesis from phylogenetic analysis is that regeneration is a basic, primordial attribute of metazoans rather than a mechanism which has evolved independently in a variety of contexts. In order to explain its absence in closely related species, it is postulated to be lost secondarily for reasons which are not understood. Our approach to this question is to compare a differentiated newt cell with its mammalian counterpart in respect of the plasticity of differentiation.