Susan C. Barnett

Schwann cell-like myelination following transplantation of an olfactory bulb-ensheathing cell line into areas of demyelination in the adult CNS

In this study we have transplanted a clonal olfactory bulb‐ensheathing cell line into focal areas of the rat spinal cord which contain demyelinated axons but neither oligodendrocytes nor astrocytes. The cell line was created by retroviral incorporation of the temperature‐sensitive Tag gene into FACS‐sorted 04+ cells from 7‐day‐old rat pup olfactory bulb. The spinal cord lesions were obtained by injecting small volumes of ethidium bromide into the dorsal white matter of spinal cord previously exposed to 40 Grays of X‐irradiation. Many of the axons were remyelinated by P0+ myelin sheaths 21 days after transplantation. Light and electron microscopy revealed cells engaging and myelinating axons in a manner highly reminiscent of Schwann cells within similar lesions. GFAP+ cells were also present within the lesion. This study provides the first in vivo evidence that olfactory bulb‐ensheathing cells are able to produce peripheral‐type myelin sheaths around axons of the appropriate diameter.

Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes

Transplanted olfactory ensheathing cells (OECs) are able to remyelinate demyelinated axons and support regrowth of transected axons after transplantation into the adult CNS. Transplanted Schwann cells (SCs) share these repair properties but have limitations imposed on their behavior by the presence of astrocytes (ACs). Because OECs exist alongside astrocytes in the olfactory bulb, we have hypothesized that they have advantages over SCs in transplant‐mediated CNS repair due to an increased ability to integrate and migrate within an astrocytic environment. In this study, we have tested this hypothesis by comparing the interactions between astrocytes and either SCs or OECs, using a range of in vitro assays. We have shown that (1) astrocytes and SCs segregate into defined non‐overlapping domains in co‐culture, whereas astrocytes and OECs freely intermingle; (2) both SCs and OECs will migrate across astrocyte monolayers, but only OECs will migrate into an area containing astrocytes; (3) SCs spend less time in contact with astrocytes than do OECs; and (4) astrocytes undergo hypertrophy when in contact with SCs, but not with OECs. Expression of N‐cadherin has been implicated as a key mediator of the failure of SCs to integrate with astrocytes. However, we found no differences in the intensity of N‐cadherin immunoreactivity between SCs and OECs, suggesting that it is not the adhesion molecule that accounts for the observed differences. In addition, the number of astrocytes expressing chondroitin sulfate proteoglycans (CSPG) is increased when astrocytes are co‐cultured with Schwann cells compared with the number when astrocytes are grown alone or with OECs. Taken together, these data support the hypothesis that OECs will integrate more extensively than Schwann cells in astrocytic environments and are therefore better candidates for transplant‐mediated repair of the damaged CNS. GLIA 32:214–225, 2000.

Induction of Potent Immune Responses by Cationic Microparticles with Adsorbed Human Immunodeficiency Virus DNA Vaccines

ABSTRACT The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8+ T-cell and antibody responses by ∼100- and ∼1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIVenv and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.

Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than schwann cells following transplantation into adult cns white matter

Both Schwann cells and olfactory ensheathing cells (OECs) create an environment favorable to axon regeneration when transplanted into the damaged CNS. However, transplanted cells can also exert an effect on the host tissue that will influence the extent to which regenerating axons can grow beyond the transplanted area and reenter the host environment. In this study equivalent numbers of Lac-Z-labeled Schwann cells and OECs have been separately transplanted into normal white matter of adult rat spinal cord and the host astrocyte response to each compared. Schwann cell transplantation resulted in a greater area of increased glial fibrillary acidic protein (GFAP) expression compared to that associated with OEC transplantation. This was accompanied by a greater increase in the expression of axon growth inhibitory chrondroitin sulfate proteoglycans (CSPGs) following Schwann cell transplantation compared to OEC transplantation. However, no differences were detected in the increased expression of the specific CSPG neurocan following transplantation of the two cell types. These results mirror differences in the interactions between astrocytes and either Schwann cells or OECs observed in tissue culture models and reveal one aspect of the complex biology of creating regeneration-promoting environments by cell transplantation where transplanted OECs have favorable properties compared to transplanted Schwann cells.

Olfactory Ensheathing Cells and CNS Regeneration: The Sweet Smell of Success?

It is our current ignorance about fundamental questions such as these that expose the deficiencies in the OEC field, where the contribution these cells can make to regenerating the CNS has raced ahead of an in-depth evaluation of their basic biology. There are a number of pressing clinically related issues for which this fundamental knowledge is required. For example, although OECs can be obtained from the human olfactory system, it is nevertheless a somewhat inaccessible region. By contrast, its close relative, the Schwann cell, can be obtained much more easily from peripheral nerve. If one knew precisely how these cells differ from Schwann cells, then it might be possible by means of relatively small steps to convert Schwann cells into “OECs,” thereby providing sufficiently large numbers of these cells from a more accessible source and increasing the feasibility of autologous transplantation. Alternatively, if more were known about the mechanisms of OEC development, then we would be better placed to exploit advances in stem cell biology to generate the numbers and purity of cells required for transplantation. Thus, the realization of the enormous potential of these cells in overcoming the regenerative limitations of the CNS will require the combined efforts of many branches of the neurosciences.‡To whom correspondence should be addressed (e-mail:rjf1000@cam.ac.uk).

Olfactory ensheathing cells and CNS repair: going solo or in need of a friend?

Therapeutic transplantation using cell-based strategies has shown potential in promoting CNS repair. A range of cell types and tissues has been examined but over the past ten years olfactory ensheathing cells (OECs) have emerged as a leading candidate. Here, an appraisal of current literature on OEC transplantation is presented in the context of the basic biology of OECs and the varied cell populations that make up the peripheral olfactory system. Despite the early, intuitive belief that pure populations of OECs would be optimal for transplantation, recent evidence suggests that a more heterogeneous cellular composition might be preferable in enhancing the level of repair. Through a consideration of the different molecular and cell biological characteristics that these diverse cell types might contribute to the repair process, specific insights will be provided into the factors that could prove crucial in determining a favourable outcome for cell transplantation.

Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, 13C–glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Olfactory ensheathing cells (OECs) and the treatment of CNS injury: advantages and possible caveats

One of the main research strategies to improve treatment for spinal cord injury involves the use of cell transplantation. This review looks at the advantages and possible caveats of using glial cells from the olfactory system in transplant‐mediated repair. These glial cells, termed olfactory ensheathing cells (OECs), ensheath the axons of the olfactory receptor neurons. The primary olfactory system is an unusual tissue in that it can support neurogenesis throughout life. In addition, newly generated olfactory receptor neurons are able to grow into the CNS environment of the olfactory bulb tissue and reform synapses. It is thought that this unique regenerative property depends in part on the presence of OECs. OECs share some of the properties of both astrocytes and Schwann cells but appear to have advantages over these and other glial cells for CNS repair. In particular, OECs are less likely to induce hypertrophy of CNS astrocytes. As well as remyelinating demyelinated axons, OEC grafts appear to promote the restoration of functions lost following a spinal cord lesion. However, much of the evidence for this is based on behavioural tests, and the mechanisms that underlie their potential benefits in transplant‐mediated repair remain to be clarified.

Identification of growth factors that promote long-term proliferation of olfactory ensheathing cells and modulate their antigenic phenotype

Olfactory ensheathing cells can develop into distinct subtypes in culture after incubation in serum‐free medium conditioned by astrocytes, which have Schwann cell–like and astrocyte‐like properties. It has not been possible so far to modulate and grow large numbers of these olfactory ensheathing cell subtypes. In this study, we have shown that astrocyte‐conditioned medium, although promoting differentiation of the two olfactory ensheathing cell types, is growth‐restrictive after 14 days, probably due to the upregulation of p16 and p27. Growth arrest can be overridden and cells maintained for a further 11 weeks, by a mitogen mix of fibroblast growth factor 2, forskolin, and heregulin (olfactory mitogen medium) combined with astrocyte‐conditioned medium. In the absence of astrocyte‐conditioned medium, combinations of the same factors can also override growth arrest but to a lesser extent. Olfactory mitogen medium combined with astrocyte‐conditioned medium upregulates O4 and low‐affinity nerve growth factor receptor expression on olfactory ensheathing cells, leading to a 100% Schwann cell–like phenotype. If cells are maintained in olfactory mitogen medium alone, or if they are treated with forskolin or fibroblast growth factor 2 diluted in serum‐free medium, O4 and low‐affinity nerve growth factor receptor expression remains at 100%, but there is also an increase in expression of E‐NCAM, the astrocyte‐like marker. Medium containing serum also overrides growth arrest, but for only 4 weeks, during which time most differentiation‐specific markers disappear. These studies have allowed us to define conditions to modulate the olfactory ensheathing cell phenotype. GLIA 37:349–364, 2002.

Olfactory ensheathing cell transplantation as a strategy for spinal cord repair--what can it achieve?

Restoring function to the injured spinal cord represents one of the most formidable challenges in regenerative medicine. Glial cell transplantation is widely considered to be one of the most promising therapeutic strategies, and several differentiated glial cell types—in particular, Schwann cells and olfactory ensheathing cells (OECs)—have been proposed as transplant candidates. In this Review, we analyze evidence from animal studies for improved functional recovery following transplantation of OECs into spinal cord injuries, and examine the mechanisms by which repair might be achieved. Data obtained using various injury models support the view that OEC transplants can promote functional recovery, but accumulating anatomical evidence indicates that although axons regenerate within a transplant, they do not cross the lesion or reconnect with neurons on the opposite side to any significant extent. Consequently, it is possible that neuroprotection and promotion of sprouting from intact fibers are the main mechanisms that contribute to functional recovery. We conclude that for the foreseeable future the clinical benefits of OEC transplants alone are likely to be modest. The future potential of cell transplantation strategies will probably depend on the success with which the transplants can be combined with other, synergistic, therapies to achieve significant regeneration of axons and re-establish functionally useful connections across a spinal cord injury.