Lars Olson
 

Lars Olson is professor at Karolinska Institute, Stockholm, Sweden e-mail: lars.olson@neuro.ki.se.

In perpetual search of better therapies for human afflictions, humankind is scouring the surrounding biosphere for plant- and animal-derived remedies. As it turns out, transplantation of living cells offers particularly rich possibilities. Cells can be grafted to replace lost cells, provide molecules such as hormones, growth factors, or signaling chemicals, or proliferated to replace tissues or even organs. They can also be used as long-term sources of these molecules. All of these applications often require that the cells be genetically modified before grafting. On page 949, Imaizumi et al.¹ describe a cell-grafting approach designed to overcome the immunological problems of xenografting¹ for axonal regeneration of spinal cord injuries. They have developed transgenic pigs armed with the gene encoding human complement-inhibitory protein (hCD59). From these pigs, they collected two important nerve fiber-ensheathing cells: olfactory ensheathing cells (OECs) and Schwann cells. When these cells were delivered into the spinal cords of rats with lesions through the dorsal column of the spinal cord, they induced axonal regeneration and remyelination, restoring impulse conduction across the severed spinal cord.

As experimental data accumulate, demonstrating the feasibility and efficacy of cell grafting strategies for diverse conditions ranging from diabetes to Parkinson's disease, the need for new sources of useful cells has become evident. In general, the transplantability of cells is directly proportional to their degree of immaturity, and inversely proportional, for immunological and other reasons, with increasing phylogenetic distance from Homo sapiens .

The most desirable source for cells to be transplanted is the body of the human recipient (or an identical twin thereof), provided that the collection of cells is not too unpleasant. As plastic surgeons and others know well, humans do possess considerable renewable sources of cells/tissues that can be grafted to serve many purposes—and without causing any immunological disturbance.

Another option, that of obtaining cells from another human being, carries all the well-known, but largely manageable, immunological complications of grafting between individuals of our own species. Such grafting also includes the possibility of obtaining immature cells, which can be collected following early elective abortions. Experimental fetal cell transplants for treatment of Parkinson's disease are an example.

In the present study, Imaizumi et al. report interesting results from experiments using the third available option, xenografting. For certain animal species, such as pigs, it appears that ethical consensus can be reached regarding suitability as cell, tissue, and organ donors for humans. However, the immunological problems with xenografts constitute a major obstacle. For instance, as old-world primates, we produce antibodies against 1,3-galactosyl-terminated glycolipids and glycoproteins, probably because our gastrointestinal tract is colonized by bacteria expressing these antigens. Pigs, on the other hand, naturally express the same antigens, for example on the lumenal side of blood vessels. Therefore, antibodies occurring naturally in the human host can mount a rapid and detrimental attack on the endothelial cells of blood vessels in a vascularized pig-to-human graft, with complement-mediated cell lysis leading to hyperacute rejection within minutes to hours².

To overcome these and other immunological problems of xenografting, the researchers used pigs expressing human CD59, a complement inhibitor, as a source of two nerve ensheathing cell types known to support regeneration, OECs and Schwann cells. The researchers then asked if these two cell types express the transgene product, if they can be grafted to another species, and if they have functional effects.

OECs are particularly interesting³. Ensheathing the nonmyelinated olfactory axons all the way from the olfactory epithelium into the CNS environment of the olfactory bulb, these cells naturally support a remarkable degree of regeneration of olfactory axons in adult individuals. Rats OECs can be harvested and grown in vitro, and when grafted to other rats will support axons of many kinds and also myelinate them, even though they do not normally produce myelin in rodents. The OECs form a peripheral type of myelin sheath, including a basement membrane, much like those formed by Schwann cells and unlike the normal oligodendrocyte-derived myelin sheaths of the CNS. Several examples have been published of the promising reparative properties of OECs when grafted to CNS lesions. They populate the engrafted area, orient themselves in a directionally organized manner, migrate into adjacent noninjured sites, and stimulate and guide axonal regeneration both in the spinal cord and in the brain^{4, 5}.

Imaizumi et al. first demonstrated that OECs and Schwann cells cultured from the transgenic pigs expressed the desired transgene product on their cell surfaces. They then made lesions through the dorsal column of the spinal cord in adult immunosuppressed rats and delivered OECs, Schwann cells, or vehicle into the lesioned area (see Fig. 1). A month later spinal cords were removed to determine whether electrical stimuli applied below the transection of the dorsal columns could evoke compound action potentials in the spinal cord above the injury. Both Schwann cells and OECs had positive effects and promoted regeneration of ascending sensory pathways, as shown by action potentials as far as 14 mm craniad to the lesion. This is compatible with the typical rate of nerve fiber growth, which is limited to about 1 mm/day by the slow rate of anterograde axonal transport. Surprisingly, the regenerated fibers appeared to conduct axon potentials even faster than their normal counterparts, suggesting effective myelination. Histological examinations demonstrated migration of the xenografted cells as well as the formation of peripheral-type myelin sheaths. In all, the transgenic pig cells behaved as predicted from similar rat-to-rat grafting experiments conducted by the same research team.

Though unthinkable only a decade or two ago, it now appears that reparative treatment for spinal cord injury may be within reach. Several different lines of experimental evidence⁶ suggest that the combined use of strategies to minimize secondary degenerative events—including application of tissue or cellular bridges across the injury zone, blockade of naturally occurring nerve growth-inhibitory mechanisms, guidance of growing axons, and chemical stimulation of axon growth and synapse formation—may eventually lead to clinically useful treatments for this severely debilitating condition. Similarly, other diseases and injuries in the human CNS may benefit from ongoing research, with the OECs as one current focus.

Studies showing that not only bone marrow, but also the adult CNS and perhaps most tissues in the body, harbor pluripotent stem cells, combined with the rapidly increasing understanding of how to harvest, grow, and differentiate such cells, have resulted in the hope that they may become clinically useful^{7, 8}. Such cells can differentiate into virtually any and all cell and tissue types, regardless of their sources. For instance, stem cells found in the adult brain can give rise to cell types from all germ layers⁹. Conversely, and therapeutically more attractive, stem cells collected, say from bone marrow, or even from a blood sample of an individual, might be used to grow specified cell types or perhaps even tissues for autografting back to the same individual. It may thus be assumed that human-derived cells—whether from each individual in need of a cellular therapy, or from a limited number of individuals from whom cell lines have been established and chosen to match different immunological requirements—will greatly diminish the need for animal-derived cells.

However, the case for xenotransplants remains strong when it comes to organs. Hearts, lungs, kidneys, and livers cannot—yet, one should probably caution—be grown from stem cells, and there is a severe shortage of human donors of these and other organs and tissues. Unfortunately, there are still many hurdles in the way. Even when grafting to the brain, regarded as having a degree of immunological privilege², pig embryonic brain tissue xenografts elicit both a humoral and a cell-mediated response leading to rejection by an adult rat within a month¹². Therefore, to increase imunotolerance, animals such as pigs will likely need need further genetical engineering. Although recently a transgenic pig heart was reported to have survived a month in a baboon¹⁰, multiple genetic changes such as those exemplified by the recently described triple-transgenic pig¹¹ will presumably be needed to obtain useful survival times. In this context, the recent announcement of the cloning of pigs is an important milestone (Nat. Biotechnol. 18, 365, 2000).

Do xenografts pose risks? Certainly. With the recent outbreak of bovine encephalopathy fresh in their memories, scientists and official bodies have urged that exceptional measures be taken to safeguard against the possible cross-species transmission of animal diseases to humans. Viral zoonotic risks include porcine endogenous retrovirus (PERV), which can infect primate cells¹³. The US Public Health Service recently stated that "all xenotransplant products pose a risk of infection and disease to humans" and that this applies to "all species", and has moved to tighten guidelines for xenograft research¹⁴. In the end, however, such precautions should help to define the safety issues and hopefully to resolve them.

Meanwhile, work such as that reported here should continue to spark interest in innovative ways to reduce immunological barriers associated with porcine xenografts, to use xenografts for treating a variety of human disorders.

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