• Wayne Murrell, Emma Sanford, Leif Anderberg, Brenton Cavanagh, and Alan Mackay-Sim.
• National Centre for Adult Stem Cell Research, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Australia. Wayne.Murrell@rr-research.no
• Spine J. 2009 Jul 1;9(7):585-94.

BackgroundIn humans, lower back pain is one of the most common causes of morbidity. Many studies implicate degeneration of intervertebral discs as the cause. In the normal intervertebral disc, the nucleus pulposus exerts a hydrostatic pressure against the constraining annulus fibrosus, which allows the disc to maintain flexibility between adjacent vertebrae, while absorbing necessary compressive forces. The nucleus pulposus performs this role because of its hydrophilic gel-like structure. The extracellular matrix of the nucleus pulposus is up to 80% hydrated, as a result of large amounts of the aggregating proteoglycan, chondroitin sulfate proteoglycan (CSPG). This proteoglycan is enmeshed in a randomly orientated network of fine collagen Type II (CT2) fibers. STUDY DESIGN AND PURPOSE: A useful adult tissue-derived stem cell is that from the olfactory mucosa, the organ of smell. These cells, accessible in humans from nasal biopsies, are multipotent and are able to make many cell types from all germ layers. They are easily grown in vitro and can be expanded to large numbers and stored frozen. These qualities indicate the potential for autologous transplantation for disc repair. In this article, using a rat model, we explore the hypothesis that olfactory stem cells can differentiate into a nucleus pulposus chondrocyte phenotype in vitro, as well as in vivo after transplantation into the injured intervertebral disc.Patient SampleFemale rats (14 weeks) were anesthetized with xylazine/ketamine. The abdominal wall was shaved and injected with local anesthetic (lidocaine) before incision. The ventral part of the lumbar spine, including two intervertebral discs, was exposed. Disc degeneration was then induced in the two exposed discs by needle aspiration of the nucleus pulposus. The prominent spina iliaca posterior superior was used as an anatomical landmark for identification of the first disc. Two weeks later, one injured intervertebral disc was exposed in a second, similar, surgery and 20,000 olfactory neurosphere-derived cells were transplanted with a 25-G needle.Outcome MeasuresIn vitro induction of nucleus pulposus chondrocyte phenotype is measured by the percentage of cells expressing CT2 and CSPG. In vivo, a successful outcome is evidence of engraftment of donor-derived cells and their expression of CT2 and CSPG.MethodsIn this article, we tested two hypotheses: the first that progenitor cells within olfactory neurospheres could be induced to express markers distinctive of the nucleus pulposus when placed in vitro in a coculture experiment. The second hypothesis tested the same induction in genetically labeled transplanted cells within damaged vertebral discs in vivo. The two markers measured are those held by current literature to engender the necessary cushioning characteristics of nucleus pulposus, CT2 and CSPG.ResultsOur experiments demonstrated virtually 100% induction of these two markers in vitro. Also, this induction was achieved in donor-derived cells after delivery to the nucleus pulposus region of animals whose discs had previously been lesioned 2 weeks before transplant.ConclusionsThese results provide a rationale for moving toward more extensive larger animal studies for assessment of regeneration before human trials where relief of symptoms can be more easily assessed.

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