Project description:Autologous stem cell therapy has potential for biologic treatment of disc degeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal stem cells (AD-MSC) are readily available. Our objectives were: 1) To develop/validate methods to harvest AD-MSC and direct them to a disc-like phenotype by three-dimensional (3D) culture and TGF-Ã3 exposure; 2) To perform gene expression profiling for human AD-MSC and annulus cells in 3D culture; 3) To test whether disc cell-AD-MSC co-culture could augment proteoglycan production. Stem cell plasticity offers potential for future biologic therapies for disc degeneration. Data indicated that human AD-MSC can successfully be manipulated in 3D culture to express gene products important in the disc ECM (types I and II collagen, chondroitin sulfate, keratin sulfate, decorin), and that co-culture of annulus cells with AD-MSC enhances proteoglycan production. Studies defined gene expression patterns of AD-MSC and human annulus cells in 3D culture, important as we explore the potential of MSC in biologic therapies for disc degeneration. Experiment Overall Design: AD-MSC were extracted from human adipose tissue, and characterized as stem cells using accepted criteria (direction into osteoblasts or chondrocytes; and cell surface marker criteria). Three AD-MSC cultures were grown in 3D with or without TGF-Ã3 for 2-3 weeks. Disc Tissue samples were obtained from surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Seven disc cultures were also grown in 3-D for 2 weeks. RNA was harvested according to instructions with the Trizol isolation method, checked for quality using the 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA USA), reverse-transcribed to double-stranded cDNA, subjected to two rounds of transcription, and hybridized to the DNA microarray in the Affymetrix Fluidics Station 400. Affymetrix human U133 X3P arrays were used. Using Genesifter, gene expression in AD-MSC with and witout TGF-B3 was compared to annulus cells. Gene expression of stem cells with TGF-Ã3 was also compared to stem cells grown in the absence of TGF-Ã3.

Project description:Although the degenerating disc is considered to be the key source of pain in patients with low back pain, the relationship between disc cells, nerves, and pain production is poorly understood. Neurotrophins are signaling molecules involved in the survival, differentiation, migration, and neurite outgrowth of central and peripheral neurons. Neurotrophins are now known to be expressed in non-neuronal tissues, including the intervertebral disc. We hypothesize that the inflammatory cytokine interleukin-1 (IL-1beta), which is produced by disc cells during degeneration, is a key element in the cascade of events involving neurotrophins. To test this, we used an in vitro experimental design which challenged 3D-cultured human annulus cells with IL-1beta and utilized microarray analysis to evaluate neurotrophin- and nerve-related gene expression profiles in treated vs. control cells. Analysis of nerve growth factor levels in conditioned media was also performed. Findings presented here support the hypothesis that proinflammatory cytokines (which are produced by disc cells during degeneration) are involved with a significant increase in the expression of neurotrophins and other nerve-related genes. Findings expand previous data on expression of neurotrophins in the degenerating disc, and provide the first documentation of expression of neurotrophin 3 and neuropilin 2. These results have direct translational relevance because they address the primary clinical issue with disc degeneration (low back pain) and open the possibility of novel analgesic therapies based on development of specific small-molecular antagonists to neurotrophins. Human disc tissue samples were obtained from surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Cultured annulus cells were grown in a 3D collagen construct with or without 10 2 pM IL-1 for a total of 14 days. Following homogenization in TRIzol reagent, total RNA was isolated and analyzed via mircoarray.

Project description:The presence of senescent cells in the aging/degenerating human disc is now well-recognized. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue, however, offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells. Here we use a novel experimental design using laser capture microdissection to selectively separately harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and then compare their gene expression with microarray analysis. An initial in vitro study using cultured human annulus cells was first performed to test whether there was any difference in identification of senescent cells using the accepted histochemical methodology vs. the immunofluoresent identification of cells positive for senescence-associated-ß-galactosidase in control cells and cells induced into stress-induced premature senescence via hydrogen peroxide exposure. No statistically significant difference was found between the 2 methods. Laser capture microdissection was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens, and microarray analysis was used to determine gene expression levels. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells. Additional genes related to cytokines, cell proliferation, and other cell processes were also identified. Disc Tissue samples were obtained from surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Tissue was fixed and paraffin embedded. Standard laser capture microdissection (LCM) techniques were used to collect senescent cells. Remaining non-senescent cells were scraped from the histology slide. Total RNA was isolated and analyzed via mircoarray. Gene expression from senescent cells was compared to non-senescent cells. Eight histological samples were used to obtain both senescent and non-senescent cells. From an additional 3 samples, only senescent cells were harvested.

Project description:Asporin, also known as periodontal ligament-associated protein 1 (PLAP1), is a member of the family of small leucine-rich proteoglycan (SLRP) family. It is present within the cartilage extracellular matrix (ECM), and is reported have a genetic association with osteoarthritis. Its D14 allele has recently been found to be associated with lumbar disc degeneration in Asian subjects. There have been no studies, however, of this gene’s normal immunohistochemical localization within the human intervertebral disc, nor of expression levels in Caucasian individuals with disc degeneration. Studies were approved by our human subjects Institutional Review Board. Methods included immunohistochemical localization of asporin in the disc of humans and the sand rat (a small rodent with spontaneous age-related disc degeneration), and Affymetrix microarray analysis of asporin gene expression in vivo and in vitro. mmunohistochemical studies of human discs revealed that some, but not all, cells of the outer annulus expressed asporin. Fewer cells in the inner annulus contained asporin, and it was rarely present in cells in the nucleus pulposus. Similar patterns were found for the presence of asporin in lumbar discs of sand rats. Substantial relative gene expression levels were seen for asporin in both disc tissue and in annulus cells grown in three-dimensional culture. More degenerate human discs (Thompson grade 4) showed higher expression levels of asporin than did less degenerate (grade 1, 2 and 3) discs, p = 0.004. In the discs of Caucasian subjects studied here, and in the sand rat, greater immunolocalization levels were found in the outer compared to inner annulus. Localization was rare in the nucleus. Gene expression studies showed greatest expression of asporin in the more degenerate human discs in vivo. Disc Tissue samples were obtained via the National Cancer Institute Cooperative Tissue Network (CHTN) as well as surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Tissue was fixed and paraffin embedded. Standard laser capture microdissection (LCM) techniques were used to collect the cells from which total RNA was isolated and analyzed via microarray. Based on the Thompson scouring system, unhealthy discs (grade 4) were compared to healthy discs (grades 2,3).

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