Cell Isolation and Culture
Following the approved guidelines set by the U.S. National Institutes of Health Office of Human Subjects Research for use of surgical waste, we obtained human intervertebral disc samples from six individuals (thirteen to sixteen years old) who had undergone discectomy for surgical management of scoliosis at the University of Virginia Hospital. Samples from this source are likely to be more normal than those obtained from patients with degenerative disc disease. Following an anterior approach to the spine, an anulotomy was performed with a number-11 blade. A sharp Cobb elevator was then used to separate the disc from the osseous end plate so that as much of the disc as possible could be removed. As the disc samples were obtained from an anterior approach, no extraneous ligaments were attached. The outer layer of anulus fibrosus, rich in fibers and low in cells, was dissected in order to avoid the contamination of ingrown cells from nerves and small blood vessels. The poorly structured, gelatinous parachordal inner anulus fibrosus lies adjacent to the nucleus pulposus, and as it is difficult to discern nucleus pulposus from anulus fibrosus, these regions were removed to rule out nucleus pulposus contamination. The remaining transition zone containing solely anulus fibrosus tissue was then cut into small pieces and digested with 0.01% collagenase (Crescent Chemical, Islandia, New York) at 37°C for two to four hours. At the end of this time, the aqueous portion was carefully removed and centrifuged at 500 g for ten minutes. The resulting cell pellet was resuspended in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, New York) with erythrocyte lysis buffer (160 mM of NH4Cl), agitated at room temperature for ten minutes, and recentrifuged to obtain a pellet. The cells were then resuspended and placed along with undigested anulus fibrosus tissue into DMEM/F12 (DMEM: Nutrient Mixture F-12; Gibco BRL) containing 10% fetal bovine serum (Gibco Invitrogen, Carlsbad, California) and 1% penicillin-streptomycin, and then were plated in a 100-mm tissue-culture dish and were maintained at 37°C in a humidified incubator with 5% CO2. Culture media was changed every other day. Cells were maintained at subconfluent levels and were passaged sequentially with use of trypsin-EDTA (Gibco BRL).
Flow Cytometry
To characterize the anulus fibrosus population, flow cytometry was performed with a BD FACSCalibur (Becton Dickinson, Franklin Lakes, New Jersey) as previously described16. Sources of antibodies are listed in a table in the Appendix. Among these antibodies are cell surface antigens expressed in adipose-derived stem cells or mesenchymal stem cells, for instance, CD29, CD49e, CD51, CD73, CD90, CD105, CD166, CD184, and Stro-1; cell surface antigens for hematopoietic and endothelial lineages, such as CD31, CD34, CD45, CD106, CD117, and CD13314,16,17; and neuronal stem cell markers nestin and neuron-specific enolase; CD24 is a surface marker for nucleus pulposus cells, but it is weakly expressed in anulus fibrosus cells18. Briefly, approximately 3 to 5 × 105 anulus fibrosus cells were stained with phycoerythrin (PE) or fluorescein isothiocyanate (FITC)-conjugated antibodies and isotype-matched controls (isotype control IgG1 [immunoglobulin G1] PE or isotype control IgG1 FITC) and were incubated in the dark for thirty minutes at 4°C. After incubation, cells were washed three times with buffer and resuspended in 0.25 mL of cold, protein-free phosphate-buffered saline with 0.25 mL cold formaldehyde (2%) solution as a preservative.
Adipogenic Differentiation
Adipogenic differentiation was induced with use of the Adipogenesis Assay Kit (Chemicon International, Temecula, California) as per the manufacturer's instructions. Briefly, second to fourth-passage 100% confluent anulus fibrosus cells were incubated with Adipogenesis Induction Media (DMEM/F12 supplemented with 10% fetal bovine serum, 0.5 M 1-methyl-3-isobutylxanthine, 10 µg/mL insulin, 1 µM dexamethasone, and 100 µM indomethacin) for six days at 37°C and 5% CO2. The Adipogenesis Initiation Media was replaced with the Adipogenesis Maintenance Media for two days. Cells were then induced with Adipogenesis Induction Media for an additional six days, Adipogenesis Maintenance Media for two days, and finally Adipogenesis Induction Media for another five days.
Osteogenic Differentiation
To induce osteogenic differentiation, second to fourth-passage 100% confluent anulus fibrosus cells were treated with osteogenic induction media for four weeks. Osteogenic induction media consists of DMEM/F12 supplemented with 0.01 µM 1,25-dihydroxyvitamin D3 (R & D Systems, Minneapolis, Minnesota), 50 µM ascorbate-2-phosphate, and 10 mM ß-glycerophosphate, as previously reported19.
Chondrogenic Differentiation
Chondrogenesis of anulus fibrosus cells was induced in a pellet (micromass) cell culture system as previously described20. Briefly, 2 × 105 anulus fibrosus cells that had undergone between two and four passages were gently centrifuged for five minutes at 500 g in a 15-mL polypropylene tube to form a pellet at the bottom of the tube and then were treated with chondrogenic induction media for three weeks. Chondrogenic induction media consisted of DMEM/F12 supplemented with 1% fetal bovine serum, 10 nM dexamethasone, 10 ng/mL transforming growth factor ß1 (BD Biosciences, Franklin Lakes, New Jersey), 1% ITS-Premix (6.25 g/mL insulin, 6.25 g/mL transferrin, 6.25 ng/mL selenous acid, 1.25 mg/mL bovine serum albumin, and 5.35 mg/mL linoleic acid; Collaborative Biomedical, Becton Dickinson, Bedford, Massachusetts), and 37.5 g/mL ascorbic-2-phosphate.
Neurogenic Differentiation
Neurogenic differentiation was induced with a multistep protocol with use of the NeuroCult NS-A Proliferation Kit and NeuroCult NS-A Differentiation Kit (STEMCELL Technologies, Vancouver, British Columbia, Canada) as per the manufacturer's instructions. Briefly, second to fourth-passage anulus fibrosus cells were seeded at a density of 5 × 104 viable cells/cm2 in 4 mL "Complete" NeuroCult NS-A Proliferation Medium in a six-well plate. Media was replenished (2 mL) at days 2, 4, and 6 after plating, and the neurosphere cultures were ready for neurogenic differentiation once the diameter of the spheres had reached 100 µm. For the neurogenic differentiation, single cells were dissociated from neurospheres at days 7 to 10. The cells were resuspended in "Complete" NeuroCult NS-A Differentiation Medium at a cell density of 1 × 105 cells/cm2 and were plated in eight-well BioCoat Poly-D-Lysine Culture Slides (Becton Dickinson). Half of the medium was replaced with fresh medium every two days, and neurogenesis was assessed on days 7 and 10. The "Complete" NeuroCult NS-A Proliferation Medium consisted of NeuroCult NS-A Basal Medium supplemented with 1/10 volume of NeuroCult NS-A Proliferation Supplements, 20 ng/mL human epidermal growth factor, 10 ng/mL human fibroblast growth factor-b, and 2 µg/mL heparin. The "Complete" NeuroCult NS-A Differentiation Medium consisted of NeuroCult NS-A Basal Medium supplemented with 1/10 volume of NeuroCult NS-A Differentiation Supplements.
Endothelial Cell Differentiation
Anulus fibrosus cells were plated in eight-well BioCoat Poly-D-Lysine Culture Slides at a density of approximately 5000 cells/cm2 and induced with Endothelial Cell Growth Medium MV 2 (PromoCell, Heidelberg, Germany), which consists of 5% fetal bovine serum, 5.0 ng/mL epidermal growth factor, 0.2 µg/mL hydrocortisone, 0.5 ng/mL vascular endothelial growth factor, 10 ng/mL basic fibroblast factor, 20 ng/mL R3 insulin-like growth factor-1, and 1 µg/mL ascorbic acid. Endothelial cell differentiation was assessed at days 3, 5, and 7.
Cytohistological Staining
Oil-Red-O Staining:
Cells were fixed with 4% formaldehyde, stained with oil-red-O solution (Chemicon International) for fifty minutes, and then counterstained with hematoxylin solution (Chemicon International) for fifteen minutes. Lipid droplet formation was quantified by ultraviolet-visible spectrometry, which revealed the OD520 value of oil red O-stained lipids in lysates.
Alizarin-Red-S Staining:
For evaluation of mineralized matrix, cells were fixed with ice-cold 70% ethanol for one hour, stained with Alizarin Red S Solution (Chemicon International) for thirty minutes, and viewed under a light microscope.
Safranin-O Staining:
Chondrogenic differentiation was evaluated after pellets were fixed with 10% buffered formalin for four hours. The samples were dehydrated by treatment with a series of graded alcohols and embedded in paraffin. Samples were then cut into 5-µm sections, rehydrated, and stained with safranin O for detection of proteoglycan.
Type-II Collagen Immunostaining and Immunofluorescence
Immunochemical staining for type-II collagen was performed with use of a collagen staining kit (Chondrex, Redmond, Washington). For immunofluorescence staining of intracellular proteins, cells growing on the glass coverslips were fixed with 4% formaldehyde for ten minutes and incubated with one of the mouse primary antibodies, including those against human aggrecan (1:50; RDI, Flanders, New Jersey), human ß-tubulin (1:150; Invitrogen, Carlsbad, California), human microtubule-associated protein 2 (1:150; Santa Cruz Biotechnology, Santa Cruz, California), human neurofilament light chain (1:150; Invitrogen), and von Willebrand factor (vWF) (1:150; Santa Cruz Biotechnology), overnight at 4°C. This was followed by fluorescein or phycoerythrin-coupled goat antimouse IgG secondary antibody for one hour. Cells were counterstained with the fluorescent dye YOYO-1 iodide (1:3000; Invitrogen) for nucleic acids.
Gene Expression Analysis
Total RNA from cultured cells or cell pellets was obtained with use of the RNeasy Mini Kit (QIAGEN, Valencia, California) according to the manufacturer's instructions. cDNA was then synthesized from the total RNA with use of the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, California) according to the manufacturer's instructions. Polymerase chain reaction was performed with iQ Supermix (Bio-Rad), and real-time polymerase chain reaction was then performed with iTaq SYBR Green Supermix with ROX (Bio-Rad). Amplification primers are listed in a table in the Appendix.
Statistical Analysis
Statistical evaluation was performed with use of the analysis-of-variance test followed by a post hoc Student t test. A p value of <0.05 was considered significant. Data are expressed as the mean and standard deviation.
Source of Funding
The authors did not receive grants or outside funding in support of this research.
Human Anulus Fibrosus Cell Expression of Mesenchymal and Neuronal Stem Cell Markers
We investigated the expression of eighteen different cell markers, including CD29, CD31, CD34, CD45, CD49e, CD51, CD73, CD90, CD105, CD106, CD117, CD133, CD166, CD184, nestin, neuron-specific enolase, and Stro-1 in human anulus fibrosus cells as well as a marker for nucleus pulposus cells, CD24. In addition, dead cells were excluded with use of 7-aminoactinomycin D, with approximately 98% of the total cells negative for 7-aminoactinomycin D, indicating very high cell viability during flow cytometric analysis. Specificity of the secondary antibody was confirmed by negatively staining cells with the PE-labeled mouse IgG alone, with no staining of >99% of the total cells. The immunophenotype of the markers in cultured anulus fibrosus cells is illustrated in Figure 1. Positively stained markers included the majority of the cell surface antigens expressed in adipose-derived stem cells and mesenchymal stem cells, i.e., CD29, CD49e, CD51, CD73, CD90, CD105, CD166, and CD184, as well as the neuronal stem cell markers nestin and neuron-specific enolase. By contrast, cell surface antigens for hematopoietic and endothelial lineages were either only partially present or no binding was identified at all. Furthermore, 37.8% and 29% of cells were positive for CD24 and Stro-1, respectively.
Adipogenesis of Human Anulus Fibrosus Cells
We next investigated adipogenesis of human anulus fibrosus cells. When cultured in the presence of adipogenic media, there was extensive formation of lipid droplets in human anulus fibrosus cells compared with unsupplemented cells as revealed by staining with oil red O (Fig. 2, A). Lipid droplet formation was quantified by ultraviolet-visible spectrometry, which revealed that the OD520 value of oil-red-O-stained lipids in lysates increased from 0.090 ± 0.002 for uninduced cells to 0.151 ± 0.007 for induced cells (p < 0.05). The induced cells also expressed the adipogenesis-specific genes, fatty acid binding protein 4, peroxisome proliferator-activated receptor-gamma 2, and lipoprotein lipase, as revealed by reverse transcription-polymerase chain reaction (Fig. 2, B) and real-time reverse transcription-polymerase chain reaction (Fig. 2, C, D, and E). Expression of these genes was markedly increased in induced cells with mRNA levels at twenty-one days that were approximately four, fifteen, and fiftyfold those at three days for peroxisome proliferator-activated receptor-gamma 2 (Fig. 2, D), lipoprotein lipase (Fig. 2, E), and fatty acid binding protein 4 (Fig. 2, C), respectively. On the other hand, in cells that were cultured in the unsupplemented medium, expression of lipoprotein lipase and peroxisome proliferator-activated receptor-gamma 2 remained unchanged, and no expression of fatty acid binding protein 4 was detected during twenty-one-day culture.
Osteogenesis of Human Anulus Fibrosus Cells
When human anulus fibrosus cells were cultured in an osteogenic medium for four weeks, an obvious increase in mineralization was revealed by alizarin-red-S staining (Fig. 3, A). Moreover, real-time reverse transcription-polymerase chain reaction (Fig. 3, B, C, and D) revealed that the osteogenic supplements stimulated expression of the osteogenesis-specific genes alkaline phosphatase, runt-related transcription factor 2, and osteocalcin. The mRNA levels at four weeks were approximately twenty and sixfold those at one week for alkaline phosphatase (Fig. 3, B) and runt-related transcription factor 2 (Fig. 3, C), respectively. Expression of osteocalcin was greatly elevated at one week after the addition of induction media and continued at a high level thereafter (Fig. 3, D). However, in uninduced cells, expression of each gene remained unchanged during four-week culture (Fig. 3, B, C, and D).
Chondrogenesis of Human Anulus Fibrosus Cells
After treatment of human anulus fibrosus cells with chondrogenic supplements for three weeks, cells showed marked staining with safranin O (Fig. 4, A) compared with human anulus fibrosus pellets that had been cultured in basal medium and were weakly stained. Immunocytochemical staining also revealed that the chondrogenesis-specific extracellular matrix proteins, aggrecan and type-II collagen, were enriched in the induced cells (Fig. 4, A). This elevated expression was also confirmed by assaying the cellular mRNA levels with use of reverse transcription-polymerase chain reaction (Fig. 4, B) and real-time reverse transcription-polymerase chain reaction (Fig. 4, C, D, and E). After induction with chondrogenic stimuli for one week, levels of both type-II collagen and aggrecan had approximately doubled and their expression continued to increase after longer periods of induction (Fig. 4, D and E). The levels of expression of aggrecan and type-II collagen were not increased in the absence of the chondrogenic stimuli. By contrast, the expression of type-I collagen increased in the absence of chondrogenic stimuli, with a large elevation occurring between weeks 1 and 2 and the levels remaining constant from week 2 to week 3 (Fig. 4, C). However, in the presence of chondrogenic stimuli, there was no significant variation in the expression levels of type-I collagen throughout the three weeks in culture (Fig. 4, C). At one week, expression of type-I collagen in the induced group was about ninefold that in the uninduced group (Fig. 4, C).
Neurogenesis of Human Anulus Fibrosus Cells
Neurogenesis of human anulus fibrosus cells was performed by initially expanding them with use of a sphere culture technique and then culturing them in the presence of neurogenic stimuli. Neurite-bearing cells were clearly visible after induction for seven or fourteen days, whereas no such signs of neuronal cells appeared when cells were cultured for seven or fourteen days in the absence of the neurogenic stimuli (Fig. 5, A). Expression of the neuronal markers ß-tubulin, microtubule-associated protein 2, and neurofilament light chain was assayed by immunofluorescent staining and was positive in the cells that had been cultured in the presence of the neurogenic stimuli (Fig. 5, B). Elevated expression of these proteins was also confirmed by reverse transcription-polymerase chain reaction analysis (Fig. 5, C). However, levels of the other neuronal markers that were assayed, i.e., glial fibrillary acidic protein, noggin, neuron-specific enolase, and nestin, were found to be similar whether the cells had or had not been cultured in the presence of the neurogenic stimuli. Interestingly, although glial fibrillary acidic protein and noggin were not expressed whether the cells were or were not stimulated, neuron-specific enolase and nestin were both expressed at very high levels in both stimulated and unstimulated cells (Fig. 5, C).
Induction of Human Anulus Fibrosus Cells to Differentiate into an Endothelial Cell Lineage
Human anulus fibrosus cells were grown in a commercially sourced, endothelial-inducing medium. The degree of differentiation into an endothelial cell lineage was assessed by assaying for the endothelial cell markers CD31 and vWF by a fluorescent immunostaining-based reverse transcription-polymerase chain reaction method and by real-time reverse transcription-polymerase chain reaction analysis. Both CD31 and vWF stained positively in induced cells, whereas they were negatively or weakly stained in noninduced cells (Fig. 6, A). By day 7 after induction, CD31 and vWF expression had increased by approximately tenfold in comparison with day 3 after induction (Fig. 6, B, C, and D).