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Stem Cells 2005;23:403-411 www.StemCells.com
© 2005 AlphaMed Press

Induction of Intervertebral Disc–Like Cells From Adult Mesenchymal Stem Cells

Eric Steck, Helge Bertram, Rainer Abel, Bohua Chen, Anja Winter, Wiltrud Richter

Division of Experimental Orthopaedics, Orthopaedic Clinic, University of Heidelberg, Germany

Key Words. Intervertebral disc • Articular cartilage • Adult bone marrow stem cells • Chondrogenic induction • Gene expression

Correspondence: Dr. Wiltrud Richter, Division of Experimental Orthopaedics, Orthopaedic Clinic, University of Heidelberg, Schlierbacher Landstr. 200a, D-69118 Heidelberg, Germany. Telephone: 49-6221-969254; Fax: 49-6221-969288; e-mail: Wiltrud.Richter{at}ok.uni-heidelberg.de


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor ß (TGFß)-mediated induction in vitro.

Bone marrow–derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGFß3 dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage–, bone–, and stem cell–relevant genes was used to quantify gene expression profiles.

After TGFß-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture.

In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Low back pain is one of the most common causes of disability, with 60% to 80% of people affected at some point during their lives. Several studies have shown that degeneration of intervertebral discs (IVDs) is one reason for a multiplicity of cases of low back pain [1–4], although the evidence for an indisputable link between clinical symptoms and IVD degeneration remains elusive. Current surgical treatments for IVD degeneration are disc excision or disc immobilization, procedures that do not repair the tissue.

One approach aiming to repair degenerated discs is tissue engineering of IVDs. Tissue engineering can be defined as the use of "living cells, manipulated through their extracellular environment or even genetically, to develop biological substitutes for implantation into the body and/or to foster the remodeling of tissue in some other active manner. The purpose is to either repair, replace, maintain, or enhance the function of a particular tissue or organ." [5]. For tissue engineering of IVD, one major aim is the identification of suitable cell populations with the capacity to generate IVD tissue.

In several animal models, application of autologous cell sources was beneficial for the regeneration of degenerated discs [6–9]. Although culture systems have been described that preserved the phenotype of native human IVD cells [10], expansion of cells is usually not possible under such conditions. However, high cell numbers are desired in cell therapy or tissue engineering approaches. Unfortunately, articular chondrocytes respond to monolayer expansion by dedifferentiation. They alter their cell morphology and matrix gene expression in comparison with native tissue [11–15], and the changes include the loss of the capacity to induce stable cartilage implants after intramuscular injection into nude mice [16]. Mesenchymal stem cells (MSCs) isolated from bone marrow aspirates provide a nearly unlimited cell source with extremely high proliferation activity [17] and the potential to differentiate into several mesenchymal cell lineages [18], including chondrogenic differentiation [19, 20]. Morphologically, articular cartilage and IVD tissue are clearly distinct, although both tissues have been described to harbor chondrocytes surrounded by extensive extracellular matrix [21]. According to collagen type 2, sox 9, aggrecan, and proteoglycan expression [22, 23], the differentiation status of IVD cells is believed to resemble that of articular chondrocytes. Intriguingly, the gene expression profile of both cell types, to our knowledge, has never been compared on the transcriptional level. Whether chondrogenic differentiation of MSCs may, thus, be suitable for generation of IVD-like or articular chondrocyte-like cells remained to be established.

In this study we aimed to identify an unlimited cell source with an expression profile resembling that of native IVD tissue. We hypothesized that extensive expansion of MSCs in monolayer followed by a TGFß-mediated induction protocol could yield such cells and be an attractive method for tissue engineering of IVD-like fibrocartilage. To our knowledge, this is the first study using cDNA array technology to characterize the gene expression profile of human IVD tissue and to compare it with expression levels in hyaline articular cartilage and MSCs after differentiation in 3D culture.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Samples
Bone marrow samples for isolation of mesenchymal stem cells were obtained from seven patients (mean age, 34.9 years; range, 8 to 78) undergoing iliac bone graft harvest (n = 4) or total hip replacement (n = 3). From all donors, rapidly growing MSC cultures could be established even from those of advanced age. No age-dependent differences regarding proliferation or differentiation potential were evident for the isolated MSCs. Normal articular cartilage from tibia plateaus from five donors (mean age, 40.6 years; range, 31 to 60) was obtained either at autopsy within 48 hours of death (n = 4) or from patients undergoing amputation for sarcoma resection of the proximal fibula (n = 1). No indication of arthritis was present in the joint samples. IVD tissue was obtained from six scoliotic patients (mean age, 13.7 years; range, 10 to 16) undergoing lumbar discectomy (L1/2, L2/3, or L4/5) and interbody fusion surgery. All IVD tissue samples were evaluated histologically by a semiquantitative histodegeneration score developed by Boos et al. [24], ranging from 0 points (unaffected tissue) to 22 points (highly degenerated), where samples from healthy 10- to 17-year-old donors score between 1 and 4 points. The scoliotic samples used in this study were scored between 2 and 5 points. The studies were approved by the local ethics committee. Informed consent was obtained from all individuals included in the study or an immediate family member.

Cell Isolation and Cultivation
MSCs were isolated from fresh bone marrow samples as described previously [25]. Briefly, cells were fractionated on a Ficoll-Paque Plus density gradient (Amersham Pharmacia, Uppsala, Sweden), and the low-density MSC-enriched fraction was washed and seeded in culture flasks in MSC expansion medium [26] containing 2% fetal calf serum, with recombinant human epidermal growth factor (Strathmann Biotech, Hamburg, Germany) and recombinant human platelet-derived growth factor BB (Sigma-Aldrich, Deisenhofen, Germany). After 24 to 48 hours, cultures were washed with phosphate buffered saline (PBS) to remove nonadherent material. During expansion in monolayer, cells were plated at a cell density of 1 to 3 x 104 cells/cm2, and medium was replaced twice a week.

IVD tissue 1 mg was digested overnight in 10 ml Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Karlsruhe, Germany) containing collagenase B (333 µg/ml) (Roche Diagnostics, Mannheim, Germany) and hyaluronidase (200 µg/ml) (Serva, Heidelberg, Germany). Isolated IVD cells were separated from digest solution by a centrifugation step at 210g for 10 minutes and cultured in monolayer in DMEM, 10% fetal calf serum (Biochrom, Berlin, Germany), and 1% penicillin/streptomycin (Biochrom) at 37°C, 5% CO2.

Induction of Differentiation
For induction of TGFß-mediated differentiation at high cell density in 3D culture, cells were seeded at 3 to 5 x 105 cells/well in a 48-well plate in TGFß containing differentiation medium (DMEM containing 4.5 g/L glucose [Gibco] supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 µg/ml selenous acid, 0.1µM dexamethasone, 0.17 mM ascorbic acid–2-phosphate, 1 mM sodium pyruvate, 0.35 mM proline, 1.25 mg/ml bovine serum albumin [BSA], and 10 µg/ml TGFß3 all additives from Sigma-Aldrich, Taufkirchen, Germany). Because of the action of TGFß3 and in the absence of an external matrix scaffold, all cells aggregated spontaneously within 1 to 2 days to form rounded spheroids of approximately 1 mm in diameter, comprising the input cells. Spheroids were moved to 96-well U-bottomed plates after 4 days to reduce the costs for medium and growth factors due to lower volume at feeding (twice weekly).

Cell Staining
For immunohistochemical staining, differentiated MSC spheroids were fixed in PBS containing 4% paraformaldehyde for 2 hours at 20°C, dehydrated in alcohol, washed in acetone, and infiltrated with paraffin. Paraffin sections (3 to 4 µm) were dried, deparaffinized using XEM-200 (Vogel, Giessen, Germany), and rehydrated in alcohol. For Safranin-O staining, sections were incubated for 10 minutes with Safranin-O (0.1% wt/vol water) (Waldeck/Chroma, Münster, Germany) and unspecific dye was removed by aqua dest. For immunostaining, sections were pretreated with 2 mg/ml hyaluronidase (Merck, Darmstadt, Germany) for 15 minutes at 37°C and subsequently with 1 mg/ml pronase (Roche Diagnostics) for 30 minutes at 37°C. Nonspecific background was blocked using PBS containing 5% BSA for 30 minutes. Sections were incubated overnight at 4°C with a monoclonal mouse anti-human type I collagen or anti-human type II collagen (both ICN Biomedicals, Aurora, OH) in PBS containing 1% BSA. After washing with Tris-buffered saline, reactivity was detected using biotinylated donkey anti-mouse secondary antibody (1:200) (Dianova, Hamburg, Germany), streptavidin-alkaline phosphatase (Dako, Glostrup, Denmark) for 30 minutes at 20°C, and fast red (F4648; Sigma-Aldrich, Bornem, Belgium) for 20 minutes at 20°C. As a negative control, only the anti-mouse secondary antibody was used and counterstained with haemalaun (Waldeck/Chroma). All sections were permanently mounted with Aquatex (Merck) and examined by light microscopy.

RNA Isolation
Total RNA was isolated from monolayer-expanded undifferentiated MSCs and monolayer-cultured IVD cells using standard guanidinium thiocyanate/phenol extraction technique (peqGOLD TriFast; peqLab, Erlangen, Germany). Four to six differentiated stem cell spheroids were pooled, minced using a polytron (Kinematica, Littau-Luzern Switzerland), and subjected to a guanidinium thiocyanate/phenol extraction. From total RNA, messenger RNA (mRNA) was isolated using oligo(dT) coupled to magnetic beads (Dynabeads; Dynal Biotech, Oslo, Norway) according to the manufacturer’s instructions. Native cartilage and IVD tissue samples were pulverized in a freezer mill, and poly-adenylated mRNA was directly isolated from the tissue lysate using oligo (dT) coupled to magnetic beads (Dynabeads; Dynal Biotech, Oslo, Norway) according to the manufacturer’s instructions.

Complementary DNA Array Production
An in house–generated cDNA array comprising 48 genes was designed and developed for special use in these experiments (Table 1Go; Fig. 1AGo). Analyzed genes included mostly cartilage-relevant and stem cell–relevant molecules, but also bone-specific and adipose tissue–specific genes, as well as housekeeping genes (RPL13A, RPS9, GAPDH, and ß-actin) and negative controls (Arabidopsis thaliana–specific genes) (Table 1Go). Selected cDNAs (size range, 400 to 850 bp) were cloned into pBluescript SK+ vector (Stratagene, Amsterdam, The Netherlands). DNA was polymerase chain reaction (PCR) amplified using vector specific primers and 50 ng of plasmid as template. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and concentrated. Standardized amounts were arrayed onto positively charged Hybond-N+ nylon filters (Amersham Pharmacia Biotech, Freiburg, Germany). Gene fragments (10 ng/dot) were spotted twice on each filter, as depicted in Figure 1DGo. The array represented the differentiation capacity of MSCs described by us [20] and others [18].


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  		Table 1. Composition of the cDNA array
 


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  		Figure 1. Evaluation of gene expression by cDNA array analysis. Representative gene expression profiles of expanded noninduced MSCs (n = 3), of spheroid cultures of MSCs (n = 7) 2 weeks after TGFß-mediated induction, of IVD cells expanded in monolayer culture (n = 3), and of native IVD tissue (n = 6). (A): Array location of genes. For nomenclature of genes, see Table 1Go. Expanded MSCs (B) expressed only a very limited number of genes. Genes showing an elevated expression after TGFß-mediated induction of MSCs (C) are marked in solid lines. The genes in ovals represent typical cartilage-relevant molecules of the extracellular matrix; the genes in boxes are relevant in hypertrophic chondrocytes or bone. Genes in dotted ovals were downregulated after chondrogenic induction. Cartilage-relevant genes (D) downregulated in cultured IVD cells compared with native IVD tissue are indicated by dotted circles; relevant genes for hypertrophic chondrocytes or bone are in dotted squares. Genes more highly expressed in native IVD tissue (E) compared with cultured IVD cells are in solid lines. Genes in dashed rectangles were exclusively found in native IVD tissue and were not expressed in any culture system used. Abbreviations: IVD, intervertebral disc; MSC, mesenchymal stem cell; neg, negative control genes from A. thaliana; TGFß, transforming growth factor ß.

 
Complementary DNA Array Hybridization
Complementary DNA (32P-dATP–labeled) probes were prepared from isolated mRNA according to the manufacturer’s protocol (SuperScript II; Life Technologies, Karlsruhe, Germany). The labeled cDNA was denatured and hybridized to cDNA arrays overnight at 68°C. Arrays were washed three times (for 30 minutes each) in 0.04 M phosphate buffer (pH 7.2)/1% sodium dodecyl sulfate at 68°C before being exposed to an imaging plate (Fuji Photo Film, Düsseldorf, Germany) for 18 hours. Images were captured on the BAS-1800 II Bio-imaging Analyzer using BAS Reader 2.26 beta software (Fuji/Raytest, Straubenhardt, Germany) and were analyzed using AIDA software (Fuji/Raytest). Expression levels in mRNA samples from different sources were normalized to the signal strength of the housekeeping genes.

Statistical Analyses
Because of the size and distribution of the samples in this expression study, the Mann-Whitney U test was chosen to evaluate significant differences in expression levels. This non-parametric two-tailed test is not based on assumptions about the distribution of expression values (e.g., normal distribution) or the equality of variance. For all tests, p ≤ .05 was considered significant. Data analysis was performed with SPSS for Windows 10.0 (SPSS Inc., Chicago).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene Expression Profiles in Mesenchymal Stem Cells After TGFß-Mediated Induction
The gene expression profile of undifferentiated in vitro expanded MSCs was characterized by expression of collagens types I and III, the small leucin-rich proteoglycans biglycan, lumican, and decorin, as well as alkaline phosphatase, osteonectin, endoglin, and chitinase 3-like 1 (Fig. 1BGo). Besides genes coding for the small leucin-rich proteoglycans and chitinase 3-like 1, none of these genes was described to be of relevance for chondrocytes.

The TGFß-mediated induction of MSCs in 3D spheroid culture resulted in induction and further upregulation of many genes typical for articular cartilage, such as collagens type II, XI, and XII, biglycan, fibromodulin, cartilage oligomeric matrix protein (COMP), proline arginine-rich end leucine-rich repeat protein (PRELP), lumican, and decorin (Fig. 1CGo, closed circles). In parallel, collagen type X, bone sialoprotein, and osteopontin were induced, which are known to be expressed in hypertrophic chondrocytes or bone [27] (Fig. 1CGo, closed boxes). In contrast, the dedifferentiation- and proliferation-associated markers chitinase 3-like 1 (cartilage glycoprotein 39, YKL40) [14] and endoglin were downregulated after induction (Fig. 1CGo, dotted circles). Transcripts for the cartilage-relevant proteins collagen type II and COMP (Fig. 1DGo, dotted circles) and the hypertrophic markers collagen type X, bone sialoprotein, and osteopontin (Fig. 1DGo, dotted squares) were undetectable in cultured IVD cells. However, all of them except collagen type X were present in native IVD tissue (Fig. 1EGo, closed boxes and circles), which also, uniquely, expressed transcripts for cartilage intermediate layer protein (CILP), chondroadherin, and osteocalcin (Fig. 1EGo, dashed squares). In sum, the data demonstrated a highly similar although not identical gene expression profile between native IVD tissue and MSC spheroids after TGFß-mediated induction and indicated that IVD cells underwent dedifferentiation during monolayer expansion in culture.

Two weeks after TGFß-mediated induction, collagen type II protein expression commenced in most cases at the surface of the spheroids (Fig. 2BGo), whereas at 4 weeks, a ring-like structure with strong collagen type I and II content surrounded the stained centre of the spheroid (Figs. 2C, 2FGo). Collagen type I protein was detectable at all time points (Figs. 2D–2FGo). This was accompanied by metachromatic staining of Safranin-O (Fig. 2IGo). Figure 2AGo shows the morphology of expanded MSCs in monolayer before TGFß-mediated induction.



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  		Figure 2. Morphological assessment of differentiated MSCs. MSCs were expanded in monolayer culture (A), and TGFß-mediated differentiation of spheroid cultures was induced for 2 weeks (B, D, E, I) or 4 weeks (C, F, G, H) before collagen type II (B, C, G) and collagen type I (E, F, H) protein was detected by immunhistochemistry. The negative control was obtained by omitting the primary antibody (D). Proteoglycan production was evident according to metachromatic staining by Safranin-O (I) 2 weeks after induction. Boxes in (C) and (F) represent areas shown at higher magnification in (G) and (H), respectively. Bars in (B) through (F) indicate 500 µm; in (G) through (I), 100 µm, respectively. These results are representative of three independent experiments. Abbreviations: MSC, mesenchymal stem cell; TGFß, transforming growth factorß.

 
TGFß-Mediated Differentiation of MSC Spheroids Yields Gene Expression Levels Closer to Native IVD Tissue Than to Hyaline Articular Cartilage
Quantitative evaluation of the gene expression profiles of native IVD tissue (n = 6) and MSC spheroids 2 weeks after TGFß-mediated induction (n = 7) revealed highly similar expression levels for most genes (Fig. 3AGo). Except for CILP, chondroadherin, and osteocalcin, which were not induced in the differentiated MSC spheroids, PRELP remained on a lower level (p < .05). On the other hand, collagen type X was unique for MSCs after induction, and lumican reached a significantly higher level (p < .05) compared with IVD tissue.



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  		Figure 3. Quantitative analysis of gene expression levels of selected genes (signal intensity above 15% in IVD tissue, except collagen type X, which was negative). Spheroid cultures of MSCs 2 weeks after TGFß-mediated induction (n = 7) were compared with IVD tissue (n = 6) (A) and articular cartilage tissue (n = 5) (B). The signal intensities were normalized to the gene expression levels of the housekeeping genes on each filter. The medians of independent experiments are shown and expressed as relative values in percent of the housekeeping genes. Abbreviations: IVD, intervertebral disc; MSC, mesenchymal stem cell; nd, not determined; TGFß, transforming growth factorß.

 
When comparing native healthy articular cartilage (n = 5) with differentiated MSC spheroids, many cartilage differentiation markers, such as PRELP, COMP, decorin, biglycan, fibromodulin, aggrecan, collagen type XI, and melanoma inhibitory activity, were expressed at considerably higher levels in articular cartilage (Fig. 3BGo). Discrepant expression was evident for CILP, osteopontin, collagen type I, and collagen type X.

In conclusion, MSC spheroids attained a gene expression profile after TGFß-mediated induction, which was quantitatively closer to native IVD tissue than to articular cartilage.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Notochordal cells are remnants of the chorda of embryonic tissue and are lost generally at the latest by the tenth year of age in humans [5]. It has been discussed that IVD degeneration, starting at an age of approximately 30 to 50 years in humans, might in part be caused by the loss of notochordal cells, which could function as stem cells regenerating affected IVD tissue [5]. According to this hypothesis, the application of TGFß-mediated predifferentiated MSCs or even of undifferentiated MSCs as described by Sakai et al. [7] might support IVD regeneration or at least decelerate the degenerative process by overcoming the limitation of self-regeneration, which is considered the main cause of degeneration-mediated functional loss of IVDs.

The data presented in this study convincingly show that MSCs can be considered a highly attractive cell source for IVD tissue-engineering projects. We were able to show that TGFß-mediated induction protocols for MSCs in 3D culture resulted in a gene expression profile highly similar to that of native IVD tissue and a molecular and histological appearance closer to that of fibrocartilage than that of hyaline articular cartilage. Although we expected that common protocols for TGFß-mediated chondrogenesis may need to be adapted to reach transcription levels close to IVD tissue, these protocols seem to be most suitable for generation of a collagen type I–rich fibrocartilaginous tissue. According to our data, TGFß-mediated chondrogenesis towards a molecular phenotype and morphological features of hyaline cartilage may demand further optimization, because shutdown of collagen type I, collagen X, bone sialoprotein, and osteopontin is desired.

In proliferating MSCs, hardly any of the cartilage-relevant genes were expressed. The high expression levels of collagen type I and osteonectin in these cells has already been described by Silva et al. [28], who analyzed the profile of gene expression of human marrow mesenchymal stem cells and identified collagen type I and osteonectin among the most abundantly expressed genes. After TGFß-mediated induction of the MSCs in spheroid culture, most of the genes analyzed with our cDNA array exhibited similar expression levels compared with IVD tissue, and only collagen type X was inadequately expressed in induced MSCs. Remarkably, upregulation of collagen type X has been reported to occur in IVD tissue at older ages [29] as well as in articular cartilage during osteoarthritis [30]. We also performed gene expression profiling of surgical IVD tissue (n = 7; mean age, 35 years) in the course of a separate study and detected enhanced collagen type X compared with scoliotic tissue (Bertram, unpublished results). The gene expression profile of such age-matched surgical samples was even more similar to the differentiated MSCs than the scoliotic tissue.

The distinct morphology of nucleus pulposus and anulus fibrosus in IVD tissue suggests that different gene expression profiles may occur in these two compartments of the disc. Because of the low cell number and the high content of extracellular matrix in the inner nucleus pulposus, we were not able to isolate sufficient mRNA from a single disc for cDNA array hybridization. Negatively charged proteoglycan residues are known to interfere with standard RNA isolation protocols, and the high proteoglycan content of inner nucleus pulposus may contribute to this result [31, 32]. Hybridization of inner nucleus samples yielded no signals above the detection threshold of the cDNA array, demonstrating that they made merely a very small contribution to the gene expression data obtained for individual discs. When analyzing differences in gene expression between anulus fibrosus and nucleus pulposus IVD tissue from three donors by quantitative real-time PCR, we identified a higher expression of aggrecan in the nucleus and stronger expression of collagen type I in the anulus. All other analyzed genes had similar expression levels or varied considerably between samples. These results support the work of Cs-Szabo et al. [22], who found approximately threefold higher levels of aggrecan in nucleus compared with anulus, whereas versican, decorin, biglycan, and fibromodulin were expressed within the same range in both IVD regions. Thus, we were unable to define a distinct molecular phenotype for nucleus and anulus cells based on analysis of individual discs, and our data should be interpreted in favor of a gene expression profile close to anulus fibrosus cells. Although the morphology of the extracellular matrix in anulus and nucleus is highly different in IVD tissue, we suppose that this is only barely represented by the gene expression levels in developed tissue.

TGFß-mediated induction of MSCs here seemed to be more promising than culturing primary IVD cells, not only because of the obstacles of extracting cells from a degenerated disc or a previously uninjured donor site. Beside their limited source and expansion capacity, IVD cells lost differentiation markers such as collagen type II and COMP during monolayer expansion. This closely resembled the situation in primary articular chondrocytes cultured in monolayer in which dedifferentiation was partially irreversible [14]. Gene expression profiles of both cultured cell types became almost indistinguishable during this dedifferentiation process despite the fact that native IVD and cartilage tissue showed obvious differences in their gene expression levels (Fig. 3Go). Thus, our results based on the expression of a large number of genes support nicely the previously described dedifferentiation process of cultured IVD cells characterized on the morphological and the biochemical level [10, 33–35]. Because MSCs can be expanded to almost unlimited cell numbers before differentiation is induced, an irreversible loss of differentiation markers should be no issue.

We conclude that TGFß-mediated induction of bone marrow–derived mesenchymal stem cells is a highly attractive source for projects aiming to engineer IVD tissue, because a high number of genes relevant for extracellular matrix components were expressed equally in quality and quantity both in IVD tissue and in induced MSC spheroids.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors wish to thank Stephanie Kadel, Katrin Götzke, and Thea Hennig for excellent technical assistance and Dr. Sven Schneider for statistical support. This work was supported by a grant from the research fund of the Stiftung Orthopädische Universitätsklinik Heidelberg and by Cytonet, Weinheim.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received May 12, 2004; accepted for publication November 15, 2004.



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