Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation

Abstract

Objective. Development of stem cell therapies for regenerating the nucleus pulposus (NP) are hindered by the lack of specific markers by which to distinguish NP cells from articular chondrocytes (ACs). The purpose of this study was to define the phenotype profile of human NP cells using gene expression profiling and to assess whether the identified markers could distinguish mesenchymal stem cell (MSC) differentiation to a correct NP cell phenotype.Methods. Affymetrix MicroArray analyses were conducted on human NP cells and ACs, and differential expression levels for several positive (NP) and negative (AC) marker genes were validated by real-time quantitative polymerase chain reaction (PCR) analysis. Novel marker gene and protein expression was also assessed in human bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AD-MSCs) following differentiation in type I collagen gels.Results. Analysis identified 12 NP-positive and 36-negative (AC) marker genes that were differentially expressed >20-fold, and for a subset of them (NPpositive genes PAX1, FOXF1, HBB, CA12, and OVOS2; AC-positive genes GDF10, CYTL1, IBSP, and FBLN1), Conclusion. This study is the first to use gene expression profiling to identify the human NP cell phenotype. Importantly, these markers can be used to determine the in vitro differentiation of MSCs to an NP-like, rather than an AC-like, phenotype. Interestingly, these results suggest that AD-MSCs may be a more appropriate cell type than BM-MSCs for use in engineering intervertebral disc tissue.Low back pain is a major public health problem in the Western World, with over 80% of the population estimated to report low back pain during their lifetime (1). This has a serious impact on economies, with total costs related to this condition exceeding $100 billion per year in the US alone (2). One of the principal causes of low back pain is degeneration of the intervertebral disc (IVD), whereby adverse changes in the matrix results in dehydration of the nucleus pulposus (NP), loss of disc height, and loss of mechanical function, leading to traumatic damage and associated pain (3-5).Current treatments are aimed at relieving symptoms of low back pain, rather than halting progression of the disease by inhibiting the underlying disease mechanisms or by restoring disc structure and function. Thus, there is an urgent need to provide more-effective treatments for IVD degeneration. Since the NP is the region of the IVD that is most affected during degeneration,

Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc

“…Compared to conventional marker genes bT and bKRT19 were rather difficult to detect, exemplified by the large number of non-detectable samples in tissues with low expression. In agreement with other studies, we found that the NP/IVD can be distinguished from AC by high expression of bPax1 and bFoxf1 [7,13]. These genes exhibited the highest differential expression in our study.…”

“…4a, b). These genes were originally identified as NP markers in comparison with AC [7]. Indeed AC showed 90.5 (bFoxf1) to 388.0 fold (bPax1) lower expression levels.…”

“…The IVD is a unique tissue that differs from Articular Cartilage (AC) in embryonic development, nutrient supply, oxygen tension and biochemical composition [3]. Moreover, whole transcriptome analyses of IVD and AC cells revealed distinct gene expression profiles for the NP, AF and AC in rat, canine, bovine and human IVDs [4][5][6][7]. These studies began to unravel the NP cell phenotype at the transcriptome level leading to a first consensus paper regarding definition of healthy young NP cells [8].…”

Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells

“…On the contrary, as a scaffold, chitosan was characterized by a low gene expression and production of extracellular matrix, as well as low cell survival. The data indicates that MSCs can be stimulated by culture conditions to express collagen type II/aggrecan in a ratio similar to that of NP cells cultured in vitro, however gene expression analyses of recently identified NP markers [35], demonstrated that MSCs compared to NP cells express these markers in a different pattern.…”

“…To improve the discrimination between cartilage-like and NP-like differentiation of MSCs in different scaffolds after 5 weeks in chondrogenic medium, we tested the expression of genes recently associated with NP phenotype [35,36], namely KER19, PAX1 and FOXF1 (Fig. 7).…”