Human MSCs are a potential cell source for regeneration of the degenerated human IVD yet the appropriate method for pre-differentiation remains unclear. This study differentiated human MSCs for 21 days, using two notochordal cell media conditions as well as chondrogenic and basal media groups. The lack of clear NP phenotypic markers led to examination of a number of outcome measures including use of gene profiling with a custom PCR array of 42 genes associated with a healthy NP phenotype, an important innovation of this study, as well as assessments of GAG, histology, and cell viability. Culture of human MSCs in NCT stimulated anabolic changes most similar to a healthy NP phenotype rather than a chondrogenic phenotype with increased proteoglycan, while NCA conditioning resulted in significant down-regulation of fibrotic genes and minimal effects on the hypertrophic gene COLX. Chondrogenic and basal groups demonstrated many similarities in gene expression compared to Day 0 (pre-culture) conditions, suggesting that micromass culture under hypoxic conditions produces a similar gene profile as MSCs cultured with Chondrogenic media.
NCT conditioning of MSCs resulted in up-regulation of SOX9
, and TGFB3
that could be associated with a healthy NP phenotype [10
]. This was corroborated at the protein level with a significant increase in GAG associated with the cell pellet as shown by the DMMB assay and Alcian blue staining despite a decrease in ACAN
at the gene level. The increase in GAGB observed for NCT relative to other media groups suggests that the cell phenotype induced with NCT was more closely an NP than chondrocytic phenotype. NCT also demonstrated an increase in matrix enzymes and IL-1B
. However, because significant increases in most anabolic genes and GAG were also observed, it is possible that the catabolic effects induced by NCT are associated with remodeling during differentiation rather than a catabolic cell phenotype [41
NCA had an anti-fibrotic effect on MSC differentiation with significant down-regulation of COL1A1
. Whilst significant increases were observed in COLX
for both NCT and C, for NCA minimal changes in the hypertrophic marker COLX
were noted. A common problem of in vitro
induced chondrogenesis is hypertrophic differentiation of MSCS with increased expression of Collagen × [29
]. Hypertrophic differentiation and calcification has also been shown during intervertebral disc degeneration [43
]. Minimal changes in COLX
expression suggests that NC cells in alginate alone may produce soluble factors capable of limiting or preventing hypertrophy and inhibiting synthesis of certain fibrous proteins. Unlike NCT, NCA had little impact on anabolic gene expression however accumulation of GAG was observed in MSCs after 21 days. These results are in contrast to a previous study that used conditioned media from NCs in alginate constructs to treat human MSCs for seven days and found increases in expression of matrix proteins [32
]. We can speculate that differences in gene expression may be due to the different time courses of the studies (7 days versus 21 days) or the differences in methods of CM generation.
A preliminary proteomics study demonstrated that NCA media conditions contained porcine alpha-2-macroglobulin, clusterin and tenascin. Intriguingly, these proteins have been implicated as cytoactive proteins that could be involved in reducing fibrous collagens, limiting matrix degradation or hypertrophy. Alpha-2-macroglobulin is an endoproteinase inhibitor present in blood and joint fluid which functions as a substrate for matrix enzymes such as ADAMTS-4 and -5 and inhibits their activity [44
]. Clusterin is known to be a multifunctional, secreted glycoprotein expressed in diverse locations, implicated in regulating complement activation and cell death in injured and degenerating tissues, and may have a cytoprotective effect on chondrocytes including NP cells [45
]. Tenascin is an extracellular matrix glycoprotein known to be abundant in the annulus of young IVDs and localized pericellularly in degenerated IVDs, and possibly could have a role in fibronectin - disc cell interactions [47
]. The biological roles of these proteins were not tested in this study, therefore, their effects are speculative and require further validation to confirm such roles.
Effects observed for C were consistent with the chondrocyte cell phenotype (a trend of increasing SOX9 and COL2 expression) at the gene and also at the protein level with GAG detected in the cell pellet. Results for B showed many similarities to that of C including the presence of GAG in the cell pellet. The only principal differences were a lack of COL2 expression and up-regulation of the phenotypic marker GPC1, growth factor TGFB3, and anti-catabolic protein TIMP1. These changes were unexpected as B was a control group. This suggests that the initial dose of TGFβ-3 for 24 hours followed by 3D culture/hypoxia for three weeks was sufficient to differentiate MSCs toward a chondrogenic phenotype. As a consequence of these unexpected findings, relative gene expression was normalized to Day 0, undifferentiated human MSCs, rather than B when examining the effects of NCA and NCT. Consequently, certain genes (that is, catabolic) were expressed at extremely low levels at Day 0 and relative expression levels are at very high orders of magnitude for all groups.
Until very recently no definitive markers of the IVD or NP cell phenotype were available, therefore markers of the chondrocyte phenotype were often used to assess MSC differentiation (for example, SOX9) [9
]. Microarray analysis of rat, bovine and canine IVD tissue has identified several candidate phenotypic markers such as Glypican, Biglycan, Keratin 19 and Laminin B1 [22
]. However, studies have also shown that species differences and degree of degeneration can influence the magnitude of expression of these genes, questioning their suitability as IVD/NP phenotypic markers [24
]. In this study, little change at the gene expression level was observed for these markers. Optimal NP phenotypic markers are a moving target as research continues to advance, and recent studies identified up to 12 NP positive and 36 negative marker genes using microarray analysis of human IVD cells, a subset of which were then examined in differentiated human MSCs [26
]. Future studies, therefore, require investigation of such markers to accurately assess differentiation of MSCs toward an NP phenotype.
The enhanced production of GAG in the NCT group suggests that NCs in their native tissue environment were able to differentiate MSCs toward an NP phenotype. At the gene level a decrease in aggrecan expression was noted however as protein data (GAG in pellets and histology) confirmed the presence of proteoglycan it is speculated that this effect may correspond to a negative feedback loop; as aggrecan has been made the cells down-regulate its gene expression, or alternately to the accumulation of GAG without production of aggrecan core protein. The increased GAG content is likely associated with greater total accumulation of GAG (as observed histologically with no differences in cell viability per group) as well as increased GAG per cell. The notochordal rich ECM is likely to influence the proteins present in the notochordal conditioned media from tissue and could be responsible for the observed effects on MSCs for this media group. Indeed the observed effects are likely a consequence of either soluble factors produced by NCs only when they are situated in their native matrix or factors derived from the matrix itself (for example, matricellular proteins). Although the SDS-PAGE and proteomic analysis of the NCT conditioned did not reveal observable differences in specific growth and differentiation factors, due largely to the masking effect of the bovine serum albumin in the samples, previous studies suggest many candidates. For example, the matricellular protein CTGF (connective tissue growth factor) has been implicated as an anabolic factor responsible for the effects mediated by NCCM on IVD biosynthesis [48
]. In this study, we observed significant down-regulation of CTGF at the gene level for all media groups. We suggest that this decrease in CTGF may represent a negative feed-back mechanism in which CTGF may have been synthesized at the beginning of culture or been present at sufficient levels in the CM.
This study used an in vitro
micromass culture system with human MSCs and porcine notochordal derived conditioned media for differentiation toward a healthy NP phenotype. This cross-species comparison was justified as human BM-MSCs are currently the most clinically relevant cell source for disc repair and porcine notochordal cells unlike human notochordal cells are readily available; however, it cannot be ruled out that species differences may have had an impact on the results obtained and is, therefore, a limitation of the study. Our goal is to use the NC conditioned media to identify proteins as therapeutic agents and not to ultimately use porcine tissue to generate the therapeutic agent. Thus, this is solely an experimental model and there would not be a cross-species concern if this approach were ultimately used clinically. Proteoglycan measurements using both the DMMB assay and histological analyses using Alcian blue demonstrated similar trends adding confidence to both measurements; however, the presence of guanidine thiocyanate in the cell pellet lysis solution may interfere with the DMMB assay and could have an effect on total GAG content [49
]. The relatively small sample size can also be considered a limitation however trends were consistent between specimens and only significant changes were discussed here. NCA and NCT demonstrated differing effects on MSC differentiation at the gene and protein level and differences may be accounted for by the native cell-matrix interactions present in NCT compared to NCA with cells cultured alone. Alternatively cell extraction for the NCA group may have affected notochordal cell phenotype therefore gene profiling pre and post conditioning would be a necessary next step to determine this. NCA and NCT also demonstrated differences with regard to proteomic analysis with proteins identified in NCA only. This could be explained by the presence of BSA in the media masking larger matricellular proteins derived from native notochordal tissue and also the different cell-matrix environment as mentioned above. These lines of inquiry as well as proteomic analysis of albumin-free conditioned media will be the subject of future studies.