Directing adult mesenchymal stem cells differentiation towards nucleus pulposus-like lineage by MAP-kinase interference coupled chondrogenic induction

Abstract

Over 80 percent of the population experience low back pain during lifetime, resulting in significant socioeconomic burden worldwide. Low back pain is associated with intervertebral disc (IVD) degeneration. The causes of IVD degeneration are thought to be multifactorial and have been linked to traumatic injury, ageing and genetic predisposition. Degenerated IVD shows decreased disc height, and a reduction of proteoglycan and water content. During maturation of human IVD, large and vacuolated primitive NP cells are gradually lost and small chondrocyte-like cells become the dominative cell type. In IVD degeneration, the gelatinous NP transforms into a cartilaginous and later fibrous tissue of compromised mechanical strength. Previous studies have reported a presence of progenitor cells in Rhesus monkey NP that express bone marrow mesenchymal stem cells markers and that a TIE2+/GD2+ subpopulation is linked to progenitor activity in human disc. To date, how to engineer NP cells or progenitor cells from bone marrow mesenchymal stem cells still remain a major challenge.

Microarray data and functional study from our group recently showed that a panel of genes are over-represented in human non-degenerative NP cells compared to degenerated NP and AF cells, and are expressed in the NP progenitor culture. Moreover, GO term study also implied an association of primitive NP cells with a low mitogen-activated protein kinase components activity. We used a cell pellet culture system to test the generation of NP progenitor-like cells from human bone marrow mesenchymal stem cells by inhibiting MEK kinase activity during TGFβ-induced chondrogenesis, a platform termed mitogen-activated protein kinase interference coupled chondrogenesis induction (MICCI). Results showed that MCCI could promote the expression of putative NP progenitor markers along with more committed NP cell markers. Moreover, we identified three different cell populations from human non-degenerative NP cell culture and isolated them according to their forms and plastic adherence. Our findings showed that a floating sphere-like population expressed higher levels of genes linked to pluripotent stem cells, NP progenitors, and quiescent stem cells. In addition, they expressed matrix genes COL3A1 and COL1A1 and transcription factor genes SOX5 and SOX6. The expression of the stem or progenitor markers was validated at protein level.

In summary, our findings suggested that modulation of MAP kinase activity during chondrogenic induction may have a potential to direct mesenchymal stem cells into a lineage that is consistent with the NP cell phenotype. Moreover, human NP cells might contain a stem/progenitor-like cell population. Further study of the MICCI platform and the stem/progenitor-like population may provide new insights to facilitate intervertebral disc tissue engineering.