Non-invasive Raman Spectroscopy and Quantitative Real-Time PCR Distinguish Among Undifferentiated Human Mesenchymal Stem Cells and Redifferentiated Nucleus Pulposus Cells and Chondrocytes .

BACKGROUND

The most common cause of lower back pain is the pathological degeneration of the nucleus pulposus (NP). Promising NP regeneration strategies involving human mesenchymal stem cells (hMSCs) would require specific markers to confirm successful differentiation into the NP lineage and to distinguish the articular cartilage (AC).

OBJECTIVE

We sought specific NP mRNA markers that are upregulated in native NP cells but not in dedifferentiated NP cells, undifferentiated hMSCs or chondrocytes. We also considered the suitability of non-invasive Raman spectroscopy to distinguish among these classes of cells.

METHOD

We used quantitative real-time PCR and Raman spectroscopy to analyse undifferentiated hMSCs in monolayers and embedded in hydrogels, and compared the results with dedifferentiated and redifferentiated human NP and AC cells.

RESULTS

The redifferentiation of NP cells induced the expression of annexin A3 (), collagen type II () and proteoglycan mRNAs, whereas the redifferentiation of AC cells only induced proteoglycan expression. Redifferentiated NP cells expressed higher levels of , , paired box 1 () and mRNA than redifferentiated AC cells. Redifferentiated NP cells and undifferentiated hMSC-TERT cells expressed similar amount of mRNA, indicating that only , and are promising markers for redifferentiated NP cells. Raman spectra clearly differed among the three cell types and highlighted their differentiation status.

CONCLUSION

We recommend , and as markers to determine the success of hMSC-based differentiation to regenerate NP cells. Raman spectroscopy can be used to determine cell type and differentiation status especially in the context of clinical trials.