Abstract
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 (ANXA3), collagen type II (COL2) and proteoglycan mRNAs, whereas the redifferentiation of AC cells only induced proteoglycan expression. Redifferentiated NP cells expressed higher levels of ANXA3, COL2, paired box 1 (PAX1) and OCT4 mRNA than redifferentiated AC cells. Redifferentiated NP cells and undifferentiated hMSC-TERT cells expressed similar amount of OCT4 mRNA, indicating that only ANXA3, COL2 and PAX1 are promising markers for redifferentiated NP cells. Raman spectra clearly differed among the three cell types and highlighted their differentiation status.Conclusion:We recommend ANXA3, COL2 and PAX1 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.
Highlights
Disorders of the intervertebral disc (IVD) are common in modern society, causing severe pain for patients and a high cost burden on national health systems [1, 2]
We found that nucleus pulposus (NP) cells clearly showed evidence of extracellular matrix (ECM) production after 21 days culture in agarose hydrogels, whereas chondrocytes produced only small amounts of ECM (Fig. 1)
We identified the mRNAs for annexin A3 (ANXA3), collagen type II (COL2) and paired box 1 (PAX1) as the most promising markers based on their preferential strong expression in redifferentiated NP cells
Summary
Disorders of the intervertebral disc (IVD) are common in modern society, causing severe pain for patients and a high cost burden on national health systems [1, 2]. The CEPs are derived from the sclerotome and consist of chondrocytes and hyaline cartilage [3]. These structures supply nutrients to and remove waste from the entire IVD, their capacity is limited by calcification. The AF is derived from the sclerotome, but contains fibroblastlike and chondrocyte-like cells [4] embedded in an ECM consisting mainly of type-I collagen with small amounts of proteoglycan (PG) or type-II collagen [5]. The NP is derived from the notochord and in humans consists of notochordal cells (NCs) until the end of the first life decade, and chondrocyte-like NP cells during adolescence and adulthood [6]. 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)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
