RESEARCH ARTICLE


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



Franziska Ehlicke1, 5, Natascha Köster1, Denise Salzig1, Peter Czermak1, 2, 3, 4, *
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Wiesenstr 14, 35390 Giessen, Germany
2 Department of Chemical Engineering, Kansas State University, Manhattan, KS 66506, USA
3 Faculty of Biology and Chemistry, Justus-Liebig-University of Giessen, Ludwigstr. 23, 35390 Giessen, Germany
4 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group Bioresources, Winchesterstr. 3, 35394 Giessen, Germany
5 Department Tissue Engineering and Regenerative Medicine, University Hospital Wuerzburg, Roentgenring 11, 97070 Wuerzburg, Germany


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© 2017 Ehlicke et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Wiesenstr 14, 35390 Giessen, Germany; Tel: +49 641-309-2551; Fax: +49 641-309-2553; E-mails: peter.czermak@kmub.thm.de; pczermak@k-state-edu


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.

Keywords: Intervertebral disc regeneration, Mesenchymal stem cells, Nucleus pulposus cells, Chondrocytes, Characterization, Distinction, Cell markers, Raman spectroscopy.