Micro-FTIR identifies osteodifferentiation markers in heterogeneous mesenchymal stem cell populations

Mesenchymal stem cells (MSCs) are often used in bone tissue engineering. However, for optimized MSC osteogenic commitment to be achieved, a deeper understanding of the cellular molecular mechanisms underlying osteodifferentiation is required. In this paper we show that this can be provided by Fourier Transform Infrared (FTIR) spectroscopy in tandem with microscopy (micro-FTIR), a methodology which may also aid the rapid early evaluation of differentiation outcomes.

We report a micro-FTIR time-course characterization of human adipose tissue mesenchymal stem cell (hAMSCs) osteodifferentiation (for 21 days), considering different independent experiments and biological replicates to account for cell heterogeneity, under both differentiation and proliferation (control) conditions. Despite extensive spectral variability in both groups, multivariate statistical methods revealed time-dependent spectral changes and potential spectral signatures of proliferation and osteodifferentiation extension. Under proliferation alone, spectral changes reflecting helical proteins and lipids were seen up to day 14, stabilizing thereafter. Osteodifferentiating cells exhibited higher inter- and intra-replicate variability and a distinct spectral time-course trajectory. Evidence was found of both helical and β-sheet protein structures, with a relative increase of the former from day 6, although masked by increasing sample variability. Other osteo-specific changes were noted in lipid moieties and at lower wavenumbers (1030-1115 cm^-1). The latter were found to relate to different degrees of hydroxyapatite maturity between and even within samples.

Specific spectral FTIR signatures have been identified as markers of the extent of hAMSC proliferation and osteodifferentiation, potentially enabling the rapid and non-invasive evaluation of hAMSCs osteodifferentiation outcome and of inter- and intra-sample variability.