Non-invasive spectral analysis of osteogenic and adipogenic differentiation in adipose derived stem cells using dark-field hyperspectral imaging technique

Mesenchymal stem cells derived from adult adipose tissue possess the ability to differentiate into adipocytes, osteocytes, and chondrocytes which in turn can be developed into adipose tissues, cartilages, and bones. This regenerative characteristics has fueled the need to define improved stem-cell analysis protocol for enabling investigation of the differentiation process efficiently, economically, and non-invasively by start-of-the art imaging modalities. Here, we have demonstrated hyperspectral microscopy-based label-free imaging approach to study ASCs at a single-cell level. ASCs has been stimulated to become osteocytes using the growth media containing β –glycerophosphate, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and dexamethasone. Further, ASCs were stimulated to form adipocytes using the growth media containing biotin, pantothenate, bovine insulin, IBMX, penicillin, rosiglitazone, and dexamethasone. In the present study, dark-field based hyperspectral Imaging (HSI) technique has been utilized to image single as well as multiple osteoblasts and adipocytes in salt media grown on the glass substrate. The spectral response of the cells at each pixel of the images were recorded in the visible-NIR range (400-900 nm). Response is stored in the three dimensional data-cube formed with two spatial dimensions and one spectral dimension. No special tagging or staining of the ASCs and derived osteoblasts, adipocytes has been done, as more likely required in traditional microscopy techniques. Incident light is diffracted at multiple angles and hence scattering response received after transmission is different even within the single cell due to sub-cellular heterogeneities present in the control and differentiating ASCs. Based on dark-field images of control and differentiated sample, we found significant structural and spectral distinctiveness at day 14 onwards for differentiated osteoblasts and at day 6 onwards for adipocytes. Fourier filtering of images provides good visual inspection of structural modifications. Spectral data from the cellular surface and intracellular markers, and secreted molecules is stored to build the spectral libraries. Matrix-assisted laser deposition/ionization (MALDI) spectrometry technique is performed on control and differentiated cells to obtain insight of sub-cellular single molecules, mineral deposits, fats, proteins, and other biological mono-constituents. In the hyperspectral images, the entire spectrum is stored within each pixel as a vector where the number of spectral bands (wavelength range) equals vector dimension and the corresponding intensity signifies the component of the individual vector. Spectral signatures from the identified lipids are then matched to the in vitro stem-cells via spectral angle mapping (SAM) algorithms. By computing angle between two pixels, remarkable spectral similarity and dissimilarity are identified between control and differentiated stem cells. Pseudo-colored differentiating maps are produced by calibrating ‘match’ threshold. Secondary validation to the HSI is provided by evaluating optical images with template-match and edge-detection algorithms as well as second-harmonic generation microscopy to investigate osteoblasts. Establishing this label-free protocol with minimum specimen preparation enables promising outcomes to overcome phototoxicity effect of traditional microscopy such as fluorescence/staining bleaching errors. The study would lead to high-throughput identification of patient specific derived cells for clinical use preventing mass rejection, and advance our understanding of the behavior of stem cellular clusters undergoing adipogenic and osteogenic differentiation.