(E-168) Label-Free Microscopy to Evaluate MSC Metabolism and Osteogenic Differentiation

Mesenchymal Stem Cells (MSCs) are renowned for their therapeutic capabilities in medicine. They are important due to their regenerative potential, immunomodulatory properties, and low immunogenicity, making them valuable candidates for tissue repair, immune-related disorders, and transplantation therapies[1]. However, the lack of FDA- approved MSC therapies is attributed to unreliable potency metrics and limited understanding about MSCs’ mechanisms of action[2]. 

Commonly used techniques to assess MSCs differentiation and function include Western blots, quantitative polymerase chain reaction, and immunohistochemistry. However, these methods are destructive. Also, regulating MSCs’ differentiation is challenging due to complex interplay between signaling pathways, the extracellular environment, and metabolic demands[2]. Several studies have reported alterations in MSCs autofluorescence traits during differentiation[3][4][5][6]. The optical redox (oxidation-reduction) ratio represents the balance between the intensities of the autofluorescent molecules: oxidized flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH)[7]. Assessing the redox ratio allows for the evaluation of cellular metabolism and osteogenic differentiation efficiency[8]. Additionally, third harmonic generation (THG) signal takes place at structural interfaces characterized by local changes in the refractive index or the third-order nonlinear susceptibility, which are inherent physical properties of materials and liquids[9].

In this study, we adopt the non-invasive, label-free differential phase-contrast microscopy and multi-photon fluorescence microscopy to rapidly and gently characterize MSCs. We demonstrate that the THG signal can be found in differentiated MSCs and differentiated MSCs exhibit a higher metabolic activity than undifferentiated MSCs. Our results evaluate MSCs’ potential success in therapeutic treatments for various metabolic and bone disorders, as well as tissue regeneration. 

MSCs were obtained from a single human bone marrow (RB183 cell line) and passaged into a T-175 flask. During passage two, the MSCs were seeded into 24-well plates. The next day, basic media (BM) was replaced with homemade osteogenic induction media (OIM), to stimulate osteogenic differentiation of the MSCs. To track MSC proliferation and differentiation, weekly phase-imaging was conducted on a portion of the same well and alizarin red S (ARS) was used to stain calcium deposits produced by differentiated MSCs. 

To evaluate the metabolic rate of MSCs, differentiated RB183 cells cultured in commercial Osteo-Max (OM), grown in a 24-well plate, and seeded at a density of 2,000 cells per well were obtained for autofluorescence imaging. OIM and OM both induced MSCs but commercial OM accelerated the induction process. For a control, undifferentiated RB183 cells seeded at a density of 10,000 cells per well were imaged with the multi-photon microscope.

Phase images were gathered from the differential phase-contrast microscope. For autofluorescence imaging, the optical setup is like what was reported previously[10]. Dichroic mirror (Semrock FF552-Di02, Semrock FF409-Di01) and filters (Semrock 571/72 nm, Semrock 457/50 nm, Semrock 356/30 nm) were used to separate the spectral channel to capture signals from FAD, NADH and third harmonic generation (THG) of MSCs, respectively. The wavelength used for autofluorescence imaging is 775nm, for THG is 1072nm. The optical redox ratio, calculated as the ratio of FAD over the amount of FAD + NADH, was determined using Python code for cells cultured in OM and BM.

Figure 2 C presents an analysis of the phase-images shown in Figure 2 A, comparing the number of red pixels across different cell culture days. An increasing red pixel count is correlated with an increasing number of differentiated MSCs. This difference in red pixel count between day 14 and day 21 is statistically significant. This indicates that a majority of MSCs proliferated and exhibited osteogenic potential during that time.

In Figure 3, the FAD, NADH, and THG signal were found in differentiated MSCs. However, in Figure 4, only the FAD and NADH were found in undifferentiated MSCs.

Figure 5 illustrates that MSCs grown in OM had a larger redox ratio of 0.51; comparatively, MSCs grown in BM had a redox ratio of 0.38. This suggests differentiated MSCs exhibit a stronger FAD fluorophore intensity and weaker NADH fluorophore intensity.

The study confirms that MSC differentiation occurs between day 14 and 21 because the ARS showed the osteogenic potential of the MSCs over time. Also, MSC differentiation changes metabolic activity and leads to a larger redox ratio. MSCs with greater osteogenic potential display heightened metabolic activity to support their elevated energy requirements during differentiation. Undifferentiated cells have a smaller redox ratio, due to increased production of reduced forms of cellular molecules. THG is not found for undifferentiated cells because unlike differentiated cells, there was no mineralization due to a lack of calcium deposits.

Furthermore, these results confirm the efficiency of osteogenic differentiation as indicated by the presence of oxidative phosphorylation, a characteristic marker of osteogenic differentiation.

In conclusion, we evaluate the osteogenic potential of MSCs using label-free differential phase-contrast microscopy and multi-photon fluorescence microscopy. We observe that differentiated MSCs have an increased metabolic rate than undifferentiated MSCs. Furthermore, mineralization was found in differentiated MSCs. This provides more methods in evaluating the efficacy for potential therapeutic treatments.

In the future, more cell lines are expected to be used to study the timing of MSC differentiation. Furthermore, to investigate how the altered metabolism found in differentiated MSCs can be manipulated to enhance MSC function, metabolic pathways such as ATP consumption should be studied.

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