There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs) for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing.

We previously reported that mesenchymal stem cells (MSCs) possess therapeutic effects in a murine model of carbon tetrachloride-induced acute liver failure. In the study, we observed that the majority of repopulated hepatocytes were of recipient origin and were adjacent to transplanted MSCs; only a low percentage of repopulated hepatocytes were from transplanted MSCs. The findings indicate that MSCs guided the formation of new hepatocytes. Exosomes are important messengers for paracrine signaling delivery. The aim of this study is to investigate the paracrine effects, in particular, the effects of exosomes from MSCs, on hepatocytes. Mature hepatocytes were isolated from murine liver by a two-step perfusion method with collagenase digestion. MSCs were obtained from murine bone marrow, and conditioned medium (CM) from MSC culture was then collected. Time-lapse imaging was used for observation of cell morphological change induced by CM on hepatocytes. In addition, expression of markers for hepatic progenitors including oval cells, intrahepatic stem cells, and hepatoblasts were analyzed. Treatment with the CM promoted the formation of small oval cells from hepatocytes; time-lapse imaging demonstrated the change from epithelial to oval cell morphology at the single hepatocyte level. Additionally, expression of EpCAM and OC2, markers of hepatic oval cells, was upregulated. Also, the number of EpCAMhigh cells was increased after CM treatment. The EpCAMhigh small oval cells possessed colony-formation ability; they also expressed cytokeratin 18 and were able to store glycogen upon induction of hepatic differentiation. Furthermore, exosomes from MSC-CM could induce the conversion of mature hepatocytes to EpCAMhigh small oval cells. In summary, paracrine signaling through exosomes from MSCs induce the conversion of hepatocytes into hepatic oval cells, a mechanism of action which has not been reported regarding the therapeutic potentials of MSCs in liver regeneration. Exosomes from MSCs may therefore be used to treat liver diseases. Further studies are required for proof of concept of this approach.

Introduction: Mesenchymal stromal cells (MSCs) are well known for their application potential in tissue engineering. We previously reported that MSCs are able to differentiate into hepatocytes in vitro. However, conventional methods for estimating the maturation of hepatic differentiation require relatively large amounts of cell samples. Raman spectroscopy (RS), a photonic tool for acquisition of cell spectra by inelastic scattering, has been recently used as a label-free single-cell detector for biological applications including phenotypic changes and differentiation of cells and diagnosis. In this study, RS is used to real-time monitor the maturation of hepatic differentiation in live MSCs. Methods: The MSCs were cultured on the type I collagen pre-coating substrate and differentiated into hepatocytes in vitro using a two-step protocol. The Raman spectra at different time points are acquired in the range 400–3000 cm^–1and analyzed by quantification methods and principle component analysis during hepatic differentiation from the MSCs. Results: The intensity of the broad band in the range 2800–3000 cm^–1 reflects the amount of glycogen within lipochrome in differentiated hepatocytes. A high correlation coefficient between the glycogen amount and hepatic maturation was exhibited. Moreover, principle component analysis of the Raman spectra from 400 to 3000 cm^–1 indicated that MSC-derived hepatocytes were close to the primary hepatocytes and were distinct from the undifferentiated MSCs. Conclusions: In summary, RS can serve as a rapid, non-invasive, real-time and label-free biosensor and reflects changes in live cell components during hepatic differentiation. The use of RS may thus facilitate the detection of hepatic differentiation and maturation in stem cells. Such an approach may substantially improve the feasibility as well as shorten the time required compared to the conventional molecular biology methods.

Corneoscleral-ring-derived extracellular vesicles represent a potential therapeutic strategy for promoting in vitro corneal wound healing. In this study, we successfully isolated and characterized extracellular vesicles from human corneolimbal tissue obtained from 42 donors, with a mean age of 51.62 ± 15.56 years. Donor-related factors such as age, corneal endothelial cell density, and underlying systemic conditions did not confound extracellular vesicle size and concentration with mean peak size of 99.52 ± 13.00 nm by nanoparticle tracking analysis. Western blotting analysis revealed positive Alix, stable expression of CD9 and CD81, and variable expression of CD63. Limbal stem cell (LSC)-associated markers, i.e., ABCG2, p63, Notch-1, and Integrin α9 were positively detected in the isolated extracellular vesicles. Notably, Integrin α9 showed stable and relatively strong expression in all samples serving a specific marker of LSC-derived extracellular vesicles. Functional assays demonstrated that LSC-derived extracellular vesicles exhibited better wound healing potency compared to extracellular vesicles derived from mesenchymal stem cells (MSCs). These findings suggest that corneoscleral-ring-derived extracellular vesicles express distinct LSC markers, including Integrin α9, and hold significant potential for application in corneal wound healing and ocular surface regeneration.