Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed "molecular rotors", in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation. In order to correlate the viscosity values with membrane lipid composition, the detailed analysis of the corresponding membrane lipid composition of differentiated cells was performed by timeof-flight secondary ion mass spectrometry. Our results directly demonstrate for the first time that differentiation of MSCs results in distinct membrane viscosities, that reflect the change in lipidome of the cells following differentiation. Membrane fluidity is considered a key parameter influencing biological function of cells, such as cell adhesion, migration and differentiation 1. These properties are of particular importance in stem cell lines, where small modifications in membrane parameters have the potential to either promote a lineage commitment or a selfrenewal 2. The plasma membrane is the interface between a cell and its environment, it directly interacts with the matrix outside the cell and is responsible for many important tasks such as signaling and mass transfer. In stem cells, its composition and properties are likely to reflect their differentiation status. However, little is known on how the viscosity parameters of different stem cell lineages can change depending on the direction of differentiation. There is evidence that the membrane fluidity substantially changes during induced pluripotent stem cells (iPS) differentiation. Generalized polarization monitoring was previously used to detect the rise of membrane rigidity during iPSC differentiation 1. Furthermore, the results in 1 potentially suggested that membrane rigidification could be transmitted to neighboring cells, resulting in the acceleration of a cells differentiation, in a wave-line fashion. It was also reported that the viscoelastic properties can predict which subpopulations of undifferentiated mesenchymal stem cells (MSCs) differentiate into osteocytes, and which would turn into adipocytes or chondroblasts. The stiffest cell populations produced more bone cells; the softest cells predominantly produced fat cells; the cells with the highest viscosity became cartilage cells 3. While cell stiffness measured in 3 is a distinctly different property to the cell membrane viscosity, both depend on membrane lipid compositions. There is evidence that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts, chondrocytes or adipocytes produces specific membrane compositions and biophysical properties,
In the presented study, we have developed a synthetic strategy allowing a gradual variation of a polylactide arms' length, which later influences the micromorphology of the scaffold surface, formed by a two-photon polymerization technique. It has been demonstrated that the highest number of cells is present on the scaffolds with the roughest surface made of the polylactide with longer arms (PLA760), and osteogenic differentiation of mesenchymal stem cells is most pronounced on such scaffolds. According to the results of biological testing, the PLA760 scaffolds were implanted into a created cranial defect in a mouse for an in vivo assessment of the bone tissue formation. The in vivo experiments have shown that, by week 10, deposition of calcium phosphate particles occurs in the scaffold at the defect site, as well as, the formation of a new bone and ingrowth of blood vessels from the surrounding tissues. These results demonstrate that the cross-linked microstructured tetrafunctional polylactide scaffolds are promising microstructures for bone regeneration in tissue engineering.
Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.
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