Monitoring membrane viscosity in differentiating stem cells using BODIPY-based molecular rotors and FLIM.

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 time-of-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.

Metabolic activity and intracellular pH in induced pluripotent stem cells differentiating in dermal and epidermal directions.

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.

Multimodal label-free imaging of living dermal equivalents including dermal papilla cells.

BACKGROUND: Despite the significant progress in the development of skin equivalents (SEs), the problem of noninvasively assessing the quality of the cell components and the collagen structure of living SEs both before and after transplantation remains. Undoubted preference is given to in vivo methods of noninvasive, label-free monitoring of the state of the SEs. Optical bioimaging methods, such as cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), and fluorescence lifetime imaging microscopy (FLIM), present particular advantages for the visualization of such SEs. METHODS: In this study, we simultaneously applied several visualization techniques for skin model examination. We investigated the structure and quality of dermal equivalents containing dermal papilla (DP) cells and dermal fibroblasts (FBs) using CP OCT, MPT, and FLIM. Both the energy metabolism of the cell components and the structuring of the collagen fibrils were addressed. RESULTS: Based on the data from the fluorescence lifetimes and the contributions of protein-bound NAD(P)H, a bias toward oxidative metabolism was indicated, for the first time, in both the DP cells and FBs on day 14 of SE cultivation. The CP OCT and MPT data also indicated that both DP cells and FBs structured the collagen gel in a similar manner. CONCLUSION: In this study, multimodal label-free imaging of the structure and quality of living dermal equivalents was implemented for the first time with the use CP OCT, MPT, and FLIM of NAD(P)H. Our data suggest that the combination of different imaging techniques provides an integrated approach to data acquisition regarding the structure and quality of dermal equivalents, minimizes the potential disadvantages of using a single method, and provides an ideal information profile for clinical and research applications.

Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation.

BACKGROUND: Metabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM. METHODS: Differentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums. 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. The cells were imaged before the induction of differentiation (day 0) and on days 7, 14, and 21 of osteogenic and chondrogenic differentiation. RESULTS: Based on the data for the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound NAD(P)H, we registered a metabolic shift toward a more glycolytic status in the process of MSC differentiation. The difference was that, in osteogenic differentiation, an increase in oxidative phosphorylation preceded the shift to the glycolytic status in the process of such MSC differentiation. The fluorescence lifetime characteristics of FAD indicated the stimulation of an unknown metabolic pathway, where protein-bound FAD participates. CONCLUSIONS: In this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.

Probing metabolic states of differentiating stem cells using two-photon FLIM.

The ability of stem cells to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology. Because traditional assessments of stem cells are destructive, time consuming, and logistically intensive, the use of a non-invasive, label-free approach to study of cell differentiation provides a powerful tool for rapid, high-content characterization of cell and tissue cultures. Here, we elucidate the metabolic changes in MSCs during adipogenic differentiation, based on the fluorescence of the metabolic co-factors NADH, NADPH, and FAD using the methods of two-photon fluorescence microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH is required. In our study we demonstrated, for the first time, an increased contribution of protein-bound NADPH in adipocytes that is associated with lipogenesis. The optical redox ratio FAD/NAD(P)H decreased during adipogenic differentiation, and that this was likely to be explained by the intensive biosynthesis of lipids and the enhanced NADPH production associated with this. Based on the data on the fluorescence lifetime contribution of protein-bound NAD(P)H, we registered a metabolic switch from glycolysis to oxidative phosphorylation in adipocytes.

Influence of mesenchymal stem cells on metastasis development in mice in vivo.

INTRODUCTION: In recent years, mesenchymal stem cells (MSCs) have been demonstrated to play an important role in carcinogenesis. However, the effect of MSCs on tumor and metastasis development and the mechanisms underlying the interaction of cancer and stem cells are not completely understood. This study investigated the effect of MSCs on breast cancer metastasis formation by using the methods of in vivo fluorescence and luminescence imaging. METHODS: MSCs were isolated from bone marrow of normal donors, characterized, and genetically labeled with luciferase (luc2). The effects of MSCs on MDA-MB-231 cancer cell proliferation were evaluated in conditioned medium from MSCs. To generate lung metastases, MDA-MB-231 cells stably expressing red fluorescent protein Turbo FP650 were injected intravenously into nude mice. On day 10 after the cancer cell injection, mice were injected via the tail vein with MSCs-luc2 cells (the MET+MSCs group). Animals that received the injection of MDA-MB-231-Turbo FP650 alone (the MET group) and no injections (the intact control group) served as controls. Fluorescence and bioluminescence imaging was performed for monitoring of the metastasis formation and MSC distribution in the recipient's body. RESULTS: We found that the proliferative activity of the cancer cells in the presence of MSC conditioned medium was lower than that of the cells grown in conventional culture medium. The metastasis formation in the MET+MSCs group was delayed in time as compared with the MET group. Macroscopic and histological examination of isolated lungs 8 weeks after cancer cell injection showed that the total number of metastases in animals of the MET+MSCs group was significantly lower. Using bioluminescence imaging in vivo, we found that MSCs-luc2 cells survived in the host animal for at least 7 weeks and re-migrated to the lung 6 to 7 weeks after injection. Immunohistochemical analysis revealed the presence of MSCs-luc2 in metastases and lung tissue. CONCLUSIONS: Long-term in vivo bioluminescence imaging of intravenously injected MSCs-luc2 cells showed distribution of MSCs to the lungs and abdominal organs within the first 2 to 3 weeks and re-migration to the lungs in weeks 6 to 7. It was found that MSCs reduced the proliferative activity of cancer cells in vitro and lung metastasis formation in mice.