Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation

Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21%) has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF), the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the "hypoxic niches" present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue

Effects of the pulsed electromagnetic field PST® on human tendon stem cells : A controlled laboratory study

Background: Current clinical procedures for rotator cuff tears need to be improved, as a high rate of failure is still observed. Therefore, new approaches have been attempted to stimulate self-regeneration, including biophysical stimulation modalities, such as low-frequency pulsed electromagnetic fields, which are alternative and non-invasive methods that seem to produce satisfying therapeutic effects. While little is known about their mechanism of action, it has been speculated that they may act on resident stem cells. Thus, the purpose of this study was to evaluate the effects of a pulsed electromagnetic field (PST\uae) on human tendon stem cells (hTSCs) in order to elucidate the possible mechanism of the observed therapeutic effects. Methods: hTSCs from the rotator cuff were isolated from tendon biopsies and cultured in vitro. Then, cells were exposed to a 1-h PST\uae treatment and compared to control untreated cells in terms of cell morphology, proliferation, viability, migration, and stem cell marker expression. Results: Exposure of hTSCs to PST\uae did not cause any significant changes in proliferation, viability, migration, and morphology. Instead, while stem cell marker expression significantly decreased in control cells during cell culturing, PST\uae-treated cells did not have a significant reduction of the same markers. Conclusions: While PST\uae did not have significant effects on hTSCs proliferation, the treatment had beneficial effects on stem cell marker expression, as treated cells maintained a higher expression of these markers during culturing. These results support the notion that PST\uae treatment may increase the patient stem cell regenerative potential

Isolation and characterization of 2 new human rotator cuff and long head of biceps tendon cells possessing stem cell-like self-renewal and multipotential differentiation capacity

BACKGROUND: Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. HYPOTHESIS: Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. STUDY DESIGN: Controlled laboratory study. METHODS: Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. RESULTS: Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types--namely, osteoblasts, adipocytes, and skeletal muscle cells. CONCLUSION: This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. CLINICAL RELEVANCE: Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration

GM1 Ganglioside Promotes Osteogenic Differentiation of Human Tendon Stem Cells

Gangliosides, the sialic acid-conjugated glycosphingolipids present in the lipid rafts, have been recognized as important regulators of cell proliferation, migration, and apoptosis. Due to their peculiar localization in the cell membrane, they modulate the activity of several key cell receptors, and increasing evidence supports their involvement also in stem cell differentiation. In this context, herein we report the role played by the ganglioside GM1 in the osteogenic differentiation of human tendon stem cells ( hTSCs). In particular, we found an increase of GM1 levels during osteogenesis that is instrumental for driving the process. In fact, supplementation of the ganglioside in the medium significantly increased the osteogenic differentiation capability of hTSCs. Mechanistically, we found that GM1 supplementation caused a reduction in the phosphorylation of the platelet-derived growth factor receptor-ss ( PDGFR-ss), which is a known inhibitor of osteogenic commitment. These results were further corroborated by the observation that GM1 supplementation was able to revert the inhibitory effects on osteogenesis when the process was inhibited with exogenous PDGF

Lipogems product treatment increases the proliferation rate of human tendon stem cells without affecting their stemness and differentiation capability

Increasing the success rate of rotator cuff healing remains tremendous challenge. Among many approaches, the possibility of activating resident stem cells in situ, without the need to isolate them from biopsies, could represent valuable therapeutic strategy. Along this line, it has been recently demonstrated that lipoaspirate product, Lipogems, contains and produces growth-factors that may activate resident stem cells. In this study, human tendon stem cells (hTSCs) from the rotator cuff were cocultured in a transwell system with the Lipogems lipoaspirate product and compared to control untreated cells in terms of cell proliferation, morphology, stem cell marker and VEGF expression, and differentiation and migration capabilities. Results showed that the Lipogems product significantly increases the proliferation rate of hTSCs without altering their stemness and differentiation capability. Moreover, treated cells increase the expression of VEGF, which is crucial for the neovascularization of the tissue during the healing process. Overall, this study supports that directly activating hTSCs with the Lipogems lipoaspirate could represent a new practical therapeutic approach. In fact, obtaining a lipoaspirate is easier, safer, and more cost-effective than harvesting cells from tendon or bone marrow biopsies, expanding them in GMP facility and then reinjecting them in the patient

Clasificación microscópica de las lesiones cutáneas producidas por Hypoderma lineatum

Trabajo presentado a la: XXVII Reunión de la Sociedad Española de Anatomía Patológica Veterinaria. (Barcelona, España, 17 al 19 Junio, 2015).Peer Reviewe