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  1. null (Ed.)
  2. Chondrocyte viability is a crucial factor in evaluating cartilage health. Most cell viability assays rely on dyes and are not applicable forin vivoor longitudinal studies. We previously demonstrated that two-photon excited autofluorescence and second harmonic generation microscopy provided high-resolution images of cells and collagen structure; those images allowed us to distinguish live from dead chondrocytes by visual assessment or by the normalized autofluorescence ratio. However, both methods require human involvement and have low throughputs. Methods for automated cell-based image processing can improve throughput. Conventional image processing algorithms do not perform well on autofluorescence images acquired by nonlinear microscopes due to low image contrast. In this study, we compared conventional, machine learning, and deep learning methods in chondrocyte segmentation and classification. We demonstrated that deep learning significantly improved the outcome of the chondrocyte segmentation and classification. With appropriate training, the deep learning method can achieve 90% accuracy in chondrocyte viability measurement. The significance of this work is that automated imaging analysis is possible and should not become a major hurdle for the use of nonlinear optical imaging methods in biological or clinical studies.

     
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  3. Background Information

    Wnt/β‐catenin signalling, in the microenvironment of pluripotent stem cells (PSCs), plays a critical role in their differentiation and proliferation. Contradictory reports on the role of Wnt/β‐catenin signalling in PSCs self‐renewal and differentiation, however, render these mechanisms largely unclear.

    Results

    Wnt/β‐catenin signalling pathway in human‐induced pluripotent stem cells (hiPSCs) was activated by inhibiting glycogen synthase kinase 3 (GSK3), driving the cells into a mesodermal/mesenchymal state, exhibiting proliferative, invasive and anchorage‐independent growth properties, where over 70% of cell population became CD 44 (+)/CD133 (+). Wnt/β‐catenin signalling activation also altered the metabolic state of hiPSCs from aerobic glycolysis to oxidative metabolism and changed their drug and oxidative stress sensitivities. These effects of GSK3 inhibition were suppressed in HIF1α‐stabilised cells.

    Conclusions

    Persistent activation of Wnt/β‐catenin signalling endows hiPSCs with proliferative/invasive ‘teratoma‐like’ states, shifting their metabolic dependence and allowing HIF1α‐stabilisation to inhibit their proliferative/invasive properties.

    Significance

    The hiPSC potential to differentiate into ‘teratoma‐like’ cells suggest that stem cells may exist in two states with differential metabolic and drug dependency.

     
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