Human bone marrow mesenchymal stem cells (hBMSCs) are widely used cell source for clinical bone regeneration. Achieving the greatest therapeutic effect is dependent on the osteogenic differentiation potential of the stem cells to be implanted. However, there are still no practical methods to characterize such potential non-invasively or previously. Monitoring cellular morphology is a practical and non-invasive approach for evaluating osteogenic potential. Unfortunately, such image-based approaches had been historically qualitative and requiring experienced interpretation. By combining the non-invasive attributes of microscopy with the latest technology allowing higher throughput and quantitative imaging metrics, we studied the applicability of morphometric features to quantitatively predict cellular osteogenic potential. We applied computational machine learning, combining cell morphology features with their corresponding biochemical osteogenic assay results, to develop prediction model of osteogenic differentiation. Using a dataset of 9,990 images automatically acquired by BioStation CT during osteogenic differentiation culture of hBMSCs, 666 morphometric features were extracted as parameters. Two commonly used osteogenic markers, alkaline phosphatase (ALP) activity and calcium deposition were measured experimentally, and used as the true biological differentiation status to validate the prediction accuracy. Using time-course morphological features throughout differentiation culture, the prediction results highly correlated with the experimentally defined differentiation marker values (R>0.89 for both marker predictions). The clinical applicability of our morphology-based prediction was further examined with two scenarios: one using only historical cell images and the other using both historical images together with the patient's own cell images to predict a new patient's cellular potential. The prediction accuracy was found to be greatly enhanced by incorporation of patients' own cell features in the modeling, indicating the practical strategy for clinical usage. Consequently, our results provide strong evidence for the feasibility of using a quantitative time series of phase-contrast cellular morphology for non-invasive cell quality prediction in regenerative medicine.
Heat shock proteins (HSPs) are recognized as significant participants in cancer immunity. We previously reported that HSP70 expression following hyperthermia using magnetic nanoparticles induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by administration of recombinant HSP70 protein into the tumor in situ. Hyperthermia was conducted using our original magnetite cationic liposomes (MCLs), which have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs and recombinant mouse HSP70 (rmHSP70) were injected into melanoma nodules in C57BL/6 mice, which were subjected to AMF for 30 min. Temperature within the tumor reached 43 degrees C and was maintained by controlling the magnetic field intensity. The combined treatment strongly inhibited tumor growth over a 30-day period and complete regression of tumors was observed in 20% (2/10) of mice. It was also found that systemic antitumor immunity was induced in the cured mice. This study suggests that novel combined therapy using exogenous HSP70 and hyperthermia has great potential in cancer treatment.
Heat shock proteins (HSPs) are recognized as significant participants in immune reactions. We previously reported that expression of HSP70 in response to hyperthermia, produced using our original magnetite cationic liposomes (MCLs), induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by hsp70 gene transfer. A human hsp70 gene mediated by cationic liposomes was injected into a B16 melanoma nodule in C57BL/6 mice in situ. At 24 hours after the injection of the hsp70 gene, MCLs were injected into melanoma nodules in C57BL/6 mice, which were subjected to an alternating magnetic field for 30 minutes. The temperature at the tumor reached 431C and was maintained by controlling the magnetic field intensity. The combined treatment strongly arrested tumor growth over a 30-day period, and complete regression of tumors was observed in 30% (3/10) of mice. Systemic antitumor immunity was induced in the cured mice. This study demonstrates that this novel therapeutic strategy combining the use of hsp70 gene therapy and hyperthermia using MCLs may be applicable to patients with advanced malignancies. Cancer Gene Therapy (2003) 10, 918-925. doi:10.1038/sj.cgt.7700648Keywords: heat shock proteins; hyperthermia; magnetite; cancer immunotherapy H yperthermia is a promising approach for cancer therapy. 1,2 However, the inevitable technical problem with hyperthermia is the difficulty in heating only the local tumor region to the intended temperature without damaging the surrounding normal tissue. Magnetic nanoparticles have been used for hyperthermia treatment in an attempt to overcome these disadvantages. 3,4 Magnetic nanoparticles generate heat in an alternating magnetic field (AMF) due to hysteresis loss. 5 We have developed magnetite cationic liposomes (MCLs) for intracellular hyperthermia. 6,7 The hyperthermic effect of MCLs was examined in an in vivo study, and complete tumor regression was observed. 8 Since some researchers have reported that heat treatment itself can enhance the immunogenicity of cancer cells, 9-12 we previously investigated hyperthermia-induced antitumor immunity in T-9 rat glioma cells in vivo. 13 This induced immunity continued for an extended period of time, and the rats treated with hyperthermia completely rejected T-9 cells as a metastasis model. Moreover, we investigated the mechanisms of antitumor immunity induced by intracellular hyperthermia with regard to the role of heat shock proteins (HSPs). 14,15 HSPs are a highly conserved group of intracellular proteins whose synthesis is increased by a large variety of stressors including heat shock. 16 As expression of HSP70 protects cells from heat-induced apoptosis, 17 HSP70 expression is considered a complicating factor in hyperthermia. On the other hand, recent reports have shown the importance of HSPs, such as HSP70, HSP90, and glucose-regulated protein 96 (gp96), in immune reactions. 18,19 A possible mechanism by which HSPs influence tumor cell immunogenicity is associated with...
BackgroundWe have developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma.MethodMCLs were injected into the osteosarcoma and then subjected to an AMF.ResultsThe tumor was heated at over 42°C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression.ConclusionThese results suggest that this treatment is effective for osteosarcoma.
Human bone marrow mesenchymal stem cells (hBMSCs) represents one of the most frequently applied cell sources for clinical bone regeneration. To achieve the greatest therapeutic effect, it is crucial to evaluate the osteogenic differentiation potential of the stem cells during their culture before the implantation. However, the practical evaluation of stem cell osteogenicity has been limited to invasive biological marker analysis that only enables assaying a single end-point. To innovate around invasive quality assessments in clinical cell therapy, we previously explored and demonstrated the positive predictive value of using time-course images taken during differentiation culture for hBMSC bone differentiation potential. This initial method establishes proof of concept for a morphology-based cell evaluation approach, but reveals a practical limitation when considering the need to handle large amounts of image data. In this report, we aimed to scale-down our proposed method into a more practical, efficient modeling scheme that can be more broadly implemented by physicians on the frontiers of clinical cell therapy. We investigated which morphological features are critical during the osteogenic differentiation period to assure the performance of prediction models with reduced burden on image acquisition. To our knowledge, this is the first detailed characterization that describes both the critical observation period and the critical number of time-points needed for morphological features to adequately model osteogenic potential. Our results revealed three important observations: (i) the morphological features from the first 3 days of differentiation are sufficiently informative to predict bone differentiation potential, both activities of alkaline phosphatase and calcium deposition, after 3 weeks of continuous culture; (ii) intervals of 48 h are sufficient for measuring critical morphological features; and (iii) morphological features are most accurately predictive when early morphological features from the first 3 days of differentiation are combined with later features (after 10 days of differentiation). Biotechnol. Bioeng. 2014;111: 1430–1439.
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