Integrating deep learning and unbiased automated high-content screening to identify complex disease signatures in human fibroblasts.

Drug discovery for diseases such as Parkinson's disease are impeded by the lack of screenable cellular phenotypes. We present an unbiased phenotypic profiling platform that combines automated cell culture, high-content imaging, Cell Painting, and deep learning. We applied this platform to primary fibroblasts from 91 Parkinson's disease patients and matched healthy controls, creating the largest publicly available Cell Painting image dataset to date at 48 terabytes. We use fixed weights from a convolutional deep neural network trained on ImageNet to generate deep embeddings from each image and train machine learning models to detect morphological disease phenotypes. Our platform's robustness and sensitivity allow the detection of individual-specific variation with high fidelity across batches and plate layouts. Lastly, our models confidently separate LRRK2 and sporadic Parkinson's disease lines from healthy controls (receiver operating characteristic area under curve 0.79 (0.08 standard deviation)), supporting the capacity of this platform for complex disease modeling and drug screening applications.

Hypothermic and cryogenic preservation of tissue-engineered human bone.

To foster translation and commercialization of tissue-engineered products, preservation methods that do not significantly compromise tissue properties need to be designed and tested. Robust preservation methods will enable the distribution of tissues to third parties for research or transplantation, as well as banking of off-the-shelf products. We recently engineered bone grafts from induced pluripotent stem cells and devised strategies to facilitate a tissue-engineering approach to segmental bone defect therapy. In this study, we tested the effects of two potential preservation methods on the survival, quality, and function of tissue-engineered human bone. Engineered bone grafts were cultured for 5 weeks in an osteogenic environment and then stored in phosphate-buffered saline (PBS) solution at 4 °C or in Synth-a-Freeze™ at -80 °C. After 48 h, samples were warmed up in a water bath at 37 °C, incubated in osteogenic medium, and analyzed 1 and 24 h after revitalization. The results show that while storage in Synth-a-Freeze at -80 °C results in cell death and structural alteration of the extracellular matrix, hypothermic storage in PBS does not significantly affect tissue viability and integrity. This study supports the use of short-term hypothermic storage for preservation and distribution of high-quality tissue-engineered bone grafts for research and future clinical applications.

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.

BACKGROUND: Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines. METHODS: In the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium). RESULTS: The results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum. CONCLUSIONS: The findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.

Biomimetic Crystallization of MnFe2O4 Mediated by Peptide-Catalyzed Esterification at Low Temperature.

Enzymes are some of the most efficient catalysts in nature. If small catalytic peptides mimic enzymes, there is potential for broad applications from catalysis for new material synthesis to drug development, due to the ease of molecular design. Recently a hydrogel-based combinatory phage display library was developed and protease-mimicking peptides were identified. Here we advanced the previous discovery to apply one of these catalytic peptides for the synthesis of bimetal oxide nanocrystals through the catalytic ester-elimination pathway. Conventional bimetal oxide crystallization usually requires high temperatures above several hundred °C; however, this catalytic peptide could grow superparamagnetic MnFe2O4 nanocrystals at 4°C. Superconducting quantum interference device (SQUID) analysis revealed that MnFe2O4 nano-crystals grown by the catalytic peptide exhibit superpara-magnetism. This study demonstrates the usefulness of protease-mimicking catalytic peptides in the field of material synthesis.