Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties.

BACKGROUND: Cells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body. METHODS: A blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested. RESULTS: Chondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties. CONCLUSIONS: A DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.

High throughput screening of mesenchymal stem cell lines using deep learning.

Mesenchymal stem cells (MSCs) are increasingly used as regenerative therapies for patients in the preclinical and clinical phases of various diseases. However, the main limitations of such therapies include functional heterogeneity and the lack of appropriate quality control (QC) methods for functional screening of MSC lines; thus, clinical outcomes are inconsistent. Recently, machine learning (ML)-based methods, in conjunction with single-cell morphological profiling, have been proposed as alternatives to conventional in vitro/vivo assays that evaluate MSC functions. Such methods perform in silico analyses of MSC functions by training ML algorithms to find highly nonlinear connections between MSC functions and morphology. Although such approaches are promising, they are limited in that extensive, high-content single-cell imaging is required; moreover, manually identified morphological features cannot be generalized to other experimental settings. To address these limitations, we propose an end-to-end deep learning (DL) framework for functional screening of MSC lines using live-cell microscopic images of MSC populations. We quantitatively evaluate various convolutional neural network (CNN) models and demonstrate that our method accurately classifies in vitro MSC lines to high/low multilineage differentiating stress-enduring (MUSE) cells markers from multiple donors. A total of 6,120 cell images were obtained from 8 MSC lines, and they were classified into two groups according to MUSE cell markers analyzed by immunofluorescence staining and FACS. The optimized DenseNet121 model showed area under the curve (AUC) 0.975, accuracy 0.922, F1 0.922, sensitivity 0.905, specificity 0.942, positive predictive value 0.940, and negative predictive value 0.908. Therefore, our DL-based framework is a convenient high-throughput method that could serve as an effective QC strategy in future clinical biomanufacturing processes.

Human Palatine Tonsils Are Linked to Alzheimer's Disease through Function of Reservoir of Amyloid Beta Protein Associated with Bacterial Infection.

Amyloid-ß (Aß)-peptide production or deposition in the neuropathology of Alzheimer's disease (AD) was shown to be caused by chronic inflammation that may be induced by infection, but the role of pathogenic-bacteria-related AD-associated Aß is not yet clearly understood. In this study, we validated the hypothesis that there is a correlation between the Aß-protein load and bacterial infection and that there are effects of bacteria, Staphylococcus aureus (S. aureus), on the Aß load in the inflammatory environment of human tonsils. Here, we detected Aß-peptide deposits in human tonsil tissue as well as tissue similar to tonsilloliths found in the olfactory cleft. Interestingly, we demonstrated for the first time the presence of Staphylococcus aureus (S. aureus) clustered around or embedded in the Aß deposits. Notably, we showed that treatment with S. aureus upregulated the Aß-protein load in cultures of human tonsil organoids and brain organoids, showing the new role of S. aureus in Aß-protein aggregation. These findings suggest that a reservoir of Aß and pathogenic bacteria may be a possible therapeutic target in human tonsils, supporting the treatment of antibiotics to prevent the deposition of Aß peptides via the removal of pathogens in the intervention of AD pathogenesis.

Potential application of human neural crest-derived nasal turbinate stem cells for the treatment of neuropathology and impaired cognition in models of Alzheimer's disease.

BACKGROUND: Stem cell transplantation is a fascinating therapeutic approach for the treatment of many neurodegenerative disorders; however, clinical trials using stem cells have not been as effective as expected based on preclinical studies. The aim of this study is to validate the hypothesis that human neural crest-derived nasal turbinate stem cells (hNTSCs) are a clinically promising therapeutic source of adult stem cells for the treatment of Alzheimer's disease (AD). METHODS: hNTSCs were evaluated in comparison with human bone marrow-derived mesenchymal stem cells (hBM-MSCs) according to the effect of transplantation on AD pathology, including PET/CT neuroimaging, immune status indicated by microglial numbers and autophagic capacity, neuronal survival, and cognition, in a 5 × FAD transgenic mouse model of AD. RESULTS: We demonstrated that hNTSCs showed a high proliferative capacity and great neurogenic properties in vitro. Compared with hBM-MSC transplantation, hNTSC transplantation markedly reduced Aß42 levels and plaque formation in the brains of the 5 × FAD transgenic AD mice on neuroimaging, concomitant with increased survival of hippocampal and cortex neurons. Moreover, hNTSCs strongly modulated immune status by reducing the number of microglia and the expression of the inflammatory cytokine IL-6 and upregulating autophagic capacity at 7 weeks after transplantation in AD models. Notably, compared with transplantation of hBM-MSCs, transplantation of hNTSCs significantly enhanced performance on the Morris water maze, with an increased level of TIMP2, which is necessary for spatial memory in young mice and neurons; this difference could be explained by the high engraftment of hNTSCs after transplantation. CONCLUSION: The reliable evidence provided by these findings reveals a promising therapeutic effect of hNTSCs and indicates a step forward the clinical application of hNTSCs in patients with AD.

Rapid Cartilage Regeneration of Spheroids Composed of Human Nasal Septum-Derived Chondrocyte in Rat Osteochondral Defect Model.

BACKGROUND: Cell-based therapies have been studied for articular cartilage regeneration. Articular cartilage defects have little treatments because articular cartilage was limited regenerative capacity. Damaged articular cartilage is difficult to obtain a successful therapeutic effect. In additionally these articular cartilage defects often cause osteoarthritis. Chondrocyte implantation is a widely available therapy used for regeneration of articular cartilage because this tissue has poor repair capacity after injury. Human nasal septum-drived chondrocytes (hNCs) from the septum show greater proliferation ability and chondrogenic capacity than human articular chondrocytes (hACs), even across different donors with different ages. Moreover, the chondrogenic properties of hNCs can be maintained after extensive culture expansion. METHODS: In this study, 2 dimensional (2D) monolayer cultured hNCs (hNCs-2D) and 3 dimensional (3D) spheroids cultured hNCs (hNCs-3D) were examined for chondrogenic capacity in vitro by PCR and immunofluorescence staining for chondrogenic marker, cell survival during cultured and for cartilage regeneration ability in vivo in a rat osteochondral defect model. RESULTS: hNCs-3D showed higher viability and more uniform morphology than 3D spheroids cultured hACs (hACs-3D) in culture. hNCs-3D also showed greater expression levels of the chondrocyte-specific marker Type II collagen (COL2A1) and sex-determining region Y (SRY)-box 9 (SOX9) than hNCs-2D. hNCs-3D also expressed chondrogenic markers in collagen. Specially, in the osteochondral defect model, implantation of hNCs-3D led to greater chondrogenic repair of focal cartilage defects in rats than implantation of hNCs-2D. CONCLUSION: These data suggest that hNCs-3D are valuable therapeutic agents for repair and regeneration of cartilage defects.

Accelerated Bone Regeneration via Three-Dimensional Cell-Printed Constructs Containing Human Nasal Turbinate-Derived Stem Cells as a Clinically Applicable Therapy.

Stem cell transplantation is a promising therapeutic strategy that includes both cell therapy and tissue engineering for the treatment of many regenerative diseases; however, the efficacy and safety of stem cell therapy depend on the cell type used in therapeutic and translational applications. In this study, we validated the hypothesis that human nasal turbinate-derived mesenchymal stem cells (hTMSCs) are a potential therapeutic source of adult stem cells for clinical use in bone tissue engineering using three-dimensional (3D) cell-printing technology. hTMSCs were cultured and evaluated for clinical use according to their cell growth, cell size, and preclinical safety and were then incorporated into a multicompositional 3D bioprinting system and investigated for bone tissue regeneration in vitro and in vivo. Finally, hTMSCs were compared with human bone marrow-derived MSCs (hBMSCs), which are the most common stem cell type used in regenerative medicine. hTMSCs from three different donors showed greater and faster cell growth than hBMSCs from two different donors when cultured. The hTMSCs were smaller in size than the hBMSCs. Furthermore, the hTMSCs did not exhibit safety issues in immunodeficient mice. hTMSCs in 3D-printed constructs (3D-hTMSC) showed much greater viability, growth, and osteogenic differentiation potential in vitro than hBMSCs in 3D-printed constructs (3D-hBMSC). Likewise, 3D-hTMSC showed better cell survival and alkaline phosphatase activity and greater osteogenic protein expression than 3D-hBMSC upon subcutaneous implantation into the dorsal region of nude mice. Notably, in an orthotopic model involving implantation into a tibial defect in rats, implantation of 3D-hTMSC led to greater bone matrix formation and enhanced bone healing to a greater degree than implantation of 3D-hBMSC. The clinically reliable evidence provided by these results is underlined by the potential for rapid tissue regeneration and ambulation in bone fracture patients implanted with 3D-hTMSC.

Magnetically actuated microrobots as a platform for stem cell transplantation.

Magnetic microrobots were developed for three-dimensional culture and the precise delivery of stem cells in vitro, ex vivo, and in vivo. Hippocampal neural stem cells attached to the microrobots proliferated and differentiated into astrocytes, oligodendrocytes, and neurons. Moreover, microrobots were used to transport colorectal carcinoma cancer cells to tumor microtissue in a body-on-a-chip, which comprised an in vitro liver-tumor microorgan network. The microrobots were also controlled in a mouse brain slice and rat brain blood vessel. Last, microrobots carrying mesenchymal stem cells derived from human nose were manipulated inside the intraperitoneal cavity of a nude mouse. The results indicate the potential of microrobots for the culture and delivery of stem cells.

Enhancing bio-hydrogen production from sodium formate by hyperthermophilic archaeon, Thermococcus onnurineus NA1.

Hyperthermophilic archaeon, Thermococcus onnurineus NA1 was reported to grow on formate producing hydrogen (H2). In this study, to sustain high H2 production rate and demonstrate the feasibility of mass production of H2, high cell density cultivation of T. onnurineus NA1 on sodium formate was employed under optimized conditions. From batch cultures, it was observed that the salinity of medium, significantly changed by the addition of formate salt and pH-adjusting agent, crucially affected cell growth and H2 production. With salinity carefully controlled between 3.7 and 4.6 %, 400 mM sodium formate was found to be an optimal initial concentration for maximizing cell growth-associated H2 production. Under optimal conditions, the repeated batch culture with cell recycling showed high cell density of OD600 of 1.7 in 3 and 30 L bioreactor, and the volumetric H2 production rate was enhanced up to 235.7 mmol L(-1) h(-1), which is one of the highest values reported to date.

Cloning, expression, and characterization of a DNA ligase from a hyperthermophilic archaeon Thermococcus sp.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed an ORF of 1689 bases encoding 562 amino acids that showed a high similarity to DNA ligases from other hyperthermophilic archaea. The ligase, which was designated TNA1_lig (Thermococcus sp. NA1 ligase), was cloned and expressed in Escherichia coli. The recombinant TNA1_lig was purified by metal affinity chromatography. The optimum ligase activity of the recombinant TNA1_lig occurred at 80 degrees C and pH 7.5. The enzyme was activated by MgCl2 and ZnCl2 but was inhibited by MnCl2 and NiCl2. Additionally, the enzyme was activated by either ATP or NAD+.