a-novel-cell-injection-method-with-minimum-invasion

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

application-live-mitochondria-staining-cell-sorting-to-purify

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...

an-automated-culture-system-for-maintaining-differentiating-human

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Kansai Medical University

Kansai Medical University Graduate School of Medicine

To enhance the therapeutic effect of growth factors, a powerful strategy is to direct their localization to damaged sites. To treat skin wounds and myocardial infarction, we selected vascular endothelial growth factor (VEGF) carrying binding affinity to collagen. A simple conjugation of a reported collagen-binding sequence and VEGF did not increase the collagen-binding affinity, indicating that the molecular interaction between the two proteins abolished collagen binding activity. Here, we present a new molecular evolution strategy, "all-in-one" in vitro selection, in which a collagen-binding VEGF (CB-VEGF) was directly identified from a random library consisting of random and VEGF sequences. As expected, the selected CB-VEGFs exhibited high binding affinity to collagen and maintained the same growth enhancement activity for endothelial cells as unmodified VEGF in solution. Furthermore, the selected CB-VEGF enhanced angiogenesis at skin wounds and infarcted myocardium. This study demonstrates that "all-in-one" in vitro selection is a novel strategy for the design of functional proteins for regenerative medicine.

"All-in-one" in vitro selection of collagen-binding vascular endothelial growth factor.

Non-genetic purification methods for pluripotent stem cell-derived hepatocyte-like cells are useful for liver regenerative therapy and pharmaceutical applications.

Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells Can Be Enriched by a Combination of Mitochondrial Content and Activated Leukocyte Cell Adhesion Molecule.

A 70-year-old man underwent off-pump coronary artery bypass grafting 28 days after his recovery from coronavirus disease 2019 (COVID-19), which was confirmed by a negative polymerase chain reaction (PCR) test result for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a nasopharyngeal swab. The PCR test result was also negative for nasopharyngeal sampling 5 days prior to the surgery. However, his redundant saphenous vein and sputum through the endotracheal tube that was taken on the operative day showed the presence of SARS-CoV-2 by PCR. Immunohistochemical analysis of Spike and Nucleoprotein of the saphenous vein showed small clusters of each antigen-positive speckle. Ultrastructural imaging of the saphenous vein showed virus-like particles. The cell-based assay suggested that the patient's serum contained a higher concentration of type-I interferons than that of healthy control sera. These observations suggest that internal viral shedding and, to some extent, innate immune responses continue after COVID-19 recovery.

Possible internal viral shedding and interferon production after clinical recovery from COVID-19: Case report.

Keratinocytes are the most abundant cells in the epidermis, and as part of the frontline immunologic defense system, keratinocytes function as a barrier to exogenous attacks. Protease-activated receptor 2 (PAR2) is expressed in human keratinocytes and activated in several inflammatory conditions, such as atopic dermatitis (AD). In this study, we demonstrated the differentiation of human induced pluripotent stem cell into keratinocytes by the improved, robust differentiation procedure and confirmed that human induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express PAR2, which is activated by external addition of the ligand peptide and trypsin. The activation of PAR2 led to the release of calcium from intracellular calcium storage, followed by the release of the proinflammatory cytokine tumor necrosis factor  Moreover, PAR2 antagonist I-191 (CAS No. 1690172-25-8) inhibited calcium release in a dose-dependent manner. This is the first study to demonstrate that iKera expresses a functional PAR2 protein. Furthermore, our results indicate crosstalk between the PAR2- and IL-4-mediated inflammatory axes in iKera, suggesting that iKera can be used as a platform for a broad range of mechanism-targeted drug screening in AD. SIGNIFICANCE STATEMENT: This is the first study to provide evidence that human induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express functional protease-activated receptor 2 (PAR2). Furthermore, this study demonstrated in iKera that the IL-4 inflammatory axis can crosstalk with the PAR2-mediated inflammatory axis in keratinocytes. To the best of our knowledge, this is the first report to indicate that iKera can be used for research and as a drug screening platform for atopic dermatitis.

Human Induced Pluripotent Stem Cell-Derived Keratinocyte-Like Cells for Research on Protease-Activated Receptor 2 in Nonhistaminergic Cascades of Atopic Dermatitis .

The technologies used to generate human induced pluripotent stem cell (iPSC) from somatic cells potentially enable the wide application of iPSC-derived differentiated cells in industrial research fields as a replacement for animals. However, as highly trained individuals are required to obtain reproducible results, this approach has limited social implementation. In the research field of iPSC, it is believed that documentable information is not enough for reproducing the quality of the differentiated cells. Therefore, automated culture machines for cell processing should make the starting of iPSC-using researches easier. We developed a programmable all-in-one automated culture machine, with dense and compact constitution that fits within a normal biosafety cabinet (200 mm wide, 233 mm height, and 110 mm depth). This instrument was fabricated using novel x-y-z-axes-rail-system, such as an overhead traveling crane, in a factory, which served as the main handling machinery. This machine enabled stable and efficient expansion of human iPSC under the feeder-free condition, without karyotype alterations, and simultaneously differentiated the cells into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. Overall, this machine would facilitate the social implementation of human pluripotent stem cells and contribute to the accumulation of sharable knowledge for the standardization of the entire handling processes of iPSC in pharmaceutical, food, and cosmetic research.

Compact automated culture machine for human induced pluripotent stem cell maintenance and differentiation.

Adenosine triphosphate (ATP) is an extracellular signaling molecule that mainly affects the pathophysiological situation in the body and can be sensed by purinergic receptors, including ionotropic P2X7. Neuronal stem cells (NSCs) remain in adult neuronal tissues and can contribute to physiological processes via activation by evoked pathophysiological situations. In this study, we revealed that human-induced pluripotent stem cell-derived NSCs (iNSCs) have ATP-sensing ability primarily via the purinergic and ionotropic receptor P2X7. Next, to develop a machine learning (ML)-based screening system for food-derived neuronal effective substances and their effective doses, we collected ATP-triggered calcium responses of iNSCs pretreated with several substances and doses. Finally, we discovered that ML was performed using composite images, each containing nine waveform images, to achieve a better ML model (MLM) with higher precision. Our MLM can correctly sort subtle unidentified changes in waveforms produced by pretreated iNSCs with each substance and/or dose into the positive group, with common mRNA expression changes belonging to the gene ontology signatures.

Machine learning discriminates P2X7-mediated intracellular calcium sparks in human-induced pluripotent stem cell-derived neural stem cells.

The parathyroid gland plays an essential role in mineral and bone metabolism. Cultivation of physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. In this study, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. Parathyroid cell differentiation occurred in accordance with embryologic development. Differentiated cells, which expressed the parathyroid hormone, adopted unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

The Induction of Parathyroid Cell Differentiation from Human Induced Pluripotent Stem Cells Promoted Via TGF-α/EGFR Signaling.

The mechanistic/mammalian target of rapamycin (mTOR) is involved in a wide range of cellular processes. However, the role of mTOR in podocytes remains unclear. In this study, we aimed to clarify the role of mTOR in podocyte differentiation from human induced pluripotent stem cells (hiPSCs) and to establish an efficient differentiation protocol for human podocytes. We generated podocytes from hiPSCs by modifying protocol. The expression of the podocyte-specific slit membrane components nephrin and podocin was measured using PCR, western blotting, flow cytometry, and immunostaining; and the role of mTOR was evaluated using inhibitors of the mTOR pathway. Nephrin and podocin were found to be expressed in cells differentiated from hiPSCs, and their expression was increased by mTOR inhibitor treatment. S6, a downstream component of the mTOR pathway, was also found to be involved in podocyte differentiation. we evaluated its permeability to albumin, urea, and electrolytes. The induced podocytes were permeable to the small molecules, but only poorly permeable to albumin. We have shown that the mTOR pathway is involved in podocyte differentiation. Our monolayer podocyte differential protocol, using an mTOR inhibitor, provides a novel in vitro model for studies of kidney physiology and pathology.

Fumiyuki Hattori

Innovative Regenerative Medicine,
Graduate School of Medicine,
Innovative Regenerative Medicine, Graduate School of Medicine

Kansai Medical University

A Novel Cell Injection Method with Minimum Invasion

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Fumiyuki Hattori

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Innovative Regenerative Medicine,Graduate School of Medicine,Innovative Regenerative Medicine, Graduate School of Medicine

Kansai Medical University

A Novel Cell Injection Method with Minimum Invasion

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Innovative Regenerative Medicine,
Graduate School of Medicine,
Innovative Regenerative Medicine, Graduate School of Medicine