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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

At our hospital, we use aprepitant for nausea and vomiting when administering highly emetic anticancer agents, according to "Guidelines for the Appropriate Use of Antiemetic Agents" given by the Japan Society of Clinical Oncology. We initiated the intravenous administration of fosaprepitant for better compliance compared with aprepitant; however, we observed phlebitis after the infusion of fosaprepitant. Therefore, we investigated measures to reduce phlebitis associated with the infusion of fosaprepitant. For the first premedication, fosaprepitant (150 mg) was dissolved in 100 mL of saline and administered for 30 minutes; 1 of 2 patients showed grade 4 phlebitis. For the modified premedication, fosaprepitant, dexamethasone, and 5- HT(3) antagonist were dissolved in 100 mL of saline and administered for 30 minutes. The modified premedication was administered to a total of 27 patients; 5 patients developed mild phlebitis (grade 1), but infusion could be continued by treating their phlebitis with a hot pack. We used a combination of dexamethasone and 5-HT(3) antagonist with fosaprepitant as a modified premedication in order to avoid drug-induced vascular damage, which resulted in the pH decreasing to 6.20-7.55 (close to neutral) and a shorter infusion time.

[Methods of preventing phlebitis induced by infusion of fosaprepitant].

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.

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.

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.

Hiromi Yamashita

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

Kansai Medical University

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

Hiromi Yamashita

.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

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