Melanocyte transplantation to skin prepared by controlled PUVA-induced sunburn-like blistering for vitiligo treatment - A pilot clinical trial.

Vitiligo is a common acquired disease of pigment loss. In lesions recalcitrant to non-invasive treatment, transplantation of cultured autologous melanocytes is an emerging choice. Conventionally, the recipient site is often prepared by laser-mediated or mechanical dermabrasion. Such preparation procedures have disadvantages including prolonged transplantation duration, long period for reepithelialization and potential scarring. We propose a method of preparing recipient sites by psoralen and controlled ultraviolet A (PUVA)-induced blistering followed by transplanting suspended melanocytes. We introduced this method in 10 patients with segmental vitiligo on their recipient site 3 to 5 days before transplantation and blistering developed in 2 to 3 days afterwards. On the day of transplantation, the blister roof could be peeled off easily without bleeding and the recipient site preparation could be completed in 20 min. The recipient site became reepithelialized within 1 week. Progressive repigmentation was observed for up to 6 months, with an average of 65.06% repigmentation in the recipient site without scarring at the end of follow-up. Hence, preparation of the recipient site by controlled PUVA-induced sunburn-like blistering can potentially facilitate melanocyte transplantation and prevent scarring.

Multi-laboratory Validation Study of the Vitrigel-Eye Irritancy Test Method as an Alternative to In Vivo Eye Irritation Testing.

Collagen vitrigel membranes (CVMs) comprising high-density collagen fibrils equivalent to in vivo connective tissues have been widely used in cell culture applications. A human corneal epithelium (hCE) model was previously developed by the Takezawa group, by culturing HCE-T cells (derived from hCE cells) on a CVM scaffold in a chamber that provided an air-liquid interface culture system. This hCE model was used to establish a new test method, known as the Vitrigel-Eye Irritancy Test (Vitrigel-EIT) method, which can be used to estimate the ocular irritation potential of test chemicals by analysing relative changes in transepithelial electrical resistance (TEER) over time. The current study was conducted in order to assess the reliability and relevance of the Vitrigel-EIT method at three participating laboratories by determining the method's within-laboratory reproducibility and between-laboratory reproducibility, as well as its capacity for distinguishing non-irritants from irritants in a bottom-up approach. The initial test sample size was found to be too low to evaluate the predictive capacity of the test method, and so it was evaluated with additional in-house data for a total of 93 test chemicals. The results showed 80-100% within-laboratory reproducibility and an excellent between-laboratory reproducibility that met the acceptance criteria of 80%. However, the method's predictive capacity for distinguishing non-irritants (test chemicals not requiring classification and labelling for eye irritation or serious eye damage, i.e. United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS) No Category) from irritants (GHS Categories 1 and 2) in a bottom-up approach was unacceptable because of false negative rates as high as 16.7%. After considerable review of the data with a view to using the method for regulatory purposes, it was determined that a more defined applicability domain, excluding test chemical solutions with a pH of 5 or less and solid test chemicals, improved the false negative rate to 4.2%. These results suggested that, within this carefully defined applicability domain, the Vitrigel-EIT method could be a useful alternative for distinguishing test chemicals that are ocular non-irritants from those that are irritants as part of a bottom-up approach.

Safety and efficacy of intracoronary artery administration of human bone marrow-derived mesenchymal stem cells in STEMI of Lee-Sung pigs-A preclinical study for supporting the feasibility of the OmniMSC-AMI phase I clinical trial.

Background: This study tested whether early left intracoronary arterial (LAD) administration of human bone marrow-derived mesenchymal stem cells (hBMMSCs, called OmniMSCs) in acute ST-segment elevation myocardial infarction (STEMI) of Lee-Sung pigs induced by 90 min balloon-occluded LAD was safe and effective. Methods and results: Young male Lee-Sung pigs were categorized into SC (sham-operated control, n = 3), AMI-B (STEMI + buffer/21 cc/administered at 90 min after STEMI, n = 6), and AMI-M [acute myocardial infarction (AMI) + hBMMSCs/1.5 × 107/administered at 90 min after STEMI, n = 6] groups. By 2 and 5 months after STEMI, the cardiac magnetic resonance imaging demonstrated that the muscle scar score (MSS) and abnormal cardiac muscle exercise score in the infarct region were significantly increased in the AMI-B than in the SC group that were significantly reversed in the AMI-M group, whereas the left ventricular ejection function by each month (from 1 to 5) displayed an opposite pattern of MSS among the groups (all p < 0.001). By 5 months, histopathological findings of infarct and fibrosis areas and isolectin-B4 exhibited an identical pattern, whereas the cellular expressions of troponin-I/troponin-T/von Willebrand factor exhibited an opposite pattern of MSS among the groups (all p < 0.001). The ST-segment resolution (>80%) was significantly earlier (estimated after 6-h AMI) in the AMI-M group than in the AMI-B group (p < 0.001). The protein expressions of inflammation (IL-1ß/TNF-α/NF-κB)/oxidative stress (NOX-1/NOX-2/oxidized protein)/apoptosis (cleaved caspase-3/cleaved PARP)/DNA damage (γ-H2AX) displayed an identical pattern to MSS among the groups, whereas the protein expressions of angiogenesis factors (SDF-1α/VEGF) were significantly and progressively increased from SC, AMI-B, to AMI-M groups (all p < 0.001). Conclusion: Early intra-LAD transfusion of OmniMSC treatment effectively reduced the infarct size and preserved LV function in porcine STEMI.

Validation study of a new reconstructed human epidermis model EPiTRI for in vitro skin irritation test according to OECD guidelines.

Following the global trend of reducing animal testing, various reconstructed human epidermis (RHE) models for skin irritation test (SIT) have been developed, verified, validated and included in OECD TG 439. We developed a new RHE called EPiTRI and a SIT method using EPiTRI (EPiTRI-SIT model) following the OECD guidelines. EPiTRI possesses morphological, biochemical and physiological properties similar to human epidermis with well-differentiated multilayered viable cells with barrier function. The EPiTRI-SIT model was tested for 20 reference chemicals in Performance Standard of OECD TG 439 (GD 220), showing good predictive capacity with 100% sensitivity, 70% specificity and 85% accuracy. EPiTRI had sensitivity in detecting di-n-propyl disulphate, as an irritant chemical (UN GHS Category 2), whereas most validated reference methods detected it as a non-irritant. An international validation study of EPiTRI-SIT was conducted in four laboratories to confirm the within- and between-laboratory reproducibility, as well as predictive capacity. The phase I/II within-laboratory and between-laboratory reproducibility was 100%/95% and 95%, respectively. The overall sensitivity, specificity and accuracy of EPiTRI-SIT was 96%, 70% and 83%, respectively, which fulfilled the OECD criteria. Thus, EPiTRI, meets the criteria of Performance Standards of OECD TG 439 (GD 220) and is suitable for screening irritating chemicals in vitro.

A reduced oxygen tension (5%) is not beneficial for maintaining human embryonic stem cells in the undifferentiated state with short splitting intervals.

BACKGROUND: Human embryos grow naturally in vivo in lower oxygen (O(2)) tension environments than atmospheric O(2) tension. Therefore, human embryonic stem cells (hESC), a derivative of embryos, will likely grow more favorably in a reduced O(2) tension. This study aimed to compare the behavior of hESC under reduced O(2) tension (5%) versus normoxia (21%). METHODS: hESC lines were cultured in different O(2) tensions and then examined for morphology, apoptosis and gene expression profiles. RESULTS: hESC grown in 5% O(2) tension were not morphologically different from hESC grown in normoxia on day 7 of the first and fourth passages. However, after prolonged culture without splitting (10-14 days), hESC colonies were thinner and looked better morphologically in 5% O(2), but the cells proliferated more slowly and their sizes were larger. At most time points, the gene expression profiles in both O(2) tensions showed no major difference in representative stemness genes (Oct-3/4, Nanog and Cripto), differentiation genes (Desmin, Nestin, alpha-fetoprotein and GDF-9) and hypoxia-related genes (HIF-1alpha and VEGF). A lower level of cyclin-D1 mRNA (suggestive of less Wnt pathway signaling on day 7 of the fourth passage) and a higher level of Desmin (suggestive of more differentiation to mesoderm, at day 7 of the first passage) were detected in 5% O(2). CONCLUSIONS: This study suggests that for routine culture of hESC with a short splitting interval (7 days), a low O(2) tension (5% O(2)) probably does not provide significant advantages over the standard 21% O(2) tension for the maintenance of an undifferentiated state by the criteria used in this study.

Blastocoel volume is related to successful establishment of human embryonic stem cell lines.

Human embryonic stem cell (hESC) banks strive to establish hESC lines from discarded or surplus human embryos. The effect of embryo quality on establishing hESC lines was investigated by observing cultures derived from 28 Taiwanese fresh surplus and donated embryos that were cultured using the whole embryo method. Cultures of hESC lines were followed for 15 months. At the blastocyst stage, 14 of the 28 embryos were graded as good quality, defined as featuring a blastocoel volume of at least half of the embryo volume. Fourteen embryos did not meet these standards on day 5. Five successful hESC lines were derived from the good quality embryos (5/14; 35.7%); these hESC cells grew for 27-60 passages. In contrast, cells from poor quality embryos all stopped growing at the second or third passage. The successful hESC exhibited typical stem cell characteristics, including the capacity for pluripotent differentiation. Embryo quality on day 5, as defined by blastocoel volume, is thus a strong predictor for successful establishment of hESC lines.

Preclinical evaluation of melanocyte transplantation by chitosan-based melanocyte spheroid patch to skin prepared by controlled sunburn blistering.

Transplantation of autologous cultured melanocytes as cell suspension has been used for the treatment of vitiligo. The recipient site is often prepared by laser-mediated dermabrasion. Such procedures encounter disadvantages including prolonged transplantation duration, unsecured cell adherence to lesional skin and potential scarring. To improve this, here we propose a method by preparing recipient sites before transplantation by psoralen and ultraviolet A (PUVA)-induced sunburn followed by transplanting cells with a chitosan-based melanocyte spheroid patch. We evaluated the method in nude mice. Application of methoxsalen-soaked filter paper on skin for 30 min followed by ultraviolet A exposure induced controlled sunburn blisters in 2 days. Upon transplantation, the blister roof could be quickly peeled off by a waxing patch. The chitosan membrane on which melanocytes were precultured into multicellular spheroids was transplanted with cells facing the skin. The chitosan patch adhered well to skin and secured the contact of melanocytes with the recipient site. One day later, melanocyte spheroids already detached from the chitosan membrane and adhered to the recipient skin. Our results suggest that the combination of chitosan-based melanocyte spheroid patch with epidermal ablation by PUVA-induced sunburn reaction can be a feasible method to facilitate melanocyte transplantation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2535-2543, 2018.

CBARA1 plays a role in stemness and proliferation of human embryonic stem cells.

Human embryonic stem cells (hESCs) are capable of unlimited self-renewal and can generate almost all of the cells in the body. Although some pluripotency factors have been identified, much remains unclear regarding the molecules and mechanisms that regulate hESC self-renewal and pluripotency. In this study, we identified a mitochondrial gene, CBARA1, that is expressed in undifferentiated hESCs and that is down-regulated rapidly after cellular differentiation. To study its role in hESCs, endogenous CBARA1 expression was knocked down using shRNA. CBARA1 knockdown in hESCs resulted in down-regulation of Oct4 and Nanog expression, attenuated cell growth, and G0/G1 phase cell cycle arrest; however, knockdown did not noticeably affect apoptosis. Taken together, these results suggest that CBARA1 is a marker for undifferentiated hESCs that plays a role in maintaining stemness, cell cycle progression, and proliferation.

Novel method of forming human embryoid bodies in a polystyrene dish surface-coated with a temperature-responsive methylcellulose hydrogel.

A temperature-responsive hydrogel composed of aqueous methylcellulose (MC) blended with distinct concentrations of PBS was prepared and characterized. The developed MC hydrogel underwent a sol-gel reversible transition upon heating or cooling at approximately 32 degrees C. This temperature-responsive hydrogel was employed to coat the surface of a polystyrene dish and used to cultivate human embryonic stem (hES) cell clumps for the formation of embryoid bodies (EBs) in liquid suspension culture (LSC-MC/PS). The conventional hanging drop culture (HDC) and LSC in the uncoated polystyrene dish (LSC-PS) or in the Corning Ultralow-Attachment plate (LSC-ULAP) were used as controls. The results indicated that LSC-PS failed to generate EBs in an efficient manner, whereas the efficiencies of EB formation observed in LSC-ULAP and LSC-MC/PS were significantly greater than in HDC. The hES cells within the EBs were shown to express molecular markers specific for representative cells from the three embryonic germ layers. These results indicated that the MC-coated dish can be used to produce a large scale of hES cell derivatives through the formation of EBs.

Production of mouse embryoid bodies with hepatic differentiation potential by stirred tank bioreactor.

Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.

Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels.

In this study, a novel yet simple method, using a thermoreversible hydrogel system coated on tissue culture polystyrene (TCPS) dishes, was developed for harvesting living cell sheets. The hydrogel system was prepared by simply pouring aqueous methylcellulose (MC) solutions blended with distinct salts on TCPS dishes at 20 degrees C. For the applications to cell culture, only those aqueous MC compositions that may form a gel at 37 degrees C were chosen for the study. It was found that the hydrogel coating composed of 8% MC blended with 10 g/L PBS (phosphate buffered saline) (the MC/PBS hydrogel, with a gelation temperature of approximately 25 degrees C) stayed intact throughout the entire course of cell culture. To improve cell attachments, the MC/PBS hydrogel at 37 degrees C was evenly spread with a neutral aqueous collagen at 4 degrees C. The spread aqueous collagen gradually reconstituted with time and thus formed a thin layer of collagen (the MC/PBS/collagen hydrogel). After cells reached confluence, a continuous monolayer cell sheet formed on the surface of the MC/PBS/collagen hydrogel. When the grown cell sheet was placed outside of the incubator at 20 degrees C, it detached gradually from the surface of the thermoreversible hydrogel spontaneously, without treating with any enzymes. The results obtained in the MTT assay demonstrated that the cells cultured on the surface of the MC/PBS/collagen hydrogel had an even better activity than those cultured on an uncoated TCPS dish. After harvesting the detached cell sheet, the remaining viscous hydrogel system is reusable. Additionally, the developed hydrogel system can be used for culturing a multilayer cell sheet. The obtained living cell sheets may be used for tissue reconstructions.