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.

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.

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.