// <![CDATA[ $('.header-date').hide();$('#titleAuthors').hide();$('#abstractHeader').hide(); // ]]> Yaqi Zhu1, 2, *, Yan Yang1, 2, *, Juanjuan Guo3, Ying Dai1, 2, Lina Ye2, Jianbin Qiu2, Zhihong Zeng2, Xiaoting Wu2, Yanmei Xing2, Xiang Long4, Xufeng Wu5, Lin Ye6, Shubin Wang4, Hui Li1, 2 1State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Basic Medical Sciences, Wuhan, Hubei 430071, China 2Shenzhen R and D Center of State Key Laboratory of Virology, Wuhan University Shenzhen Institute, Shenzhen, Guangdong 518057, China 3Xiangyang No.1 People’s Hospital, Xiangyang, Hubei 441000, China 4Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036 5Hubei Maternal and Child Health Hospital, Wuhan, Hubei 430070, China 6Shenzhen Eye Hospital, Shenzhen, Guangdong 518040, China *These authors contributed equally to this work Correspondence to: Hui Li, email: somhli@whu.edu.cn Keywords: three dimension (3D), ex vivo, human normal vaginal epithelial cells (HNVEC), air-liquid interface (ALI) culture, HSV-2 infection model Received: November 14, 2016 Accepted: January 13, 2017 Published: January 27, 2017

We have established an in vitro HSV-2 acute infection model with Human cervical epithelial (HCE cells, the primary target and natural host cells for HSV-2) to investigate the role of TLRs-mediated innate immune response to HSV-2. In current study, we found that HSV-2 infection induced activity of NF-kB reporter and expression of cytokines are TLR4-dependent using approaches with shRNA and TLR4 antagonist. Knockdown experiments demonstrated that the adaptor molecules MyD88 and Mal of the TLRs signaling pathway are required in the HSV-2 induced TLR4-dependent NF-kB activation in HCE cells. Western blot assay suggested that knockdown of TLR4 decreased the phosphorylation of IRAK1 and inhibitor of NF-kB (IkB-α) upon HSV-2 infection. Finally, decreased expression of either TLR4 or MyD88/Mal alone or both significantly abolished productions of IL-6 and IFN-β by ELISA analysis. Taken together, our results from the in vitro infection model reveal for the first time that there exists the pathway via TLR4-Mal/MyD88-IRAK1-NF-kB axis in human cervical epithelial cells in response to HSV-2 infection.

Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO₂, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery.

Background: There is unmet need for effective therapies of intrauterine adhesions (IUAs) that are common cause of menstrual disturbance and infertility, since current clinical procedures do not improve prognosis for patients with moderate to severe IUA, with a recurrence rate of 23–50%. Stem cell-based therapy has emerged as a therapeutic option with unsolved issues for IUA patients in the past few years. Primary endometrial epithelial cells for cell therapy are largely hampered with the extremely limited proliferation capacity of uterine epithelial cells. This study was to evaluate whether IUA is curable with conditionally reprogrammed (CR) endometrial epithelial cells.Methods: Mouse endometrial epithelial cells (MEECs) were isolated from C57BL female mice, and long-term cultures of MEECs were established and maintained with conditional reprogramming (CR) method. DNA damage response analysis, soft agar assay, and matrigel 3D culture were carried out to determine the normal biological characteristics of CR-MEECs. The tissue-specific differentiation potential of MEECs was analyzed with air–liquid interface (ALI) 3D culture, hematoxylin and eosin (H&E) staining, Masson’s trichrome and DAB staining, immunofluorescence assay. IUA mice were constructed and transplanted with CR-MEECs. Repair and mechanisms of MEECs transplantation in IUA mice were measured with qRT-PCR, Masson’s trichrome, and DAB staining.Results: We first successfully established long-term cultures of MEECs using CR approach. CR-MEECs maintained a rapid and stable proliferation in this co-culture system. Our data confirmed that CR-MEECs retained normal biological characteristics and endometrium tissue-specific differentiation potential. CR-MEECs also expressed estrogen and progesterone receptors and maintained the exquisite sensitivity to sex hormones in vitro. Most importantly, allogeneic transplantation of CR-MEECs successfully repaired the injured endometrium and significantly improved the pregnancy rate of IUA mice.Conclusions: Conditionally reprogrammed physiological endometrial epithelial cells provide a novel strategy in IUA clinics in a personalized or generalized manner and also serve as a physiological model to explore biology of endometrial epithelial cells and mechanisms of IUA.