Hehong Shanghai Biotechnology Ltd.

Shanghai, China

Hehong Shanghai Biotechnology Ltd.

Shanghai, China
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Lai D.,Shanghai JiaoTong University | Cheng W.,Shanghai JiaoTong University | Liu T.,Shanghai JiaoTong University | Jiang L.,Hehong Shanghai Biotechnology Ltd. | And 3 more authors.
Cellular Reprogramming | Year: 2010

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by coculture with mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of animal sera such as fetal bovine serum (FBS). Here, we use primary human amnion epithelial cells (hAECs) as feeder cells and human umbilical cord blood serum (CBS) as a replacement for FBS to support undifferentiated growth of hESCs. The 5∼10-fold higher expression levels of ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT were found in hESCs grown on hAECs compared with that on MEFs as measured by quantitative real-time polymerase chain reaction (PCR). By immunofluorescence, the expresisons of Oct-4 and Nanog is also higher in cells grown on hAECs than those on MEFs. Importantly, the ES cells grown on hAECs exhibit normal karyotypes on passage 25, thus ruling out the possibility of transformation. Using flow cytometry analysis, we show that both the ES cells grown on hAECs and MEFs have the same cell cycle distribution pattern. Further, hESCs cultured on hAECs for at least 20 passages could differentiate into three germ layers via teratoma formation. In addition, chromatin immunoprecipitation assay revealed that histone H3 is highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus and the Oct-4 locus in hESCs grown on hAECs. Conversely, hESCs grown on MEFs show histone deacetylation and H3-K4 demethylation. Taken together, these results suggest that hAECs supplemented with 10% CBS are suitable for hESC culture, and that this method may prove to be valuable for use in future regenerative therapies. Copyright 2010, Mary Ann Liebert, Inc.

Liu T.,Tongji University | Liu T.,Shanghai JiaoTong University | Xu F.,Tongji University | Du X.,Tongji University | And 8 more authors.
Molecular and Cellular Biochemistry | Year: 2010

Multi-drug resistance is an important element which leads to ineffectiveness of chemotherapeutics. To identify subpopulations of cancerous prostate cells with mutli-drug resistance and cancer stem-cell properties has recently become a major research interest. We identified a subpopulation from the prostate cancer cell line 22RV1, which have high surface expression of both CD117 and ABCG2. We found this subpopulation of cells termed CD117 +/ABCG2+ also overexpress stem cells markers such as Nanog, Oct4, Sox2, Nestin, and CD133. These cells are highly prolific and are also resistant to treatment with a variety of chemotherapeutics such as casplatin, paclitaxel, adriamycin, and methotrexate. In addition, CD117 +/ABCG2+ cells can readily establish tumors in vivo in a relatively short time. To investigate the mechanism of aggressive tumor growth and drug resistance, we examined the CpG islands on the ABCG2 promoter of CD117+/ABCG2+ cells and found they were remarkably hypomethylated. Furthermore, chromatin immunoprecipitation assays revealed high levels of both histone 3 acetylation and H3K4 trimethylation at the CpG islands on the ABCG2 promoter. Our these data suggest that CD117+/ABCG2 + cells could be reliably sorted from the human prostate cancer cell line 22RV1, and represent a valuable model for studying cancer cell physiology and multi-drug resistance. Furthermore, identification and study of these cells could have a profound impact on selection of individual treatment strategies, clinical outcome, and the design or selection of the next generation of chemotherapeutic agents. © 2010 Springer Science+Business Media, LLC.

Lai D.,Shanghai JiaoTong University | Wang F.,Shanghai JiaoTong University | Chen Y.,Shanghai JiaoTong University | Wang C.,Hehong Shanghai Biotechnology Ltd. | And 4 more authors.
Cancer Immunology, Immunotherapy | Year: 2012

Ovarian cancer comprises a small population of cancer stem cells (CSCs) that are responsible for tumor maintenance and resistant to cancer therapies, it would be desirable to develop a therapy that could selectively target ovarian CSCs. Recently, cellular immune-based therapies have improved the prognosis of cancer patients clinically. In this study, we isolated a subset of ovarian cancer sphere cells that possess CSC properties and explored the cell cytotoxicity of γδ T cells to ovarian cancer sphere cells using a transwell cocultured cell system. The proliferation rate of the cancer sphere cells decreased to 40% after cocultured with γδ T cells. The γδ T cells increased the sensitivity of SK-OV-3 sphere cells to chemotherapeutic drugs. After the treatment of γδ T cells, the expression of stem cell marker genes decreased in sphere cells, while the expression of HLA-DR antigen on tumor cells was increased in a time-dependent manner. Further, γδ T cells induced G2/M phase cell cycle arrest and subsequent apoptosis in SK-OV-3 sphere cells. Xenograft mouse models demonstrated that γδ T cells dramatically reduced the tumor burden. Notably, the level of IL-17 production significantly increased after cocultured with γδ T cells. We conclude that γδ T cells may efficiently kill ovarian CSCs through IL-17 production and represent a promising immunotherapy for ovarian cancer. © 2011 Springer-Verlag.

Lai D.,Shanghai JiaoTong University | Chen Y.,Shanghai JiaoTong University | Wang F.,Shanghai JiaoTong University | Jiang L.,Hehong Shanghai Biotechnology Ltd. | Wei C.,Fudan University
Cellular Reprogramming | Year: 2012

Human embryonic stem cells maintained on human amniotic epithelial cells (hESCs hAEC) are better preserved in an undifferentiated state and express pluripotency genes Oct4, Nanog, and Sox2 at higher levels compared with growth on mitotically inactivated mouse embryonic fibroblasts (hESCs MEF). Here we report that this correlates with the absence of the tumor suppressor and metabolic balancer gene, LKB1 expression in hESCs hAEC. RNA interference knockdown of LKB1 in hESCs MEF resulted in upregulation of pluripotency marker genes of Oct4 and Nanog, while downregulation of differentiation markers (Runx1, AFP, GATA, Brachyury, Sox17 and Nestin). As in somatic cells, LKB1 controls p21/WAF1 expression by promoter binding in hESCs MEF. Our results suggested that the absence of LKB1-mediated signaling is an important determinant of feeder cell-mediated support of hESC renewal. © 2012, Mary Ann Liebert, Inc.

Ma L.,Fudan University | Lai D.,Shanghai JiaoTong University | Liu T.,Shanghai JiaoTong University | Cheng W.,Shanghai JiaoTong University | Guo L.,Hehong Shanghai Biotechnology Ltd.
Acta Biochimica et Biophysica Sinica | Year: 2010

One emerging model for the development of drug-resistant tumors utilizes a pool of self-renewing malignant progenitors known as cancer stem cells (CSCs) or cancer-initiating cells (CICs). The purpose of this study was to propagate such CICs from the ovarian cancer cell line SKOV3. The SKOV3 sphere cells were selected using 40.0 μmol/l cisplatin and 10.0 μmol/l paclitaxel in serum-free culture system supplemented with epidermal growth factor, basic fibroblast growth factor, leukemia inhibitory factor, and insulin or standard serum-containing system. These cells formed non-adherent spheres under drug selection (cisplatin and paclitaxel) and serum-free culture system. The selected sphere cells are more resistant to cisplatin, paclitaxel, adriamycin, and methotrexate. Importantly, the sphere cells have the properties of self-renewal, with high expression of the stem cell genes Nanog, Oct4, sox2, nestin, ABCG2, CD133, and the stem cell factor receptor CD117 (c-kit). Consistently, flow cytometric analysis revealed that the sphere cells have a much higher percentage of CD133 +/CD117 +-positive cells (71) than differentiated cells (33). Moreover, the SKOV3 sphere cells are more tumorigenic. Furthermore, cDNA microarray and subsequent ontological analyses revealed that a large proportion of the classified genes were related to angiogenesis, extracellular matrix, integrin-mediated signaling pathway, cell adhesion, and cell proliferation. The subpopulation isolation from the SKOV3 cell line under this culture system offers a suitable in vitro model for studying ovarian CSCs in terms of their survival, self-renewal, and chemoresistance, and for developing therapeutic drugs that specifically interfere with ovarian CSCs. © The Author 2010.

Liu T.,Tongji University | Liu T.,Shanghai JiaoTong University | Cheng W.,Shanghai JiaoTong University | Liu T.,Hehong Shanghai Biotechnology Ltd. | And 7 more authors.
Acta Biochimica et Biophysica Sinica | Year: 2010

Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5′ of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

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