rho kinase inhibitor br Author contribution The following ar

Author contribution The following are the supplementary data related to this article.
Acknowledgments We thank Catherine Bresse for her valuable assistance in the statistical analysis. This work was supported by the CIRM grant # RL1-00636-1.
Introduction The capacity of human embryonic stem rho kinase inhibitor (hESCs) for self-renewal, propagation, and maintenance of the pluripotent state in vitro offers the potential to utilize hESC technology for therapy of many severe human diseases as well as cell-based assays (Klimanskaya et al., 2005; Mallon et al., 2006; Rosler et al., 2004; Thomson et al., 1998; Xu et al., 2001). However, many negative factors contribute to current inefficient culture systems for hESCs, which have limited the implementation of such a therapy. The ineffectiveness of hESC culture systems is due to (i) low plating efficiency when cells are seeded as single cells or small clumps (Androutsellis-Theotokis et al., 2006; Watanabe et al., 2007), (ii) very low recovery rates when cells are thawed after cryopreservation (Li et al., 2009), and (iii) acquired heterogeneous cellular states owing to various cellular stresses under excessive apoptotic or differentiation signals (Adewumi et al., 2007; Hartung et al., 2010). In addition, xenogeneic contaminants from any non-human feeder cells or foreign components of the culture system may also impede future clinical application (Mallon et al., 2006). To solve these problems, we need to establish a robust and reliable system for hESC culture and assay. Standard colony-aggregated culture exhibits slow expansion and often gives rise to heterogeneous cells (Adewumi et al., 2007; Hartung et al., 2010) and frequent chromosomal abnormalities (Baker et al., 2007; Draper et al., 2004; Lefort et al., 2008; Maitra et al., 2005; Spits et al., 2008). Hence, a non-colony type culture is preferable. The use of JAK inhibitor I (JAKi) and the Rho-kinase inhibitor Y-27632 (ROCKi) has been shown to significantly improve single-cell plating efficiency in both neural stem cell and hESC cultures (Androutsellis-Theotokis et al., 2006; Chen et al., 2010; Li et al., 2009; Ohgushi et al., 2010; Pakzad et al., 2010; Watanabe et al., 2007). Intuitively, hESCs with high single-cell plating efficiency using these small molecules could enable us to propagate the cells in a single-cell based non-colony type monolayer (NCM) culture, which would greatly improve the current culture conditions. Various defined substrates have been reported to support hESC culture as colonies under feeder- or xeno-free conditions (Klim et al., 2010; Mallon et al., 2006; Melkoumian et al., 2010; Rodin et al., 2010; Villa-Diaz et al., 2010). The use and characterization of a NCM method as an independent culture system for the maintenance of undifferentiated hESC lines under defined substrate conditions have not been reported. In this study, we report such a hESC culture system for facilitating pluripotent stem cell growth and assays.
Materials and methods
Results and discussion
Conclusions The following are the supplementary data related to this article.
Disclosure of potential conflicts of interest
Acknowledgments The NIH Stem Cell Unit is supported by NIH funds. We thank Dr. Joshua Chenoweth for the discussion, Dr. Peter Andrews for providing antibodies described in Supplemental information, Dr. Guokai Chen for the BC1 line, and Dr. Tianmin Ivy Zhang and Dr. Qi Zheng (Applied StemCell Inc.) for conducting the teratoma assays.
Introduction Glioblastomas, the most common and malignant primary brain tumor, have a dismal prognosis, despite modern refinement of diagnostic techniques and treatment strategy including surgery and radio/chemotherapy. Median survival of the patients with glioblastoma is generally about a year from the time of diagnosis and this has not significantly improved for more than three decades (DeAngelis, 2001; Stewart, 2002; Stupp et al., 2005). As a novel treatment strategy, gene therapies using the herpes simplex virus-thymidine kinase (HSVtk) gene and ganciclovir (GCV) were clinically tested (Rainov, 2000; Ram et al., 1997). However, the results were unsatisfactory partly due to limited migratory activity of the vector-producing cells derived from fibroblasts, which could not cover all of the glioma cells that widely infiltrated into the surrounding brain tissues. To improve the migratory activity of the effector cells, use of the neural stem cells and bone marrow stromal cells (BMSCs), that display extensive tropism for brain lesions including gliomas, has been introduced (Aboody et al., 2000; Benedetti et al., 2000; Li et al., 2007). In the previous study, we demonstrated that established intracranial glioma in the rat brain was successfully treated by intratumoral injection of BMSCs transduced with HSVtk gene (BMSCtk cells) followed by intraperitoneal GCV administration (Amano et al., 2009). This is mainly due to a very potent “bystander effect”, where tumor cells that are not transduced with HSVtk gene are eliminated when mixed with HSVtk-expressing cells by GCV administration (Culver et al., 1992), as well as an active tumor tracking ability of BMSCs (Lee et al., 2003; Nakamizo et al., 2005). In fact, we have demonstrated a surprisingly potent bystander effect between BMSCtk and C6 rat glioma cells (Amano et al., 2009). These observations suggest that “BMSCtk therapy” is promising as a novel clinical treatment strategy for glioblastomas.