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Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However,hPS cells cultured in vitro exhibit a high degree of heterogeneity,presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures,necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution,with single-cellular and sub-cellular analysis at high resolutions,generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1,reflecting a less pluripotent state,while cells within the first pluripotent layer are more likely to be in G2,possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes,and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly,we demonstrate that our pipeline can robustly handle high-content,high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall,the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide,and more generally,multi-scale spatial configuration. View Publication

Recent evidence suggests that pancreatic cancer and other solid tumors contain a subset of tumorigenic cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. Sorafenib (SO) is a promising new multikinase inhibitor for treatment of advanced kidney and liver cancers. We report here targeting of pancreatic cancer stem cells (CSC) by SO and the development of a strategy to enhance this effect. Although SO administration diminished clonogenicity,spheroid formation,aldehyde dehydrogenase 1 (ALDH1) activity,growth on immunodeficient mice,proliferation,and angiogenesis and induced apoptosis,we observed SO-induced activation of NF-kappaB associated with survival and regrowth of spheroids. For enhanced elimination of CSC characteristics by SO,we cotreated cells with sulforaphane (SF). This broccoli isothiocyanate was recently described to eliminate pancreatic CSCs by downregulation of NF-kappaB activity without inducing toxic side effects. On combination treatment,SF completely eradicated SO-induced NF-kappaB binding,which was associated with abrogated clonogenicity,spheroid formation,ALDH1 activity,migratory capacity,and induction of apoptosis. In vivo,combination therapy reduced the tumor size in a synergistic manner. This was due to induction of apoptosis,inhibition of proliferation and angiogenesis,and downregulation of SO-induced expression of proteins involved in epithelial-mesenchymal transition. Our data suggest that SF may be suited to increase targeting of CSCs by SO. View Publication

A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O),along with Sox2,Klf4 and c-Myc (SKM). In addition,M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs,including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here,we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast,the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs,M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition,in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment. View Publication

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by an expansion of the CAG-repeat in ATXN3. In this study,induced pluripotent stem cells (iPSCs) were generated from SCA3 patient dermal fibroblasts by electroporation with episomal plasmids encoding L-MYC,LIN28,SOX2,KLF4,OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype,were free of integrated episomal plasmids,expressed pluripotency markers,could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. Potentially,this iPSC line could be a useful tool for the investigation of SCA3 disease mechanisms. View Publication

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterised monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs),confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs,providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition,we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs),normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency,and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. This article is protected by copyright. All rights reserved. View Publication

BACKGROUND Massive liquid crystal droplets have been found during embryonic development in more than twenty different tissues and organs,including the liver,brain and kidney. Liquid crystal deposits have also been identified in multiple human pathologies,including vascular disease,liver dysfunction,age-related macular degeneration,and other chronic illnesses. Despite the involvement of liquid crystals in such a large number of human processes,this phenomenon is poorly understood and there are no in vitro systems to further examine the function of liquid crystals in biology. RESULTS We report the presence of tubular birefringent structures in embryoid bodies (EBs) differentiated in culture. These birefringent tubular structures initiate at the EB surface and penetrated the cortex at a variety of depths. Under crossed polarized light,these tubules are seen as a collection of birefringent Maltese crosses and tubules with birefringent walls and a non-birefringent lumen. The fluidity of these birefringent structures under pressure application led to elongation and widening,which was partially recoverable with pressure release. These birefringent structures also displayed heat triggered phase transition from liquid crystal to isotropic status that is partially recoverable with return to ambient temperature. These pressure and temperature triggered changes confirm the birefringent structures as liquid crystals. The first report of liquid crystal so early in development. CONCLUSION The structure of the liquid crystal tubule network we observed distributed throughout the differentiated embryoid bodies may function as a transportation network for nutrients and metabolic waste during EB growth,and act as a precursor to the vascular system. This observation not only reveals the involvement of liquid crystals earlier than previously known,but also provides a method for studying liquid crystals in vitro. View Publication