技术资料

Based on knowledge of early embryo development,where anterior neural ectoderm (ANE) development is regulated by native inhibitors of bone morphogenic protein (BMP) and Nodal/Activin signaling,most published protocols of human embryonic stem cell differentiation to ANE have demonstrated a crucial role for Smad signaling in neural induction. The drawbacks of such protocols include the use of an embryoid body culture step and use of polypeptide secreted factors that are both expensive and,when considering clinical applications,have significant challenges in terms of good manufacturing practices compliancy. The use of small molecules to direct differentiation of pluripotent stem cells toward a specified lineage represents a powerful approach to generate specific cell types for further understanding of biological function,for understanding disease processes,for use in drug discovery,and finally for use in regenerative medicine. We therefore aimed to find controlled and reproducible animal-component-free differentiation conditions that would use only small molecules. Here,we demonstrate that pluripotent stem cells can be reproducibly and efficiently differentiated to PAX6(+) (a marker of neuroectoderm) and OCT4(-) (a marker of pluripotent stem cells) cells with the use of potent small inhibitors of the BMP and Activin/Nodal pathways,and in animal-component-free conditions,replacing the frequently used Noggin and SB431542. We also show by transcript analysis,both at the population level and for the first time at the single-cell level,that differentiated cells express genes characteristic for the development of ANE,in particular for the development of the future forebrain. View Publication

Neuron-specific in vitro screening strategies have the potential to accelerate the evaluation of chemicals for neurotoxicity. We examined neurite outgrowth as a measure of neuronal response with a commercially available rat cortex progenitor cell model,where cells were exposed to a chemical during a period of cell differentiation. In control cultures,the fraction of beta-III-tubulin positive neurons and their neurite length increased significantly with time,indicating differentiation of the progenitor cells. Expression of glial fibrillary acidic protein,an astrocyte marker,also increased significantly with time. By seeding progenitor cells at varying densities,we demonstrated that neurite length was influenced by cell-cell spacing. After ten days,cultures seeded at densities of 1000 cells/mm(2) or lower had significantly shorter neurites than cultures seeded at densities of 1250 cells/mm(2) or higher. Progenitor cells were exposed to lithium,a neuroactive chemical with diverse modes of action. Cultures exposed to 30 mmol/L or 10 mmol/L lithium chloride (LiCl) had significantly lower metabolic activity than control cultures,as reported by adenosine triphosphate content,and no neurons were observed after ten days of exposure. Cultures exposed to 3 mmol/L,1 mmol/L,or 0.3 mmol/L LiCl,which encompass lithium's therapeutic range,had metabolic activity similar to control cultures. These cultures exhibited concentration-dependent decreases in neurite outgrowth after ten days of LiCl exposure. Neurite outgrowth results were relatively robust,regardless of the evaluation methodology. This work demonstrates that measurement of neurite outgrowth in differentiating progenitor cell cultures can be a sensitive endpoint for neuronal response under non-cytotoxic exposure conditions. View Publication

Bromodomains are readers of the epigenetic code that specifically bind acetyl-lysine containing recognition sites on proteins. Recently the BET family of bromodomains has been demonstrated to be druggable through the discovery of potent inhibitors,sparking an interest in protein-protein interaction inhibitors that directly target gene transcription. Here,we assess the druggability of diverse members of the bromodomain family using SiteMap and show that there are significant differences in predicted druggability. Furthermore,we trace these differences in druggability back to unique amino acid signatures in the bromodomain acetyl-lysine binding sites. These signatures were then used to generate a new classification of the bromodomain family,visualized as a classification tree. This represents the first analysis of this type for the bromodomain family and can prove useful in the discovery of inhibitors,particularly for anticipating screening hit rates,identifying inhibitors that can be explored for lead hopping approaches,and selecting proteins for selectivity screening. View Publication

Sox2 (sex-determining region Y-box protein 2) is a transcription factor regulating pluripotency in embryonic stem cells. Sox2 is aberrantly expressed in breast and other cancers,though its biological significance remains widely unexplored. To understand the significance of this aberrancy,we assessed the transcription activity of Sox2 in two Sox2-expressing breast cancer cell lines,MCF7 and ZR751,using a lentiviral Sox2 GFP reporter vector. Surprisingly,Sox2 transcription activity,as measured by GFP expression encoded in a Sox2 reporter construct,was detectable only in a small subset of cells in both cell lines. Purification of GFP+ cells (cells with Sox2 activity) and GFP- cells (cells without Sox2 activity) was enriched for two phenotypically distinct cell populations in both MCF7 and ZR751 cell lines. Specifically,GFP+ cells formed significantly more colonies in methylcellulose and more mammospheres in vitro compared to GFP- cells. These phenotypic differences are directly linked to Sox2 as siRNA knockdown of Sox2 in GFP+ cells abolished these abilities. To provide a mechanistic explanation to our observations,we performed gel shift and chromatin immunoprecipitation studies; Sox2 was found to bind to its DNA binding consensus sequence and the promoters of Cyclin D1 and Nanog (two known Sox2 downstream targets) only in GFP+ cells. GFP+ cells also up-regulated CD49f,phospho-GSK3$$,and $$-catenin. In summary,we have identified two novel phenotypically distinct cell subsets in two breast cancer cell lines based on their differential Sox2 transcription activity. We demonstrate that Sox2 transcription activity,and not its protein expression alone,underlies the tumorigenicity and cancer stem cell-like phenotypes in breast cancers. View Publication

Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that β-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly,Wnt/β-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of β-catenin from the embryonic endothelium stage (using VE-cadherin-Cre recombinase),but not from embryonic hematopoietic cells (using Vav1-Cre),precludes progression of mutant cells toward the hematopoietic lineage; however,these mutant cells still contribute to the adult endothelium. Together,those findings indicate that Wnt/β-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos. View Publication

We developed a protocol of in vitro differentiation of human embryonic stem cells into three-dimensional structures histologically and molecularly similar to the developing retina. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions,the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (ptextless0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC,E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2),at concentrations that selectively block I(K1) (50-100 µM),failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied,but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data,RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells,but robust expression of I(Kr.) In contrast to recently reported studies,our data point to major deficiencies of hiPSC-CM,with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus,efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications. View Publication

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. View Publication

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and its efficacy has been tested in pre-clinical models by delivering it systemically as a purified ligand or via engineered stem cells (SC). However,about 50% of tumor lines are resistant to TRAIL and overcoming TRAIL resistance in aggressive tumors,such as glioblastoma-multiforme (GBM),and understanding the molecular dynamics of TRAIL-based combination therapies are critical to broadly use TRAIL as a therapeutic agent. In this study,we developed death receptor (DR)4/5-reporters that offer an imaging-based platform to identify agents that act in concert with a potent,secretable variant of TRAIL (S-TRAIL) by monitoring changes in DR4/5 expression. Utilizing these reporters,we show a differential regulation of DR4/5 when exposed to a panel of clinically relevant agents. A histone deacetylase inhibitor,MS-275,resulted in upregulation of DR4/5 in all GBM cell lines,and these changes could be followed in real time both in vitro and in vivo in mice bearing tumors and they correlated with increased TRAIL sensitivity. To further assess the dynamics of combinatorial strategies that overcome resistance of tumors to SC released S-TRAIL,we also engineered tumor cells to express live-cell caspase-reporters and SCs to express S-TRAIL. Utilizing DR4/5 and caspase reporters in parallel,we show that MS-275 sensitizes TRAIL-resistant GBM cells to stem cell (SC) delivered S-TRAIL by changing the time-to-death in vitro and in vivo. This study demonstrates the effectiveness of a combination of real-time reporters of TRAIL-induced apoptosis pathway in evaluating the efficacy of SC-TRAIL-based therapeutics and may have implications in targeting a broad range of cancers. View Publication

The combination of chronic hypoxia and treatment of rats with the vascular endothelial growth factor (VEGF) receptor blocker,SU5416,induces pulmonary angio-obliteration,resulting in severe pulmonary arterial hypertension (PAH). Inflammation is thought to contribute to the pathology of PAH. Allergic inflammation caused by ovalbumin (OVA) immunization causes muscularization of pulmonary arteries,but not severe PAH. Whether disturbance of the immune system and allergic inflammation in the setting of lung endothelial cell apoptosis causes PAH is unknown. We investigated the effects of OVA-allergic inflammation on the development of PAH initiated by VEGF blockade-induced lung endothelial cell apoptosis. OVA-immunized rats were treated with SU5416 to induce pulmonary vascular endothelial cell apoptosis. The combination of OVA and SU5416 treatment resulted in severe angio-obilterative PAH,accompanied by increased IL-6 expression in the lungs. c-Kit(+) and Sca-1(+) cells were found in and around the lung vascular lesions. Pan-caspase inhibiton,dexamethasone treatment,and depletion of B-lymphocytes using an anti-CD20 antibody suppressed this remodeling. OVA immunization also increased lung tissue hypoxia-induced factor-1α and VEGF expression. Our results also suggest that the increased expression of hypoxia-induced factor-1α and IL-6 induced by the allergic lung inflammation may be a component of the pathogenesis of PAH. View Publication

The mammary gland undergoes significant remodeling during pregnancy and lactation,which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here,we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4,SOX2,NANOG,known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts,a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium,they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore,hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer,with potential use in bioengineering and tissue regeneration. View Publication

Aromatase inhibitors (AIs) are an effective therapy in treating estrogen receptor-positive breast cancer. Nonetheless,a significant percentage of patients either do not respond or become resistant to AIs. Decreased dependence on ER-signaling and increased dependence on growth factor receptor signaling pathways,particularly human epidermal growth factor receptor 2 (EGFR2/HER2),have been implicated in AI resistance. However,the role of growth factor signaling remains unclear. This current study investigates the possibility that signaling either through HER2 alone or through interplay between epidermal growth factor receptor 1 (EGFR/HER1) and HER2 mediates AI resistance by increasing the tumor initiating cell (TIC) subpopulation in AI-resistant cells via regulation of stem cell markers,such as breast cancer resistance protein (BCRP). TICs and BCRP are both known to be involved in drug resistance. Results from in vitro analyses of AI-resistant versus AI-sensitive cells and HER2-versus HER2+ cells,as well as from in vivo xenograft tumors,indicate that (1) AI-resistant cells overexpress both HER2 and BCRP and exhibit increased TIC characteristics compared to AI-sensitive cells; (2) inhibition of HER2 and/or BCRP decrease TIC characteristics in letrozole-resistant cells; and (3) HER2 and its dimerization partner EGFR/HER1 are involved in the regulation of BCRP. Overall,these results suggest that reducing or eliminating the TIC subpopulation with agents that target BCRP,HER2,EGFR/HER1,and/or their downstream kinase pathways could be effective in preventing and/or treating acquired AI resistance. View Publication