技术资料

The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells,including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy,we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs,we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells,and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Bone remodeling,a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs),is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature,osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence,anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²�?�/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones,along with significantly more OBs and fewer multinuclear OCs. In vitro,although Camkk2�?�/�?� mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs,bone marrow cells from Camkk2�?�/�?� mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective,cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further,we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand,genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target,nuclear factor of activated T cells,cytoplasmic (NFATc1). Moreover,in vivo administration of STO-609 results in increased OBs and diminished OCs,conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall,our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis. View Publication

The vertebrae mesoderm is a source of cells that forms a variety of tissues,including the heart,vasculature,and blood. Consequently,the derivation of various mesoderm-specific cell types from human embryonic stem cells (hESCs) has attracted the interest of many investigators owing to their therapeutic potential in clinical applications. However,the need for efficient and reliable methods of differentiation into mesoderm lineage cell types remains a significant challenge. Here,we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) is an essential first step toward efficient generation of the mesoderm. Under chemically defined conditions without additional growth factors/cytokines,short-term GSK inhibitor (GSKi) treatment effectively drives differentiation of hESCs into the primitive streak (PS),which can potentially commit toward the mesoderm when further supplemented with bone morphogenetic protein 4. Further analysis confirmed that the PS-like cells derived from GSKi treatment are bipotential,being able to specify toward the endoderm as well. Our findings suggest that the bipotential,PS/mesendoderm-like cell population exists only at the initial stages of GSK-3 inhibition,whereas long-term inhibition results in an endodermal fate. Lastly,we demonstrated that our differentiation approach could efficiently generate lateral plate (CD34(+)KDR(+)) and paraxial (CD34(-)PDGFRα(+)) mesoderm subsets that can be further differentiated along the endothelial and smooth muscle lineages,respectively. In conclusion,our study presents a unique approach for generating early mesoderm progenitors in a chemically directed fashion through the use of small-molecule GSK-3 inhibitor,which may be useful for future applications in regenerative medicine. View Publication

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Pluripotent stem cells hold great promise for regenerative medicine,due to their unlimited self-renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non-human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed,get relatively old (≤ 13 years),are small in size,are relatively cost-effective and have a high genetic proximity to the human. In particular,diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane-permeable,chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However,the potent small molecules IDE-1 and IDE-2 were not able to induce definitive endoderm-like cells when ESCs from the common marmoset were treated with these compounds,whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus,differences between the species of human ESCs and ESCs of this non-human primate might be a useful model to further evaluate the exact mode of action of these compounds. View Publication

BACKGROUND Brain metastases are most common in adults with lung cancer,predicting uniformly poor patient outcome,with a median survival of only months. Despite their frequency and severity,very little is known about tumorigenesis in brain metastases. METHODS We applied previously developed primary solid tumor-initiating cell models to the study of brain metastases from the lung to evaluate the presence of a cancer stem cell population. Patient-derived brain metastases (n = 20) and the NCI-H1915 cell line were cultured as stem-enriching tumorspheres. We used in vitro limiting-dilution and sphere-forming assays,as well as intracranial human-mouse xenograft models. To determine genes overexpressed in brain metastasis tumorspheres,we performed comparative transcriptome analysis. All statistical analyses were two-sided. RESULTS Patient-derived brain metastasis tumorspheres had a mean sphere-forming capacity of 33 spheres/2000 cells (SD = 33.40) and median stem-cell frequency of 1/60 (range = 0-1/141),comparable to that of primary brain tumorspheres (P = .53 and P = .20,respectively). Brain metastases also expressed CD15 and CD133,markers suggestive of a stemlike population. Through intracranial xenotransplantation,brain metastasis tumorspheres were found to recapitulate the original patient tumor heterogeneity. We also identified several genes overexpressed in brain metastasis tumorspheres as statistically significant predictors of poor survival in primary lung cancer. CONCLUSIONS For the first time,we demonstrate the presence of a stemlike population in brain metastases from the lung. We also show that NCI-H1915 tumorspheres could be useful in studying self-renewal and tumor initiation in brain metastases. Our candidate genes may be essential to metastatic stem cell populations,where pathway interference may be able to transform a uniformly fatal disease into a more localized and treatable one. View Publication

Induced pluripotent stem (iPS) cells hold the potential to revolutionize regenerative medicine through their capacity to generate cells of diverse lineages for future patient-specific cell-based therapies. To facilitate the transition of iPS cells to clinical practice,a variety of technologies have been developed for transgene-free pluripotency reprogramming. We recently reported efficient iPS cell generation from human fibroblasts using synthetic modified mRNAs. Here we describe a stepwise protocol for the generation of modified mRNA-derived iPS cells from primary human fibroblasts,focusing on the critical parameters including medium choice,quality control,and optimization steps needed for synthesizing modified mRNAs encoding reprogramming factors and introducing these into cells over the course of 2-3 weeks to ensure successful reprogramming. The protocol described herein is for reprogramming of human fibroblasts to pluripotency; however,the properties of modified mRNA make it a powerful platform for protein expression,which has broad applicability in directed differentiation,cell fate specification and therapeutic applications. View Publication

The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis,we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth,but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF,Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes,resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly,approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage,which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore,some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively,to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML. View Publication

An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent,and pluripotent stem cells are maintained by replacing FGF2-containing media daily,while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding,however,results in significant variation in growth factor levels due to FGF2 instability,which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers,increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures,so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings. View Publication

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes,mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture,hES cells expressing cell-surface NGFR protein (CD271,p75NTR) were isolated by immunoaffinity adsorption,and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression,examined by quantitative RT-PCR,found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR,SNAI1,NTRK3,SOX9,and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer,mRNAs typifying adult stromal stem cells were detected,including BMI1,KIT,NES,NOTCH1,and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1,B3GNT7,PTDGS,and ALDH3A1 were upregulated. mRNA for keratocan (KERA),a cornea-specific proteoglycan,was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate,a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells,therefore,may provide a renewable source of material for development of treatment of corneal stromal opacities. View Publication

Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development,which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently,representative DNA biomarkers become equally important in pre-clinical studies. However,it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here,we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres,adherent cell cultures,and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes,and new actionable mutations such as the loss of PIK3R1,and reveals clear patient-to-patient differences. In contrast,for each patient,we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly,we observe 96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles,we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests,at a remarkable resolution,the genome-wide conservation of a patient's tumor genetics in various pre-clinical models,and therefore supports their use for the development and testing of personalized targeted therapies. View Publication