技术资料

PURPOSE: This first-in-human dose-escalation trial evaluated the safety,tolerability,maximal-tolerated dose (MTD),dose-limiting toxicities (DLT),pharmacokinetics,pharmacodynamics,and preliminary clinical activity of pictilisib (GDC-0941),an oral,potent,and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). PATIENTS AND METHODS: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily,initially on days 1 to 21 every 28 days and later,using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET,and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. RESULTS: Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea,rash,and fatigue,whereas the DLT was grade 3 maculopapular rash (450 mg,2 of 3 patients; 330 mg,1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed textgreater90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC textgreater20 htextperiodcenteredμmol/L. Significant increase in plasma insulin and glucose levels,and textgreater25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria,respectively. CONCLUSION: Pictilisib was safely administered with a dose-proportional pharmacokinetic profile,on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily. View Publication

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. View Publication

BACKGROUND We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4,Sox2,c-Myc,and Klf4). In this study,we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential. METHODS Mature microRNAs (mir-302a-d,369-3p and 5p,and mir-200c,as needed) were introduced into colon cancer cells (DLD-1,RKO,and HCT116) using lipofection. RESULTS The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog,Oct3/4,SOX2,and Klf4,as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers,indicating that reprogramming of the cancer cells was partially successful. Moreover,we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo,accompanied by increased expression of the tumor suppressor genes p16 (ink4a) and p21 (waf1) . miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil,possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1,RKO,and HCT116 cells exhibited reduced c-Myc expression,in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors. CONCLUSIONS Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application,because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression. View Publication

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover,we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining,transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling. View Publication

BACKGROUND:Emerging studies of human pluripotent stem cells (hPSCs) raise new prospects for neurodegenerative disease modeling and cell replacement therapies. Therefore,understanding the mechanisms underlying the commitment of neural progenitor cells (NPCs) is important for the application of hPSCs in neurodegenerative disease therapies. It has been reported that epigenetic modifications of histones play important roles in neural differentiation,but the exact mechanisms in regulating hPSC differentiation towards NPCs are not fully elucidated.RESULTS:We demonstrated that suppression of histone deacetylases (HDACs) promoted the differentiation of hPSCs towards NPCs. Application of HDAC inhibitors (HDACi) increased the expression of neuroectodermal markers and enhanced the neuroectodermal specification once neural differentiation was initiated,thereby leading to more NPC generation. Similarly,the transcriptome analysis showed that HDACi increased the expression levels of ectodermal markers and triggered the NPC differentiation related pathways,while decreasing the expression levels of endodermal and mesodermal markers. Furthermore,we documented that HDAC3 but not HDAC1 or HDAC2 was the critical regulator participating in NPC differentiation,and knockdown of HDAC3's cofactor SMRT exhibited a similar effect as HDAC3 on NPC generation.CONCLUSIONS:Our study reveals that HDACs,especially HDAC3,negatively regulate the differentiation of hPSCs towards NPCs at an earlier stage of neural differentiation. Moreover,HDAC3 might function by forming a repressor complex with its cofactor SMRT during this process. Thus,our findings uncover an important epigenetic mechanism of HDAC3 in the differentiation of hPSCs towards NPCs. View Publication

Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt,correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart,we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb,with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency,consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites,we observed a high degree of gene excisions and inversions,which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency,deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments,particularly in human iPSC. View Publication

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here,neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1,Sox2,Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains,respectively,of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes,epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together,these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays. View Publication

BACKGROUND Induced pluripotent stem cells (iPSCs) hold tremendous potential,both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development. OBJECTIVE We sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children,a highly relevant and easily accessible tissue for pediatric populations. METHODS We performed detailed comparative analysis on the transcriptomes and methylomes of NECs,iPSCs derived from NECs (NEC-iPSCs),and embryonic stem cells (ESCs). RESULTS NEC-iPSCs express pluripotent cell markers,can differentiate into all 3 germ layers in vivo and in vitro,and have a transcriptome and methylome remarkably similar to those of ESCs. However,residual DNA methylation marks exist,which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro,suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming,which are indicative of possible roles in airway epithelium development. CONCLUSION NECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients,and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory. View Publication

Early cognitive deficits in Alzheimer's disease (AD) seem to be correlated to dysregulation of glutamate receptors evoked by amyloid-beta (Aβ) peptide. Aβ interference with the activity of N-methyl-d-aspartate receptors (NMDARs) may be a relevant factor for Aβ-induced mitochondrial toxicity and neuronal dysfunction. To evaluate the role of mitochondria in NMDARs activation mediated by Aβ,we followed in situ single-cell simultaneous measurement of cytosolic free Ca(2+)(Cai(2+)) and mitochondrial membrane potential in primary cortical neurons. Our results show that direct exposure to Aβ + NMDA largely increased Cai(2+) and induced immediate mitochondrial depolarization,compared with Aβ or NMDA alone. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Cai(2+) evoked by Aβ or NMDA,suggesting that mitochondria control Ca(2+) entry through NMDARs. However,incubation with rotenone did not preclude mitochondrial Ca(2+) (mitCa(2+)) retention in cells treated with Aβ. Aβ-induced Cai(2+) and mitCa(2+) rise were inhibited by ifenprodil,an antagonist of GluN2B-containing NMDARs. Exposure to Aβ + NMDA further evoked a higher mitCa(2+) retention,which was ameliorated in GluN2B(-/-) cortical neurons,largely implicating the involvement of this NMDAR subunit. Moreover,pharmacologic inhibition of endoplasmic reticulum (ER) inositol-1,4,5-triphosphate receptor (IP3R) and mitCa(2+) uniporter (MCU) evidenced that Aβ + NMDA-induced mitCa(2+) rise involves ER Ca(2+) release through IP3R and mitochondrial entry by the MCU. Altogether,data highlight mitCa(2+) dyshomeostasis and subsequent dysfunction as mechanisms relevant for early neuronal dysfunction in AD linked to Aβ-mediated GluN2B-composed NMDARs activation. View Publication

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis,and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein,we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly,TRAIL treatment induced RANKL mRNA expression in these cells. In addition,TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition,siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus,our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However,limited information is available regarding the immunologic features of iPSCs. In this study,expression of MHC and T cell co-stimulatory molecules in hiPSCs,and the effects on activation,proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation,hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ,TNF-α and IL-17,hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10,and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity,which may result from their induction of IL-10-secreting Treg. View Publication

The Mesenchymal-to-Epithelial Transition (MET) has been recognized as a crucial step for successful reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Thus,it has been demonstrated,that the efficiency of reprogramming can be enhanced by promoting an epithelial expression program in cells,with a concomitant repression of key mesenchymal genes. However,a detailed characterization of the epithelial transition associated with the acquisition of a pluripotent phenotype is still lacking to this date. Here,we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming,as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6-12. days after electroporation,in synchrony with the upregulation of early pluripotency markers,and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT) which takes place during mammalian gastrulation. View Publication