Scientific Resources

Insulin-like growth factor 1 (IGF-1) enhances cellular proliferation and reduces apoptosis during the early differentiation of bone marrow derived mesenchymal stem cells (BMSCs) into neural progenitor-like cells (NPCs) in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). BMSCs were differentiated in three groups of growth factors: (A) EGF + bFGF,(B) EGF + bFGF + IGF-1,and (C) without growth factor. To unravel the molecular mechanisms of the NPCs derivation,microarray analysis using GeneChip miRNA arrays was performed. The profiles were compared among the groups. Annotated microRNA fingerprints (GSE60060) delineated 46 microRNAs temporally up-regulated or down-regulated compared to group C. The expressions of selected microRNAs were validated by real-time PCR. Among the 46 microRNAs,30 were consistently expressed for minimum of two consecutive time intervals. In Group B,only miR-496 was up-regulated and 12 microRNAs,including the let-7 family,miR-1224,miR-125a-3p,miR-214,miR-22,miR-320,miR-708,and miR-93,were down-regulated. Bioinformatics analysis reveals that some of these microRNAs (miR-22,miR-214,miR-125a-3p,miR-320 and let-7 family) are associated with reduction of apoptosis. Here,we summarize the roles of key microRNAs associated with IGF-1 in the differentiation of BMSCs into NPCs. These findings may provide clues to further our understanding of the mechanisms and roles of microRNAs as key regulators of BMSC-derived NPC maintenance. View Publication

BACKGROUND: Epithelial-mesenchymal transition (EMT) is involved in important malignant features of cancer cells,like invasion,metastatic potential,anti-apoptotic and stem-cell like phenotypes. Among several transcription factors,SNAI2/SLUG is supposed to play an essential role for EMT. METHODS: Paraffin embedded tumor samples from 63 patients with metastatic non-small cell lung cancer,enrolled in a randomized phase II trial,were prospectively collected,53 samples qualified for further analysis. Automated RNA extraction from paraffin and RT-quantitative PCR was used for evaluation of SNAI2/SLUG,estrogen receptor 1 (ESR1) and matrix-metalloproteinases (MMP) mRNA expression. RESULTS: Clinical features like age,gender,performance status,histological subtype and stage were similarly distributed among SNAI2/SLUG positive and negative patients. SNAI2/SLUG was significantly,inversely correlated with ESR1 mRNA expression (p textless 0.0001). In contrast,MMP2 (p = 0.387),MMP7 (p = 0.396) and MMP9 mRNA expression (p = 0.366) did not correlate with SNAI2/SLUG. Patients with high SNAI2/SLUG expression (grouped by median expression) had a worse outcome. Median overall survival in patients with high SNAI2/SLUG expression was 5.7 months versus 11.6 months with low SNAI2/SLUG expression (p = .038). Inversely,patients with high ESR1 expression (grouped by median expression) had an improved median OS with 10.9 months vs. 5.0 months in the low expression group (p = .032). In multivariate analysis,SNAI2/SLUG2 (p = .022) and ESR1 (p = .017) separately were independent prognostic factors for survival. CONCLUSION: SNAI2/SLUG is prognostic of patients' outcome. The strong inverse correlation with ESR1 indicates a significant impact of estrogen receptor pathway regarding these malignant features. View Publication

BACKGROUND Accumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases,such as cancer. The lncRNA,CCDC26,is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients. RESULTS We found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26,gene knockdown (KD) was performed using short hairpin RNAs (shRNAs),and four KD clones,in which CCDC26 expression was suppressed to 1% of its normal level,were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA),indicating that transcriptional gene suppression (TGS),not post-transcriptional suppression,was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant,which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast,enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor,KIT,hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03,a KIT-specific inhibitor,eliminated the increased survival of KD clones in the absence of serum. CONCLUSIONS We suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered. View Publication

Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE),derived from the C terminus of human thrombin,inhibits proinflammatory responses in vitro and in vivo,but the mode of action is unclear. In this study,we show that GKY25,apart from binding bacterial LPS,also interacts directly with monocytes and macrophages in vitro,ex vivo,and in vivo. Moreover,GKY25 inhibits TLR4- and TLR2-induced NF-κB activation in response to several microbe-derived agonists. Furthermore,GKY25 reduces LPS-induced phosphorylation of MAPKs p38α and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25,based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-κB activity and proinflammatory cytokine production in monocytes and macrophages. View Publication

Regeneration of human cartilage is inherently inefficient; an abundant autologous source,such as human induced pluripotent stem cells (hiPSCs),is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks,with an overall efficiency textgreater90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression,extracellular matrix production,and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition,2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance,autologous nature,and potential to generate articular-like cartilage rather than fibrocartilage. In addition,hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. View Publication

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study,we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging,we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next,we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain,we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective,potent and secretable variant of a TRAIL,S-TRAIL,and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore,the incorporation of pro-drug converting enzyme,herpes simplex virus thymidine kinase,into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications. View Publication

Protein synthesis regulation via mammalian target of rapamycin complex 1 (mTORC1) signaling pathway has key roles in neural development and function,and its dysregulation is involved in neurodevelopmental disorders associated with autism and intellectual disability. mTOR regulates assembly of the translation initiation machinery by interacting with the eukaryotic initiation factor eIF3 complex and by controlling phosphorylation of key translational regulators. Collybistin (CB),a neuron-specific Rho-GEF responsible for X-linked intellectual disability with epilepsy,also interacts with eIF3,and its binding partner gephyrin associates with mTOR. Therefore,we hypothesized that CB also binds mTOR and affects mTORC1 signaling activity in neuronal cells. Here,by using induced pluripotent stem cell-derived neural progenitor cells from a male patient with a deletion of entire CB gene and from control individuals,as well as a heterologous expression system,we describe that CB physically interacts with mTOR and inhibits mTORC1 signaling pathway and protein synthesis. These findings suggest that disinhibited mTORC1 signaling may also contribute to the pathological process in patients with loss-of-function variants in CB.European Journal of Human Genetics advance online publication,22 April 2015; doi:10.1038/ejhg.2015.69. View Publication

The extent,regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific CtextgreaterU RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins,including many that are involved in pathogenesis of viral diseases. APOBEC3A,which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons,mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of CtextgreaterU RNA editing in mammals. View Publication

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections,and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2),which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs,HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However,HERVK is transcriptionally silenced by the host,with the exception of in certain pathological contexts such as germ-cell tumours,melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations,together with transactivation by OCT4 (also known as POU5F1),synergistically facilitate HERVK expression. Consequently,HERVK is transcribed during normal human embryogenesis,beginning with embryonic genome activation at the eight-cell stage,continuing through the emergence of epiblast cells in preimplantation blastocysts,and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably,we detected HERVK viral-like particles and Gag proteins in human blastocysts,indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product,the HERVK accessory protein Rec,in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection,suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover,Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy,indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development. View Publication

Endothelial cells (ECs) exist in different microenvironments in vivo,including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study,a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic,anti-inflammatory,arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h,as demonstrated by qRT-PCR for arterial markers EphrinB2,CXCR4,Conexin40 and Notch1,as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore,the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype,and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications. View Publication

BACKGROUND Acute myeloid leukemia (AML) is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs),which contribute to the progression,recurrence and therapeutic resistance of leukemia. However,the mechanisms underlying the maintenance of LSCs drug resistance have not been fully defined. In this study,we attempted to elucidate the mechanisms of LSCs drug resistance. METHODS We performed reverse phase protein arrays to analyze the expression of anti-apoptotic proteins in the LSC-enriched leukemia cell line KG-1a. Immuno-blotting,cell viability and clinical AML samples were evaluated to verify the micro-assay results. The characteristics and transcriptional regulation of survivin were analyzed with the relative luciferase reporter assay,mutant constructs,chromatin immuno-precipitation (ChIP),quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR),and western blotting. The levels of Sp1,c-Myc,phospho-extracellular signal-regulated kinase (p-ERK),phospho-mitogen and stress-activated protein kinase (p-MSK) were investigated in paired CD34+ and CD34- AML patient samples. RESULTS Survivin was highly over-expressed in CD34 + CD38- KG-1a cells and paired CD34+ AML patients compared with their differentiated counterparts. Functionally,survivin contributes to the drug resistance of LSCs,and Sp1 and c-Myc concurrently regulate levels of survivin transcription. Clinically,Sp1 and c-Myc were significantly up-regulated and positively correlated with survivin in CD34+ AML patients. Moreover,Sp1 and c-Myc were further activated by the ERK/MSK mitogen-activated protein kinase (MAPK) signaling pathway,modulating survivin levels. CONCLUSION Our findings demonstrated that ERK/MSK/Sp1/c-Myc axis functioned as a critical regulator of survivin expression in LSCs,offering a potential new therapeutic strategy for LSCs therapy. View Publication

INTRODUCTION Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of four factors--OCT4,SOX2,KLF4,and cMYC--via integrating vectors. Here,we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV). METHODS We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit. RESULTS After verification of OCT4 expression,we assessed the ability of iPSC reprogramming. The reprogramming vector cocktail with the OCT4-expressing MV vector and SOX2-,KLF4-,and cMYC-expressing lentiviral vectors efficiently transduced human skin fibroblasts and formed iPSC colonies. Reverse transcription-polymerase chain reaction and immunostaining confirmed induction of endogenous pluripotency-associated marker genes,such as SSEA-4,TRA-1-60,and Nanog. Pluripotency of derived clones was confirmed by spontaneous differentiation into three germ layers,teratoma formation,and guided differentiation into beating cardiomyocytes. CONCLUSIONS MV vectors can induce efficient nuclear reprogramming. Given the excellent safety record of MV vaccines and the translational capabilities recently developed to produce MV-based vectors now used for cancer clinical trials,our MV vector system provides an RNA-based,non-integrating gene transfer platform for nuclear reprogramming that is amenable for immediate clinical translation. View Publication