ApoE maintains neuronal integrity via microRNA and H3K27me3-mediated repression.

ApoE regulates neurogenesis, although how it influences genetic programs remains elusive. Cortical neurons induced from isogenic control and ApoE-/- human neural stem cells (NSCs) recapitulated key transcriptomic signatures of in vivo counterparts identified from single-cell human midbrain. Surprisingly, ApoE expression in NSC and neural progenitor cells (NPCs) is not required for differentiation. Instead, ApoE prevents the over-proliferation of non-neuronal cells during extended neuronal culture when it is not expressed. Elevated miR-199a-5p level in ApoE-/- cells lowers the EZH1 protein and the repressive H3K27me3 mark, a phenotype rescued by miR-199a-5p steric inhibitor. Reduced H3K27me3 at genes linked to extracellular matrix organization and angiogenesis in ApoE-/- NPC correlates with their aberrant expression and phenotypes in neurons. Interestingly, the ApoE coding sequence, which contains many predicted miR-199a-5p binding sites, can repress miR-199a-5p without translating into protein. This suggests that ApoE maintains neurons integrity through the target-directed miRNA degradation of miR-199a-5p, imparting the H3K27me3-mediated repression of non-neuronal genes during differentiation.

Microglia-secreted TNF-α affects differentiation efficiency and viability of pluripotent stem cell-derived human dopaminergic precursors.

Disease is a neurodegenerative disorder characterised by the progressive loss of dopaminergic cells of the substantia nigra pars compacta. Even though successful transplantation of dopamine-producing cells into the striatum exhibits favourable effects in animal models and clinical trials; transplanted cell survival is low. Since every transplant elicits an inflammatory response which can affect cell survival and differentiation, we aimed to study in vivo and in vitro the impact of the pro-inflammatory environment on human dopaminergic precursors. We first observed that transplanted human dopaminergic precursors into the striatum of immunosuppressed rats elicited an early and sustained activation of astroglial and microglial cells after 15 days' post-transplant. This long-lasting response was associated with Tumour necrosis factor alpha expression in microglial cells. In vitro, conditioned media from activated BV2 microglial cells increased cell death, decreased Tyrosine hydroxylase-positive cells and induced morphological alterations on human neural stem cells-derived dopaminergic precursors at two differentiation stages: 19 days and 28 days. Those effects were ameliorated by inhibition of Tumour necrosis factor alpha, a cytokine which was previously detected in vivo and in conditioned media from activated BV-2 cells. Our results suggest that a pro-inflammatory environment is sustained after transplantation under immunosuppression, providing a window of opportunity to modify this response to increase transplant survival and differentiation. In addition, our data show that the microglia-derived pro-inflammatory microenvironment has a negative impact on survival and differentiation of dopaminergic precursors. Finally, Tumour necrosis factor alpha plays a key role in these effects, suggesting that this cytokine could be an interesting target to increase the efficacy of human dopaminergic precursors transplantation in Parkinson's Disease.

cGMP-grade human iPSC-derived retinal photoreceptor precursor cells rescue cone photoreceptor damage in non-human primates.

BACKGROUND: Retinal regenerative therapies hold great promise for the treatment of inherited retinal degenerations (IRDs). Studies in preclinical lower mammal models of IRDs have suggested visual improvement following retinal photoreceptor precursors transplantation, but there is limited evidence on the ability of these transplants to rescue retinal damage in higher mammals. The purpose of this study was to evaluate the therapeutic potential of photoreceptor precursors derived from clinically compliant induced pluripotent stem cells (iPSCs). METHODS: Photoreceptor precursors were sub-retinally transplanted into non-human primates (Macaca fascicularis). The cells were transplanted both in naïve and cobalt chloride-induced retinal degeneration models who had been receiving systemic immunosuppression for one week prior to the procedure. Optical coherence tomography, fundus autofluorescence imaging, electroretinography, ex vivo histology and immunofluorescence staining were used to evaluate retinal structure, function and survival of transplanted cells. RESULTS: There were no adverse effects of iPSC-derived photoreceptor precursors on retinal structure or function in naïve NHP models, indicating good biocompatibility. In addition, photoreceptor precursors injected into cobalt chloride-induced retinal degeneration NHP models demonstrated an ability both to survive and to mature into cone photoreceptors at 3 months post-transplant. Optical coherence tomography showed restoration of retinal ellipsoid zone post-transplantation. CONCLUSIONS: These findings demonstrate the safety and therapeutic potential of clinically compliant iPSC-derived photoreceptor precursors as a cell replacement source for future clinical trials.

Polyphyllin VII induces fibroblasts apoptosis via the ERK/JNK pathway.

Hypertrophic scar (HS) is a pathological scar that often occurs in burn patients. Its histology is characterized by the excessive proliferation of fibroblasts (FB) and excessive accumulation of extracellular matrix (ECM). Inhibition of proliferation and activation of FB is essential for the treatment of HS. The crude extracts of traditional Chinese medicines have beneficial therapeutic effects on HS besides possessing fewer side effects and being easily available. Polyphyllin VII (PP7) is an isoprene saponin isolated from Rhizoma paridis. It has a pro-apoptotic effect on cancer cells. In the present study, we demonstrate that PP7 exerts a significant inhibitory effect on hypertrophic scar fibroblasts (HSFs) in vitro. We also demonstrate that PP7 considerably induces the apoptosis of HSFs and inhibits their activity. Our data show that the PP7-induced HSFs cell apoptosis was mainly due to the enhanced expression of apoptotic genes (Bax, Caspase-3, Caspase-9) and decreased expression of Bcl-2. Moreover, PP7 treatment also enhances the expression of JNK, but that of extracellular protein kinases (ERK) was reduced, and induces apoptosis through ERK/JNK pathways. Thus, PP7 can be used as a drug to prevent the formation of HS.

A structurally integrated water environmental modeling system based on dual object structure.

The GIS-based water quantity and water quality model is widely used to provide decision-making supports for water resource and water quality management. However, the existing integration patterns of GIS and model system mainly depend on data communication between themselves which may lead to low operating efficiency and time-consuming model setup. In this paper, a generalized data structure (dual object structure (DOS)) which can store the data of GIS objects and model objects together is proposed and realized for the first time, avoiding frequent data communication during the period of numerical simulation and result expression, realizing the fusion of GIS objects and model objects at the data structure level, improving the operating efficiency of the system. Finally, the water quantity and water quality modeling software (digital basin simulation system (DBSS)) based on DOS was developed by using C++ language. The software has been applied successfully in large-scale river basins of China, and one of the cases was demonstrated to show the application process and the outstanding results.

The PARK10 gene USP24 is a negative regulator of autophagy and ULK1 protein stability.

Recent studies indicate a causative relationship between defects in autophagy and dopaminergic neuron degeneration in Parkinson disease (PD). However, it is not fully understood how autophagy is regulated in the context of PD. Here we identify USP24 (ubiquitin specific peptidase 24), a gene located in the PARK10 (Parkinson disease 10 [susceptibility]) locus associated with late onset PD, as a novel negative regulator of autophagy. Our data indicate that USP24 regulates autophagy by affecting ubiquitination and stability of the ULK1 protein. Knockdown of USP24 in cell lines and in human induced-pluripotent stem cells (iPSC) differentiated into dopaminergic neurons resulted in elevated ULK1 protein levels and increased autophagy flux in a manner independent of MTORC1 but dependent on the class III phosphatidylinositol 3-kinase (PtdIns3K) activity. Surprisingly, USP24 knockdown also improved neurite extension and/or maintenance in aged iPSC-derived dopaminergic neurons. Furthermore, we observed elevated levels of USP24 in the substantia nigra of a subpopulation of idiopathic PD patients, suggesting that USP24 may negatively regulate autophagy in PD.Abbreviations: Bafilomycin/BafA: bafilomycin A1; DUB: deubiquitinating enzyme; iPSC: induced pluripotent stem cells; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; nt: non-targeting; PD: Parkinson disease; p-ATG13: phospho-ATG13; PtdIns3P: phosphatidylinositol 3-phosphate; RPS6: ribosomal protein S6; SNPs: single nucleotide polymorphisms; TH: tyrosine hydroxylase; USP24: ubiquitin specific peptidase 24.

Comprehensive investigation of key biomarkers and pathways in hepatitis B virus-related hepatocellular carcinoma.

Objective: Our study is aim to explore potential key biomarkers and pathways in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) using genome-wide expression profile dataset and methods. Methods: Dataset from the GSE14520 is used as the training cohort and The Cancer Genome Atlas dataset as the validation cohort. Differentially expressed genes (DEGs) screening were performed by the limma package. Gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), gene ontology, the Kyoto Encyclopedia of Genes and Genomes, and risk score model were used for pathway and genes identification. Results: GSEA revealed that several pathways and biological processes are associated with hepatocarcinogenesis, such as the cell cycle, DNA repair, and p53 pathway. A total of 160 DEGs were identified. The enriched functions and pathways of the DEGs included toxic substance decomposition and metabolism processes, and the P450 and p53 pathways. Eleven of the DEGs were identified as hub DEGs in the WGCNA. In survival analysis of hub DEGs, high expression of PRC1 and TOP2A were significantly associated with poor clinical outcome of HBV-related HCC, and shown a good performance in HBV-related HCC diagnosis. The prognostic signature consisting of PRC1 and TOP2A also doing well in the prediction of HBV-related HCC prognosis. The diagnostic and prognostic values of PRC1 and TOP2A was confirmed in TCGA HCC patients. Conclusions: Key biomarkers and pathways identified in the present study may enhance the comprehend of the molecular mechanisms underlying hepatocarcinogenesis. Additionally, mRNA expression of PRC1 and TOP2A may serve as potential diagnostic and prognostic biomarkers for HBV-related HCC.

Diagnostic and prognostic biomarkers of Human Leukocyte Antigen complex for hepatitis B virus-related hepatocellular carcinoma.

Background: Hepatitis B virus infection had been identified its relationship with liver diseases, including liver tumors. We aimed to explore diagnostic and prognostic values between the Human Leukocyte Antigen (HLA) complex and hepatocellular carcinoma (HCC). Methods: We used the GSE14520 dataset to explore diagnostic and prognostic significance between HLA complex and HCC. A nomogram was constructed to predict survival probability of HCC prognosis. Gene set enrichment analysis was explored using gene ontologies and metabolic pathways. Validation of prognostic values of the HLA complex was performed in the Kaplan-Meier Plotter website. Results: We found that HLA-C showed the diagnostic value (P <0.0001, area under curve: 0.784, sensitivity: 93.14%, specificity: 62.26%). In addition, HLA-DQA1 and HLA-F showed prognostic values for overall survival, and HLA-A, HLA-C, HLA-DPA1 and HLA-DQA1 showed prognostic values for recurrence-free survival (all P ≤ 0.05, elevated 0.927, 0.992, 1.023, 0.918, 0.937 multiples compared to non-tumor tissues, respectively). Gene set enrichment analysis found that they were involved in antigen processing and toll like receptor signalling pathway, etc. The nomogram was evaluated for survival probability of HCC prognosis. Validation analysis indicated that HLA-C, HLA-DPA1, HLA-E, HLA-F and HLA-G were associated with HCC prognosis of overall survival (all P ≤ 0.05, elevated 0.988 and 0.997 multiples compared to non-tumor tissues, respectively). Conclusion: HLA-C might be a diagnostic and prognostic biomarker for HCC. HLA-DPA1 and HLA-F might be prognostic biomarkers for HCC.

Perturbations in RhoA signalling cause altered migration and impaired neuritogenesis in human iPSC-derived neural cells with PARK2 mutation.

Mutations in parkin, encoded by the PARK2 gene, causes early-onset familial Parkinson's disease (PD), but dysfunctional parkin has also been implicated in sporadic PD. By combining human isogenic induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) and a novel large-scale mass spectrometry based proteomics and post-translational modification (PTM)-omics approach, we have mapped changes in protein profiles and PTMs caused by parkin deficiency in neurons. Our study identifies changes to several proteins previously shown to be dysregulated in brains of sporadic PD patients. Pathway analysis and subsequent in vitro assays reveal perturbations in migration and neurite outgrowth in the PARK2 KO neurons. We confirm the neurite defects using long-term engraftment of neurons in the striatum of immunosuppressed hemiparkinsonian adult rats. The GTP-binding protein RhoA was identified as a key upstream regulator, and RhoA activity was significantly increased in PARK2 KO neurons. By inhibiting RhoA signalling the migration and neurite outgrowth phenotypes could be rescued. Our study provides new insight into the pathogenesis of PD and demonstrates the broadly applicable potential of proteomics and PTMomics for elucidating the role of disease-causing mutations.

Clustered microRNAs hsa-miR-221-3p/hsa-miR-222-3p and their targeted genes might be prognostic predictors for hepatocellular carcinoma.

Objective: MicroRNAs (miRNAs) have been explored in malignancies. We investigated the functions of clustered miRNAs hsa-miR-221/222-3p in hepatocellular carcinoma (HCC). Methods: Human miRNA tissue atlas website was determined expression levels in liver tissue. Four databases, TarBase, miRTarBase, miRecords and miRPathDB, were found experimentally validated target genes of clustered miRNAs. TargetScanHuman was predicted target genes. The STRING website was depicted protein-protein interaction (PPI) networks. The OncoLnc website analyzed prognostic values for hsa-miR-221/222-3p and their target genes. The MCODE plugin calculated modules of PPI networks. Receiver operating characteristic (ROC) curves were predicted 1, 3, and 5 years prognostic values. Results: Expression of clustered miRNAs was high in liver tissues. A total of 1577 target genes were identified. Enrichment analysis showed that target genes were enriched mainly in cancer, Wnt signaling and ErbB signaling pathways. Two modules were calculated using PPI networks. Has-miR-221-3p was not associated with prognosis (P = 0.401). Has-miR-222-3p and target genes ESR1, TMED7, CBFB, ETS2, UBE2J1 and UBE2N of the clustered miRNAs were associated with HCC survival (all P < 0.05). Has-miR-222-3p, CBFB, and UBE2N showed good performance of ROC in prognosis prediction at 1, 3, and 5 years (all area under curves > 0.600). Conclusion: Has-miR-222-3p and target genes, especially CBFB, UBE2N, may serve as prognostic predictors for HCC.

Integrated analysis of competing endogenous RNA network revealing potential prognostic biomarkers of hepatocellular carcinoma.

Objective: The goal of our study is to identify a competing endogenous RNA (ceRNA) network using dysregulated RNAs between HCC tumors and the adjacent normal liver tissues from The Cancer Genome Atlas (TCGA) datasets, and to investigate underlying prognostic indicators in hepatocellular carcinoma (HCC) patients. Methods: All of the RNA- and miRNA-sequencing datasets of HCC were obtained from TCGA, and dysregulated RNAs between HCC tumors and the adjacent normal liver tissues were investigated by DESeq and edgeR algorithm. Survival analysis was used to confirm underlying prognostic indicators. Results: In the present study, we constructed a ceRNA network based on 16 differentially expressed genes (DEGs), 7 differentially expressed microRNAs and 34 differentially expressed long non-coding RNAs (DELs). Among these dysregulated RNAs, three DELs (AP002478.1, HTR2A-AS1, and ERVMER61-1) and six DEGs (enhancer of zeste homolog 2 [EZH2], kinesin family member 23 [KIF23], chromobox 2 [CBX2], centrosomal protein 55 [CEP55], cell division cycle 25A [CDC25A], and claspin [CLSPN]) were used for construct a prognostic signature for HCC overall survival (OS), and performed well in HCC OS (adjusted P<0.0001, adjusted hazard ratio = 2.761, 95% confidence interval = 1.838-4.147). Comprehensive survival analysis demonstrated that this prognostic signature may be act as an independent prognostic indicator of HCC OS. Functional assessment of these dysregulated DEGs in the ceRNA network and gene set enrichment of this prognostic signature suggest that both were enriched in the biological processes and pathways of the cell cycle, cell division and cell proliferation. Conclusions: Our current study constructed a ceRNA network for HCC, and developed a prognostic signature that may act as an independent indicator for HCC OS.

Evaluation of the diagnostic ability of laminin gene family for pancreatic ductal adenocarcinoma.

A poor outcome for pancreatic ductal adenocarcinoma (PDAC) patients is still a challenge worldwide. The aim of our study is to investigate the potential of key laminin subunits for being used both as a diagnostic and prognostic biomarker for PDAC patients. We evaluated the mRNA expression and prognostic value of laminin gene family in PDAC tissues using online public databases. Moreover, the relationship between key laminin subunits in PDAC blood cells and circulating tumor cells (CTCs) and the distinguishing ability of joint serum levels with carbohydrate antigen 19-9 (CA19-9) was analyzed. Two key differentially expressed subunits (LAMA3 and LAMC2) that are associated with prognosis of PDAC patients were found to show a potential for distinguishing between PDAC and non-tumor tissues. LAMA3 and LAMC2 expression were found to be positively related with CTC quantity in PDAC blood (R=0.628, p=0.029; R=0.776, p=0.003, respectively) using IgG chips. Furthermore, serum LAMC2 levels offered significant improvement over using CA19-9 alone for the discrimination of PDAC. Joint serum LAMC2 and CA19-9 levels increased the net benefit proportion in early stage/operational PDAC patients. Using integrated profiling, we identified LAMA3 and LAMC2 as potential therapeutic targets and prognostic markers for PDAC, for which further validation is warranted.

Genetic variants and Expression of Cytochrome p450 Oxidoreductase Predict Postoperative Survival in Patients with Hepatitis B Virus-Related Hepatocellular Carcinoma.

Our current study investigates the prognostic values of genetic variants and mRNA expression of cytochrome p450 oxidoreductase (POR) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). A total of 19 candidate single nucleotide polymorphisms (SNPs) located in the exons of POR were genotyped using Sanger sequencing from 476 HBV-related HCC patients who underwent hepatectomy between 2003 and 2013. The mRNA expression of POR in 212 patients with HBV-related HCC was obtained from GSE14520 dataset. Survival analysis was performed to investigate the association of POR variants and mRNA expression with overall survival (OS) and recurrence-free survival (RFS). Nomograms were used to predict the prognosis of HBV-related HCC patients. Gene set enrichment analysis (GSEA) was used to investigate the mechanism of POR in HBV-related HCC prognosis. The polymorphism POR-rs1057868 was significantly associated with HBV-related HCC OS (CT/TT vs. CC, hazard ratio [HR] = 0.69, 95% confidence interval [CI] = [0.54, 0.88], P = 0.003), but not significantly associated with RFS (CT/TT vs. CC, P = 0.378). POR mRNA expression was also significantly associated with HBV-related HCC OS (high vs. low, HR = 0.61, 95% CI = [0.38, 0.97], P = 0.036), but not significantly associated with the RFS (high vs. low, P = 0.201). Two nomograms were developed to predict the HBV-related HCC OS. Furthermore, GSEA suggests that multiple gene sets were significantly enriched in liver cancer survival and recurrence, as well as POR-related target therapy in the liver. In conclusion, our study suggests that POR-rs1057868 and mRNA expression may serve as a potential postoperative prognosis biomarker in HBV-related HCC.

Diagnostic and prognostic value of mRNA expression of phospholipase C ß family genes in hepatitis B virus­associated hepatocellular carcinoma.

Four phospholipase C ß (PLCB) isoforms, PLCB1, PLCB2, PLCB3 and PLCB4, have been previously investigated regarding their roles in the metabolism of inositol lipids and cancer. The present study aimed to explore the association between PLCB1­4 and hepatocellular carcinoma (HCC). Data from 212 patients with hepatitis B virus­associated HCC were used to analyze the diagnostic and prognostic significance of PLCB genes in. A nomogram predicted the survival probability. Gene set enrichment analysis explored gene ontology terms and the metabolic pathways associated with PLCB genes. Validation of the prognostic values of PLCB genes was performed using the Gene Expression Profiling Interactive Analysis website. PLCB1 and PLCB2 were revealed to have diagnostic value for HCC (0.869 and 0.836 area under the curve, respectively; both P≤0.05). The combination analysis of these genes had an advantage over each alone (0.905 PLCB1 and PLCB2, and 0.877 PLCB1 and PLCB3 area under the curve; P≤0.05). PLCB1 was associated with overall survival (OS) and recurrence­free survival (RFS; adjusted P=0.002 and P=0.001, respectively). A nomogram predicted survival probability of patients with HCC at 1, 3­ and 5­years. Gene set enrichment analysis indicated that PLCB1 and PLCB2 are involved in the cell cycle, cell division and the PPAR signaling pathway, among other functions. Validation using GEPIA revealed that PLCB1 and PLCB2 were associated with OS and PLCB1 and PLCB4 were associated with RFS. PLCB1 and PLCB2 exhibited diagnostic value for HCC and their combination had an advantage over each individually. PLCB1 has OS and RFS prognostic value for patients with HCC.

Identification of prognostic biomarkers for patients with hepatocellular carcinoma after hepatectomy.

Hepatocellular carcinoma (HCC) is a lethal malignancy with high morbidity and mortality rates worldwide. The identification of prognosis­associated biomarkers is crucial to improve HCC patient survival. The present study aimed to explore potential predictive biomarkers for HCC. Differentially expressed genes (DEGs) were analyzed in the GSE36376 dataset using GEO2R. Hub genes were identified and further investigated for prognostic value in HCC patients. A risk score model and nomogram were constructed to predict HCC prognosis using the prognosis­associated genes and clinical factors. Pearson's correlation was employed to show interactions among hub genes. Gene enrichment analysis was performed to identify detailed biological processes and pathways. A total of 71 DEGs were obtained and seven (ADH4, CYP2C8, CYP2C9, CYP8B1, SLC22A1, TAT and HSD17B13, all adjusted P≤0.05) of the 10 hub genes were identified as prognosis­related genes for survival analysis in HCC patients, including alcohol dehydrogenase 4 (class II), pi polypeptide (ADH4), cytochrome p450 family 2 subfamily C member 8 (CYP2C8), cytochrome P450 family 2 subfamily C member 9 (CYP2C9), cytochrome P450 family 8 subfamily B member 1 (CYP8B1), solute carrier family 22 member 1 (SLC22A1), tyrosine aminotransferase (TAT) and hydroxysteroid 17­ß dehydrogenase 13 (HSD17B13). The risk score model could predict HCC prognosis and the nomogram visualized gene expression and clinical factors of probability for HCC prognosis. The majority of genes showed significant Pearson's correlations with others (41 Pearson correlations P≤0.01, four Pearson correlations P>0.05). GO analysis revealed that terms such as 'chemical carcinogenesis' and 'drug metabolism­cytochrome P450' were enriched and may prove helpful to elucidate the mechanisms of hepatocarcinogenesis. Hub genes ADH4, CYP2C8, CYP2C9, CYP8B1, SLC22A1, TAT and HSD17B13 may be useful as predictive biomarkers for HCC prognosis.

Identification of Potential Prognostic Long Non-Coding RNA Biomarkers for Predicting Survival in Patients with Hepatocellular Carcinoma.

BACKGROUND/AIMS: The aim of the current study was to identify potential prognostic long non-coding RNA (lncRNA) biomarkers for predicting survival in patients with hepatocellular carcinoma (HCC) using The Cancer Genome Atlas (TCGA) dataset and bioinformatics analysis. METHODS: RNA sequencing and clinical data of HCC patients from TCGA were used for prognostic association assessment by univariate Cox analysis. A prognostic signature was built using stepwise multivariable Cox analysis, and a comprehensive analysis was performed to evaluate its prognostic value. The prognostic signature was further evaluated by functional assessment and bioinformatics analysis. RESULTS: Thirteen differentially expressed lncRNAs (DELs) were identified and used to construct a single prognostic signature. Patients with high risk scores showed a significantly increased risk of death (adjusted P < 0.0001, adjusted hazard ratio = 3.522, 95% confidence interval = 2.307-5.376). In the time-dependent receiver operating characteristic analysis, the prognostic signature performed well for HCC survival prediction with an area under curve of 0.809, 0.782 and 0.79 for 1-, 3- and 5-year survival, respectively. Comprehensive survival analysis of the 13-DEL prognostic signature suggested that it serves as an independent factor in HCC, showing a better performance for prognosis prediction than traditional clinical indicators. Functional assessment and bioinformatics analysis suggested that the prognostic signature was associated with the cell cycle and peroxisome proliferator-activated receptor signaling pathway. CONCLUSIONS: The novel lncRNA expression signature identified in the present study may be a potential biomarker for predicting the prognosis of HCC patients.

Distinct Diagnostic and Prognostic Values of Minichromosome Maintenance Gene Expression in Patients with Hepatocellular Carcinoma.

Background: The aim of the present study was to identify diagnostic and prognostic values of minichromosome maintenance (MCM) gene expression in patients with hepatocellular carcinoma (HCC). Methods: The biological function of the MCM genes were investigated by bioinformatics analysis. The diagnostic and prognostic values of the MCM genes were investigated by using the data of HCC patients from the GSE14520 and The Cancer Genome Atlas (TCGA) databases. Results: Bioinformatics analysis of the MCM genes substantiated that MCM2-7 genes were significantly enriched in DNA replication and cell cycle, and co-expressed with each other. These genes also co-expressed in HCC tumor tissue in both the GSE14520 and TCGA cohort. We also observed that the expression of the MCM2-7 genes was increased in tumor tissue, and diagnostic receiver operating characteristic analysis of MCM2-7 indicated that these genes could serve as sensitive diagnostic markers in HCC. Survival analysis in the GSE14520 cohort suggested that expression of MCM2, MCM4, MCM5, and MCM6 were significantly associated with hepatitis B virus-related HCC overall survival (OS). However, none of the MCM genes were associated with recurrence-free survival in the GSE14520 cohort. The validation cohort of TCGA suggested that the expression of MCM2, MCM6, and MCM7 were significantly correlated with HCC OS. Conclusion: Our study indicated that MCM2-7 genes may be potential diagnostic biomarkers in patients with HCC. Among them, MCM2 and MCM6 may serve as potential prognostic biomarkers for HCC.

Illustrating the potency of current Good Manufacturing Practice-compliant induced pluripotent stem cell lines as a source of multiple cell lineages using standardized protocols.

BACKGROUND AIMS: We have previously reported the generation of a current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) line for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of this or any other clinically compliant iPSC line METHODS: Using a stock at passage 20 prepared from the cGMP-compliant working cell bank (WCB), we tested differentiation into therapeutically relevant cell types of the three germ layers using standardized but generic protocols. Cells that we generated include (i) neural stem cells, dopaminergic neurons and astrocytes; (ii) retinal cells (retinal pigment epithelium and photoreceptors); and (iii) hepatocyte, endothelial and mesenchymal cells. To confirm that these generic protocols can also be used for other iPSC lines, we tested the reproducibility of our methodology with a second clinically compliant line RESULTS: Our results confirmed that well-characterized iPSC lines have broad potency, and, despite allelic variability, the same protocols could be used with minimal modifications with multiple qualified lines. In addition, we introduced a constitutively expressed GFP cassette in Chr13 safe harbor site using a standardized previously described method and observed no significant difference in growth and differentiation between the engineered line and the control line indicating that engineered products can be made using a standardized methodology CONCLUSIONS: We believe that our demonstration that multiple products can be made from the same WCB and that the same protocols can be used with multiple lines offers a path to a cost-effective strategy for developing cellular products from iPSC lines.

Generation of Transplantable Retinal Photoreceptors from a Current Good Manufacturing Practice-Manufactured Human Induced Pluripotent Stem Cell Line.

Retinal degeneration often results in the loss of light-sensing photoreceptors, which leads to permanent vision loss. Generating transplantable retinal photoreceptors using human somatic cell-derived induced pluripotent stem cells (iPSCs) holds promise to treat a variety of retinal degenerative diseases by replacing the damaged or dysfunctional native photoreceptors with healthy and functional ones. Establishment of effective methods to produce retinal cells including photoreceptors in chemically defined conditions using current Good Manufacturing Practice (cGMP)-manufactured human iPSC lines is critical for advancing cell replacement therapy to the clinic. In this study, we used a human iPSC line (NCL-1) derived under cGMP-compliant conditions from CD34+ cord blood cells. The cells were differentiated into retinal cells using a small molecule-based retinal induction protocol. We show that retinal cells including photoreceptors, retinal pigmented epithelial cells and optic cup-like retinal organoids can be generated from the NCL-1 iPSC line. Additionally, we show that following subretinal transplantation into immunodeficient host mouse eyes, retinal cells successfully integrated into the photoreceptor layer and developed into mature photoreceptors. This study provides strong evidence that transplantable photoreceptors can be generated from a cGMP-manufactured human iPSC line for clinical applications. Stem Cells Translational Medicine 2018;7:210-219.

Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications.

We have recently described manufacturing of human induced pluripotent stem cells (iPSC) master cell banks (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Reports, 5(4), 647-659, 2015). In this manuscript, we describe the detailed characterization of the two iPSC clones generated using this process, including whole genome sequencing (WGS), microarray, and comparative genomic hybridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibration material and with a reporter subclone and lines made by a similar process from different donors. We believe that iPSCs are likely to be used to make multiple clinical products. We further believe that the lines used as input material will be used at different sites and, given their immortal status, will be used for many years or even decades. Therefore, it will be important to develop assays to monitor the state of the cells and their drift in culture. We suggest that a detailed characterization of the initial status of the cells, a comparison with some calibration material and the development of reporter sublcones will help determine which set of tests will be most useful in monitoring the cells and establishing criteria for discarding a line.