Data for whole and mitochondrial proteome of human embryonic stem cells.

The data presented here pertain to the research article entitled "Proteome Analysis of Human Embryonic Stem Cell Organelles" (Shekariet al., 2017 [1]). In the present article we endeavour to locate new proteins and pathways in human embryonic stem cells (hESCs) by mass spectrometry and bioinformatics analysis. We have analyzed total and mitochondrial proteins extracted from three biological replicates of the hESC H9 cell line according to mass spectrometry proteomics and bioinformatics investigations.

Proteome analysis of human embryonic stem cells organelles.

As the functions of proteins are associated with their cellular localization, the comprehensive sub-cellular proteome knowledge of human embryonic stem cells (hESCs) is indispensable for ensuring a therapeutic effect. Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices. Among ten enriched signaling pathways highlighted in our results, non-canonical Wnt signaling were analyzed in greater detail. The functions of three novel hESC membrane proteins (ERBB4, GGT1 and ZDHHC13) have been assessed in terms of pluripotency. Our report is the most comprehensive for organellar proteomics of hESCs. SIGNIFICANCE: Mass spectrometric identification of proteins using a TripleTOF 5600 from nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomal fractions highlighted the significance of the non-canonical Wnt signaling in human embryonic stem cells.

Substrate-mediated commitment of human embryonic stem cells for hepatic differentiation.

Human embryonic stem cell (hESC)-derived endodermal cells are of interest for the development of cellular therapies to treat disorders such as liver failure. The soluble form of activin A (Act) has been widely used as an in vitro inducer of definitive endoderm (DE). In this study, we have developed a nanofibrous poly (ɛ-caprolactone) substrate, biofunctionalized with Act, for directed differentiation of hESCs into DE. Bioconjugation of Act on nanofibrous meshes was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunostaining. In order to investigate the bioactivity of immobilized Act (iAct), hESCs were cultivated on the Act-conjugated nanofibers for five days. The nanofibers with covalent iAct significantly increased expression levels of the endodermal markers SOX17, FOXA2, and CXCR4, compared with physically adsorbed Act (aAct) or without Act (noAct). In addition, iAct retained its bioactivity after storage for five days in the absence of cell seeding. The capability of cultivated cells to generate the DE-derived lineage was evaluated through further differentiation of seeded cells into hepatocyte-like cells (HLCs). Interestingly, the iAct sample showed a higher level of hepatic markers compared to the aAct sample. We also demonstrated that iAct in the presence of soluble Act (sAct) could improve the conventional protocol to generate HLCs from hESCs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2861-2872, 2016.

Screening of common CYP1B1 mutations in Iranian POAG patients using a microarray-based PrASE protocol.

PURPOSE: The gene coding cytochrome P4501B1 (CYP1B1) has been shown to be a major cause of primary congenital glaucoma in the Iranian population. More recently it was shown to also be important in juvenile-onset open angle glaucoma (JOAG). We aimed to further investigate the role of CYP1B1 in a larger cohort of primary open angle glaucoma (POAG) patients which included late-onset patients. We also aimed to set up a microarray based protocol for mutation screening with an intent of using the protocol in a future population level screening program. METHODS: Sixty three POAG patients, nine affected family members, and thirty three previously genotyped primary congenital glaucoma (PCG) patients were included in the study. Clinical examination included slit lamp biomicroscopy, IOP measurement, gonioscopic evaluation, fundus examination, and measurement of perimetry. G61E, R368H, R390H, and R469W were screened by a protocol that included multiplexed allele specific amplification in the presence of a protease (PrASE), use of sequence tagged primers, and hybridization to generic arrays on microarray slides. The entire coding sequences of CYP1B1 and myocilin (MYOC) genes were sequenced in all individuals assessed by the microarray assay to carry a mutation. Intragenic single nucleotide polymorphism (SNP) haplotpes were determined for mutated alleles. RESULTS: Genotypes assessed by the array-based PrASE methodology were in 100% concordance with sequencing results. Seven mutation carrying POAG patients (11.1%) were identified, and their distribution was quite skewed between the juvenile-onset individuals (5/21) as compared to late-onset cases (2/42). Four of the seven mutation carrying Iranian patients harbored two mutated alleles. CYP1B1 mutated alleles in Iranian PCG and POAG patients shared common haplotypes. MYOC mutations were not observed in any of the patients. CONCLUSIONS: The PrASE approach allowed reliable simultaneous genotyping of many individuals. It can be an appropriate tool for screening common mutations in large sample sizes. The results suggest that CYP1B1 is implicated in POAG among Iranians, notably in the juvenile-onset form. Contrary to POAG patients studied in other populations, many mutation harboring Iranian patients carry two mutated alleles. We propose an explanation for this observation.