Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

ABSTRACT: Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue-culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with Inter-Î±-inhibitor (IÎ±I), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IÎ±I efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IÎ±I-supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. SNP genotyping with Illumina HumanOmniExpressExome-array version 8v1-2_A, encompassing >964.000 single nucleotide polymorphism (SNP)-markers, were performed by the SNP&SEQ technology Platform in Uppsala (www.genotyping.se), according to the manufacturerâ??s instructions. Each of the four hPS cell lines (i.e. H181, H207, HUES1 and K2C) were analyzed in three separate SNP-array experiments for the 12 samples described here. A first analysis was performed after 2-5 passages of growth for initial reference (4 samples), and subsequently, two analyses were performed per cell line after 16-21 passages in E8:VN or E8:IÎ±I culture (8 samples). The CN-calls of the autosomal chromosomes were illustrated using the Nexus-software.

ORGANISM(S): Homo sapiens

SUBMITTER: Sara Pijuan-GalitÃ³

PROVIDER: E-GEOD-82103 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: de novo sequencing and comparative analysis of the bark transciptomes of Hevea brasiliensis induced without ethephon (C), with ethephon for 8 hours (E8) and 24 hours (E24) to identify the genes and pathways related to the stimulation of rubber production by ethylene. The goals of this study are to reveal the molecular mechanism behind the stimulation of rubber production by ethylene. Methods: Bark RNA was extracted using the TRIzolÂ® Reagent (Invitrogen) and two cDNA libraries, H (healthy rubber trees) and T (TPD-affected trees), were prepared using the mRNA-Seq 8 sample prep Kit (Illumina). The libraries were deep sequenced using Illumina HiSeqTM 2000 (Illumina Inc., San Diego, CA, USA). Raw reads produced from sequencing machines were resorted to de novo assembly and gene annotation. Results: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 hours (E8) and 24 hours (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. Conclusions: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree. De novo sequencing of the transcriptomes of C (bark without ethephon application), E8 (bark with 1.5%-ethephon treatment for 8 hours) and E24 (bark with 1.5%-ethephon treatment for 24 hours) rubber trees was conducted using Illumina HiSeq 2000.

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Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

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