Characterization of the human ESC transcriptome by hybrid sequencing.

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, full-length mRNA isoforms are not captured. On the other hand, third-generation sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.

Derivation of GMP-compliant integration-free hiPSCs using modified mRNAs.

The clinical use of human induced pluripotent stem cells (hiPSCs) and the development of patients-specific gene and cell therapies rely on the development of fast, reliable, and integration-free methods of derivation of pluripotent stem cells from somatic tissues. Here we describe an integration-free protocol for the rapid derivation of hiPSCs from dermal fibroblasts using modified mRNAs. This method is inexpensive, highly efficient, and makes use of reagents that are xeno-free and chemically defined and can therefore be adopted by any Good Manufacturing Practice (GMP) facility.