A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells.

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.

Promotion of direct reprogramming by transformation-deficient Myc.

Induced pluripotent stem cells (iPSCs) are generated from mouse and human fibroblasts by the introduction of three transcription factors: Oct3/4, Sox2, and Klf4. The proto-oncogene product c-Myc markedly promotes iPSC generation, but also increases tumor formation in iPSC-derived chimeric mice. We report that the promotion of iPSC generation by Myc is independent of its transformation property. We found that another Myc family member, L-Myc, as well as c-Myc mutants (W136E and dN2), all of which have little transformation activity, promoted human iPSC generation more efficiently and specifically compared with WT c-Myc. In mice, L-Myc promoted germline transmission, but not tumor formation, in the iPSC-derived chimeric mice. These data demonstrate that different functional moieties of the Myc proto-oncogene products are involved in the transformation and promotion of directed reprogramming.

The organization of histone H3 modifications as revealed by a panel of specific monoclonal antibodies.

Histone modifications play critical roles in the epigenetic regulation of gene expression and in the maintenance of genome integrity. Acetylation and methylation of histone H3 are particularly important in gene activation and silencing. We generated and characterized a panel of mouse monoclonal antibodies that specifically recognize different modifications on K4, K9, and K27 residues on histone H3. By using these antibodies for chromatin immunoprecipitation and immunoblotting, we analyzed the relationship between different modifications in nearby nucleosomes in human cells. Within a few nucleosome neighbors, trimethyl-K4 was associated with acetyl-K27, rather than with dimethyl-K4 and acetyl-K9, consistent with their co-localization on active promoters. Furthermore, simultaneous immunofluorescence using directly-labeled antibodies revealed that di- and tri-methylation on K4 was diminished during replicative senescence. These highly-reliable and fully-characterized monoclonal antibodies may facilitate future epigenomic studies on healthy and diseased cells.

Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.

Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc(-) iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.

A novel histone exchange factor, protein phosphatase 2Cgamma, mediates the exchange and dephosphorylation of H2A-H2B.

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A-H2B, we identified protein phosphatase (PP) 2C gamma subtype (PP2Cgamma/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A-H2B. The disruption of PP2Cgamma in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cgamma-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.

Sedative and anticonvulsant activities of goodyerin, a flavonol glycoside from Goodyera schlechtendaliana.

Goodyerin is a flavonol glycoside isolated from the whole plants of Goodyera schlechtendaliana which has been used as a substitute for the crude drug, Anoectochilus formosanus. The pharmacological properties of goodyerin were assayed for effects on spontaneous locomotor activity, on pentobarbital-induced hypnosis, and on anticonvulsant activity against picrotoxin-induced seizures in rodents. Goodyerin exhibited a significant and dose-dependent sedative and anticonvulsant effect.