Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Cx31 is assembled and trafficked to cell surface by ER-Golgi pathway and degraded by proteasomal or lysosomal pathways

Gap junctions, consisting of connexins, allow the exchange of small molecules (<1 kD) between adjacent cells, thusproviding a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Connexin 31 is amember of the connexin family. Mutations on connexin 31 are associated with erythrokeratodermia variabilis, hearingimpairment and peripheral neuropathy. However, the pathological mechanism for connexin 31 mutants in these diseasesare still unknown. In this study, we analyzed the assembly, trafficking and metabolism of connexin 31 in HeLa cellsstably expressing connexin 31. Calcein transfer assay showed that calcein transfer was inhibited when cells weretreated with Brefeldin A or cytochalasin D, but not when treated with nocodazole or α-glycyrrhetinic acid, suggestingthat Golgi apparatus and actin filaments, but not microtubules, are crucial to the trafficking and assembly of connexin31, as well as the formation of gap junction intercellular communication by connexin 31. Additionally, α-glycyrrhetinicacid did not effectively inhibit gap junctional intercellular communication formed by connexin 31. Pulse-chase assayrevealed that connexin 31 had a half-life of about 6 h. Moreover, Western blotting and fluorescent staining demonstratedthat in HeLa cells stably expressing connexin 31, the amount of connexin 31 was significantly increased after these cellswere treated with proteasomal or lysosomal inhibitors. These findings indicate that connexin 31 was rapidly renewed,and possibly degraded by both proteasomal and lysosomal pathways.