Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.

Mutations of nuclear localization signals in mNANOG generate dominant negative mutants

Mouse NANOG plays a critical role in maintaining self-renewal and pluripotency of embryonic stem cells.Yet,the precise mechanism of how mNANOG functions is still less known.Here,we report that mouse NANOG has two nuclear localization signals(NLS,RKQKMR and RMKCKR) which are responsible for the nuclear localization and transcriptional activity in the conserved homeobox domain.NLS mutants of mouse NANOG generate 3 mutants that are localized throughout the cells and lose the transactivation function.We further prove that all three NLS mutants may interact with the wild-type mouse NANOG like NANOG dimerization itself and inhibit the wild-type mouse NANOG activity,acting as dominant negative mutants.The NLS mutants of mouse NANOG may also inhibit activity of oct4 promoter in pluripotent cells,indicating that the NLS mutants can affect the endogenous mouse NANOG function in vivo.These data suggest that the NLS mutants of mouse NANOG may be used as a tool to regulate NANOG activity in pluripotent cells.

PX domain and CD domain play different roles in localization and vacuolation of Sorting Nexin 10

Sorting nexins (SNXs) are PX domain containing proteins and essential for intracellular protein sorting, trafficking and signal transduction. The PX domains of SNXs can bind to various phosphorelated phosphoinositides (PIs) and target the host proteins to endosomes. Recently, we have reported that overexpression of SNX10 in mammalian cells could induce giant vacuoles. In this study, we aimed to identify regions in SNX10 critical for the vacuolation activity. We found that both the PX domain and the CD1 region were essential for vacuolation. We provided evidence that the PX domain was able to specifically bind to PtdIns(3)P and target SNX10 to endosomes. A mutation in the β1 region of the PX domain (V15A) disrupted the PtdIns(3)P binding ability and the endosomal localization of SNX10. However, correct subcellular localization alone was not sufficient for SNX10 to induce vacuoles. We found that the CD1 region, which was not required for the localization, was indispensable for the vacuolation activity of SNX10. In summary, both the PX domain and the CD1 region are necessary for SNX10 to induce vacuoles but they play different roles in this process.

The N-terminal domain is a transcriptional activation domain required for Nanog to maintain ES cell self-renewal

Nanog is a transcription factor identified by its ability to maintain the self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF). Nanog protein contains an N-terminal domain (ND), a DNA-binding homeobox domain (HD) and a C-terminal domain (CD). We previously reported that the CD in Nanog is a transcriptional activation domain essential for the in vivo function of Nanog. Here we demonstrated that the ND in Nanog is also functionally important. Deletion of the ND reduces the transcriptional activity of Nanog on either artificial reporters or native Nanog promoters. This truncated Nanog is also less effective in regulating the endogenous Nanog target genes. Furthermore, the ND truncation disrupted the ability of Nanog to maintain ES cell self-renewal as well. We found that the ND is not required for the nuclear localization of Nanog. These results suggest that the regulation of endogenous pluripotent genes such as oct3/4 and rex-1 is required for the in vivo function of Nanog.