Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR (MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane is a potential strategy to improve opioid analgesic therapies.

Morphine-induced analgesia and antinociceptive tolerance are known to be modulated by interaction between δ-opioid receptors (DORs) and μ-opioid receptors (MORs) in the pain pathway. However, evidence for expression of DORs in nociceptive small-diameter neurons in dorsal root ganglia (DRG) and for coexistence of DORs with MORs and neuropeptides has recently been challenged. We now report, using in situ hybridization, single-cell PCR, and immunostaining, that DORs are widely expressed not only in large DRG neurons but in small ones and coexist with MORs in peptidergic small DRG neurons, with protachykinin-dependent localization in large dense-core vesicles. Importantly, both DOR and MOR agonists reduce depolarization-induced Ca 2+ currents in single small DRG neurons and inhibit afferent C-fiber synaptic transmission in the dorsal spinal cord. Thus, coexistence of DORs and MORs in small DRG neurons is a basis for direct interaction of opioid receptors in modulation of nociceptive afferent transmission and opioid analgesia.

Opioid and tachykinin systems are involved in modulation of pain transmission in the spinal cord. Regulation of surface opioid receptors on nociceptive afferents is critical for opioid analgesia. Plasma-membrane insertion of delta-opioid receptors (DORs) is induced by stimulus-triggered exocytosis of DOR-containing large dense-core vesicles (LDCVs), but how DORs become sorted into the regulated secretory pathway is unknown. Here we report that direct interaction between protachykinin and DOR is responsible for sorting of DORs into LDCVs, allowing stimulus-induced surface insertion of DORs and DOR-mediated spinal analgesia. This interaction is mediated by the substance P domain of protachykinin and the third luminal domain of DOR. Furthermore, deletion of the preprotachykinin A gene reduced stimulus-induced surface insertion of DORs and abolished DOR-mediated spinal analgesia and morphine tolerance. Thus, protachykinin is essential for modulation of the sensitivity of nociceptive afferents to opioids, and the opioid and tachykinin systems are directly linked by protachykinin/DOR interaction.

The breakthrough of induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific iPSCs may become a renewable source of autologous cells for cell therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood. Using a humanized mouse model (denoted Hu-mice) reconstituted with a functional human immune system, we demonstrate that most teratomas formed by autologous integration-free hiPSCs exhibit local infiltration of antigen-specific T cells and associated tissue necrosis, indicating immune rejection of certain hiPSC-derived cells. In this context, autologous hiPSC-derived smooth muscle cells (SMCs) appear to be highly immunogenic, while autologous hiPSC-derived retinal pigment epithelial (RPE) cells are immune tolerated even in non-ocular locations. This differential immunogenicity is due in part to abnormal expression of immunogenic antigens in hiPSC-derived SMCs, but not in hiPSC-derived RPEs. These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration.

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.3D hydrogels | neuronal migration and maturation | 3D RTT modeling N euronal migration and maturation is a key step in brain development. Defects in this process have been implicated in many disorders, including autism (1) and schizophrenia (2). Thoroughly understanding how neural progenitor cell (NPC) migration is affected in neurodevelopmental disorders requires a means of dissecting the process using cells with genetic alterations matching those in patients. Existing in vitro assays of migration generally involve measurement of cell movement across a scratch or gap or through a membrane toward a chemoattractant in 2D culture systems. Although widely used, such assays may not accurately reveal in vivo differences, as neuronal migration is tightly regulated by physical and chemical cues in the extracellular matrix (ECM) that NPCs encounter as they migrate.In vitro 3D culture systems offer a solution to these limitations (3-7). Compared with 2D culture, a 3D arrangement allows neuronal cells to interact with many more cells (4); this similarity to the in vivo setting has been shown to lengthen viability, enhance survival, and allow formation of longer neurites and more dense networks in primary neurons in uniform matrices or aggregate culture (8, 9). Indeed, 3D culture systems have been used to study nerve regeneration, neuronal and glial development (10-12), and amyloid-β and tau pathology (13). Thus, measuring neuronal migration through a soft 3D matrix would continue this trend toward using 3D systems to study neuronal development and pathology.We sought to develop a 3D assay to examine potential migration and neuronal maturation defects in Rett syndrome (RTT), a genetic neurodevelopmental disorder that affects 1 in 10,000 children in the United States and is caused by mutations in the X-linked methyl-CpG-binding protein-2 (MECP2) gene (14). Studies using induced pluripotent stem cells (iPSCs) from RTT patients in traditiona...

Oct4 is critical to maintain the pluripotency of human embryonic stem cells (hESCs), however, the underlying mechanism remains to be fully understood. Here we report that silencing of Oct4 in hESCs leads to the activation of tumor suppressor p53, inducing the differentiation of hESCs since acute disruption of p53 in p53 conditional knockout (p53CKO) hESCs prevents the differentiation of hESCs after Oct4 depletion. We further discovered that the silencing of Oct4 significantly reduces the expression of Sirt1, a deacetylase known to inhibit p53 activity and the differentiation of ESCs, leading to increased acetylation of p53 at lysine 120 and 164. The importance of Sirt1 in mediating Oct4-dependent pluripotency is revealed by the finding that the ectopic expression of Sirt1 in Oct4-silenced hESCs prevents p53 activation and hESC differentiation. In addition, employing knock-in approach, we revealed that the acetylation of p53 at lysine 120 and 164 is required for both stabilization and activity of p53 in hESCs. In summary, our findings reveal a novel role of Oct4 in maintaining the pluripotency of hESCs by suppressing pathways that induce differentiation. Considering that p53 suppresses pluripotency after DNA damage response in ESCs, our findings further underscore the stringent mechanism to coordinate DNA damage response pathways and pluripotency pathways in order to maintain the pluripotency and genomic stability of hESCs.

Voltage-gated Na+ channel (Nav) 1.8 contributes to the majority of the Na+ current that underlies the depolarizing phase of action potentials. Nav1.8 is mainly distributed intracellularly and its current amplitude can be enhanced by the β3 subunit. However, little is known about the mechanisms underlying its intracellular retention and the effects mediated by the β3 subunit. Here, we show that the β3 subunit promotes surface expression of Nav1.8 by masking its endoplasmic reticulum (ER)-retention/retrieval signal. The RRR motif in the first intracellular loop of Nav1.8 is responsible for retaining Nav1.8 in the ER and restricting its surface expression. The β3 subunit facilitates surface expression of Nav1.8. The intracellular C-terminus of the β3 subunit interacts with the first intracellular loop of Nav1.8 and masks the ER-retention/retrieval signal. Mutation of the RRR motif results in a significant increase in surface expression of Nav1.8 and abolishes the β3-subunit-mediated effects. Thus, the β3 subunit regulates surface expression of Nav1.8 by antagonizing its ER-retention/retrieval signal. These results reveal a novel mechanism for the effect of the Na+ channel β subunits on the α subunits.