Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Tongji University

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.

Proliferation rate of somatic cells affects reprogramming efficiency.

La d&eacute;couverte de cellules souches (iPS) pluripotentes induites fournit non seulement de nouvelles approches pour la th&eacute;rapie de remplacement de cellules, mais aussi de nouvelles voies pour le d&eacute;pistage des drogues. Toutefois, le m&eacute;canisme de l&#39;ind&eacute;fini et l&#39;efficacit&eacute; relativement faible de reprogrammation ont limit&eacute; l&#39;application des cellules iPS. Dans le but d&#39;optimiser la condition reprogrammation, nous avons observ&eacute; inattendue que Retrait de c-Myc de l&#39;Oct-4, 2-Sox, Klf-4 et combinaison de c-Myc (OSKM) grandement am&eacute;lior&eacute; la g&eacute;n&eacute;ration de cellules iPS. Les cellules iPS g&eacute;n&eacute;r&eacute;s sans c-Myc atteint caract&eacute;ristiques saillantes pluripotentes et &eacute;taient capables de produire des souris &agrave; terme par le biais de compl&eacute;mentation t&eacute;traplo&iuml;de. Nous avons observ&eacute; qui a forc&eacute; l&#39;expression de c-Myc induit l&#39;expression de nombreux g&egrave;nes impliqu&eacute;s dans le contr&ocirc;le du cycle cellulaire et un &eacute;tat d&#39;hyperprolif&eacute;ration des fibroblastes embryonnaires de souris durant le stade pr&eacute;coce de la reprogrammation. Ce renforc&eacute; la prolif&eacute;ration des fibroblastes embryonnaires de souris corr&eacute;l&eacute;e n&eacute;gativement &agrave; l&#39;efficacit&eacute; globale de reprogrammation. En appliquant de petites mol&eacute;cules inhibitrices de la prolif&eacute;ration des cellules au stade pr&eacute;coce de la reprogrammation, nous &eacute;tions en mesure d&#39;am&eacute;liorer l&#39;efficacit&eacute; de la g&eacute;n&eacute;ration de cellules iPS m&eacute;di&eacute;e par l&#39;OSKM. Nos donn&eacute;es ont d&eacute;montr&eacute; que le taux de prolif&eacute;ration de la cellule somatique joue un r&ocirc;le crucial dans la reprogrammation. Ralentir la prolif&eacute;ration des cellules originales peut &ecirc;tre b&eacute;n&eacute;fique pour l&#39;induction de cellules iPS.

Taux de prolif&eacute;ration des cellules somatiques affecte l&#39;efficacit&eacute; reprogrammation.

La scoperta di staminali pluripotenti indotte (iPS) fornisce non solo nuovi approcci per la terapia sostitutiva delle cellule, ma anche nuovi modi per lo screening di stupefacenti. Tuttavia, il meccanismo definito e relativamente bassa efficienza della riprogrammazione hanno limitato l&#39;applicazione delle cellule iPS. Nel tentativo di ottimizzare ulteriormente la condizione di riprogrammazione, inaspettatamente abbiamo osservato che rimozione di c-Myc da Oct-4, Sox-2, Klf-4 e c-Myc (OSKM) combinazione notevolmente migliorata la generazione di cellule iPS. Le cellule iPS generate senza c-Myc ha raggiunto le caratteristiche salienti pluripotenti e furono in grado di produrre topi di pieno-termine attraverso tetraploidi complementazione. Abbiamo osservato che ha costretto l&#39;espressione di c-Myc induce l&#39;espressione di molti geni coinvolti nel controllo del ciclo cellulare e uno stato di iperproliferazione dei fibroblasti embrionali del mouse durante la fase iniziale di riprogrammazione. Questa aumentata proliferazione dei fibroblasti embrionali del mouse correlata negativamente per l&#39;efficienza complessiva di riprogrammazione. Applicando piccole molecole inibitori della proliferazione delle cellule nella fase precoce della riprogrammazione, siamo stati in grado di migliorare l&#39;efficienza della generazione di cellule iPS mediata da OSKM. I nostri dati hanno dimostrato che il tasso di proliferazione della cellula somatica svolge un ruolo critico nella riprogrammazione. Rallentare la proliferazione delle cellule originali potrebbe essere vantaggioso per l&#39;induzione di cellule iPS.

Tasso di proliferazione delle cellule somatiche influisce sulla efficienza di riprogrammazione.

G protein-coupled receptor 3 (GPR3) is an orphan G protein-coupled receptor (GPCR) predominantly expressed in mammalian brain and oocytes. GPR3 plays important roles in these two organs and is known as a Gαs-coupled receptor-activated constitutively in cells. However, the signal transduction pathway and pharmacological function of GPR3 remain unclear because of the lack of a specific ligand. By use of a human embryonic kidney 293 cell line stably expressing FLAG-GPR3-green fluorescent protein, a chemical screening for GPR3 ligands was performed using homogeneous time-resolved fluorescence cAMP assay. Diphenyleneiodonium chloride (DPI) was identified as a novel agonist of GPR3 with weak or no cross-reactivity with other GPCRs. DPI was further characterized to activate several GPR3-mediated signal transduction pathways, including Ca(2+) mobilization, cAMP accumulation, membrane recruitment of β-arrestin2, and receptor desensitization. Parallel studies revealed that the activity of DPI is much more pronounced than sphingosine 1-phosphate, a previously reported GPR3 agonist. Our study identified a novel and specific agonist of GPR3, which provides a useful tool for further study of this orphan GPCR. 

Identification of a novel small-molecule agonist for human G protein-coupled receptor 3.

The revolutionary induced pluripotent stem cell (iPSC) technology provides a new means for cell replacement therapies and drug screening. Small molecule compounds have been found extremely useful to improve the generation of iPSCs and understand the reprogramming mechanism. Here we report the identification of a novel chemical, CYT296, which improves OSKM-mediated induction of iPSCs for >10 folds and enables efficient reprogramming with only Oct4 in combination with other small molecules. The derived iPSCs are genuinely pluripotent and support the development of two 'All-iPSC' mice by tetraploid complementation. CYT296 profoundly impacts heterochromatin formation without affecting cell viability. MEFs treated with CYT296 exhibit de-condensed chromatin structure with markedly reduced loci containing heterochromatin protein 1α (HP1α) and H3K9me3, which is very similar to the chromatin configuration in embryonic stem cells (ESCs). Given that an open chromatin structure serves as a hallmark of pluripotency and has to be acquired to fulfill reprogramming, we propose that CYT296 might facilitate this process by disrupting condensed chromatin, thereby creating a more favorable environment for reprogramming. In agreement of this idea, shRNA targeting HP1α also promotes the generation of iPSCs. Thus current findings not only provide a novel chemical for efficient iPSC induction, but also suggest a new approach to regulate somatic cell reprogramming by targeting chromatin de-condensation with small molecules. 

Small molecule compound induces chromatin de-condensation and facilitates induced pluripotent stem cell generation.

Methuselah regulates longevity via dTOR: a pathway revealed by small-molecule ligands.

G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth, death, movement, transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study, we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol, we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially, the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted, whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4, knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together, our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine.

CXCR4 activation promotes differentiation of human embryonic stem cells to neural stem cells.

G protein-coupled receptors (GPCRs) are important modulators of many physiological functions and excellent drug targets for many diseases. However, to study the functions of endogenous GPCRs is still a challenging task, partially due to the low expression level of GPCRs and the lack of highly potent and selective GPCR antibodies. Overexpression or knock-in of tagged GPCRs, or knockout of specific GPCRs in mice, are common strategies used to study the in vivo functions of these receptors. However, generating separate mice carrying tagged GPCRs or conditional alleles for GPCRs is labor intensive, and requires additional breeding costs. Here we report the generation of mice carrying an HA-tagged DOR (delta opioid receptor) flanked by LoxP sequences at the endogenous DOR locus using a single recombination step, aided by the TALEN system. These animals can be used directly to study the expression, localization, protein-protein interaction and signal transduction of endogenous DOR using anti-HA antibodies. By crossing with mice expressing tissue-specific Cre, these mice can also generate offspring with DOR knockout within specific tissues. These mice are powerful tools to study the in vivo functions of DOR. Furthermore, the gene modification strategy could also be used to study the functions of many other GPCRs.

One-step generation of mice carrying a conditional allele together with an HA-tag insertion for the delta opioid receptor.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease with chronic inflammatory demyelination of the CNS. Experimental autoimmune encephalomyelitis (EAE) is an important animal model to study MS, with many pathological phenomena similar to MS. Th17 cells are important regulators of EAE and MS pathogenesis. Most cytokines needed for Th cell development are secreted by APCs, such as dendritic cells (DCs). Consequently, MS could be improved by inhibiting cytokine secretion from DCs. In this study, we reported that chlorzoxazone could ameliorate EAE pathogenesis via inhibiting IL-6 production by DCs. The EAE signs in the chlorzoxazone-treated group of mice were relieved, which was mainly manifested as lower clinical scores, a decrease in the number of immune cells, and a reduction of demyelination in the CNS. Moreover, the proportion of Th17 cells in the spleen and CNS decreased significantly. In vitro experiments showed that chlorzoxazone treatment significantly reduced DC-derived IL-6 production. In the DC-T cell coculture experiment, significantly decreased Th17 differentiation was observed after chlorzoxazone treatment. In addition, mass spectrometric analysis was performed to elucidate the mechanism by which chlorzoxazone affected EAE and DC function. We showed that the effect of chlorzoxazone on inhibiting the secretion of IL-6 by DCs may be mediated via the AMP-activated protein kinase pathway. Overall, our study elucidated the key role of chlorzoxazone in regulating EAE pathogenesis and suggested that it might be used as a new drug for MS patients.

Chlorzoxazone Alleviates Experimental Autoimmune Encephalomyelitis via Inhibiting IL-6 Secretion of Dendritic Cells.

Emerging data have highlighted the coexistence of multiple sclerosis (MS) and Alzheimer's disease (AD), both of which are common central nervous system degenerative diseases with a heavy burden on patients, their families, and society. However, it is unclear how MS progresses under an AD pathological background. We aimed to address the question of how MS progresses under an AD pathological background. We induced the experimental autoimmune encephalomyelitis (EAE) model of MS in two types of AD mouse models, Tg6799 and APP/PS1 mice. We found that, compared with wild-type mice, the clinical symptoms of EAE were significantly ameliorated in APP/PS1 mice but not in Tg6799 mice. Moreover, a much lower level of serum Aβ was observed in Tg6799 mice. EAE clinical symptoms in Tg6799 and C57BL/6J mice were ameliorated by intraperitoneal injection of Aβ42. Peripheral administration of Aβ42 peptides was able to inhibit Th17 development in vivo, which is likely to occur through the inhibition of IL-6 production in dendritic cells. Our findings revealed that AD and EAE could coexist in the same mouse, and Aβ residing in peripheral circulation likely plays an anti-inflammatory role in preventing EAE progression. These findings reveal the potential benefit of Aβ, one of the supervillains of AD, at least in certain contexts.

Qiuhong Hua

Putuo People's Hospital,
Shanghai Key Laboratory of Signaling and Disease Research,
School of Life Sciences and Technology,
Putuo People's Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology

Tongji University

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis