Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells.

The complex neuromuscular network that controls body movements is the target of severe diseases that result in paralysis and death. Here, we report the development of a robust and efficient self-organizing neuromuscular junction (soNMJ) model from human pluripotent stem cells that can be maintained long-term in simple adherent conditions. The timely application of specific patterning signals instructs the simultaneous development and differentiation of position-specific brachial spinal neurons, skeletal muscles, and terminal Schwann cells. High-content imaging reveals self-organized bundles of aligned muscle fibers surrounded by innervating motor neurons that form functional neuromuscular junctions. Optogenetic activation and pharmacological interventions show that the spinal neurons actively instruct the synchronous skeletal muscle contraction. The generation of a soNMJ model from spinal muscular atrophy patient-specific iPSCs reveals that the number of NMJs and muscle contraction is severely affected, resembling the patient's pathology. In the future, the soNMJ model could be used for high-throughput studies in disease modeling and drug development. Thus, this model will allow us to address unmet needs in the neuromuscular disease field.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity.

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-ß1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-ß1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-ß1 and reduced their proliferative capacity in a TGF-ß1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker.

CD105 (endoglin)-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities.

Administration of in vitro expanded mesenchymal stromal cells (MSCs) represents a promising therapy for regenerative medicine and autoimmunity. Both mouse and human MSCs ameliorate autoimmune disease in syn-, allo- and xenogeneic settings. However, MSC preparations are heterogeneous which impairs their therapeutic efficacy and endorses variability between experiments. This heterogeneity has also been a main hurdle in translating experimental MSC data from mouse models to human patients. The objective of the present manuscript has been to further characterize murine MSCs (mMSCs) with the aim of designing more efficient and specific MSC-based therapies. We have found that mMSCs are heterogeneous for endoglin (CD105) expression and that this heterogeneity is not due to different stages of MSC differentiation. CD105 is induced on a subpopulation of mMSCs early upon in vitro culture giving rise to CD105(+) and CD105(-) MSCs. CD105(+) and CD105(-) mMSCs represent independent subpopulations that maintain their properties upon several passages. CD105 expression on CD105(+) mMSCs was affected by passage number and cell confluency while CD105(-) mMSCs remained negative. The CD105(+) and CD105(-) mMSC subpopulations had similar growth potential and expressed almost identical mMSC markers (CD29(+)CD44(+)Sca1 (+) MHC-I(+) and CD45(-)CD11b(-)CD31(-)) but varied in their differentiation and immunoregulatory properties. Interestingly, CD105(-) mMSCs were more prone to differentiate into adipocytes and osteocytes and suppressed the proliferation of CD4(+) T cells more efficiently compared to CD105(+) mMSCs. Based on these studies we propose to redefine the phenotype of mMSCs based on CD105 expression.

Nicotinamide inhibits the lysosomal cathepsin b-like protease and kills African trypanosomes.

Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease.

Mesenchymal stem cells expressing vasoactive intestinal peptide ameliorate symptoms in a model of chronic multiple sclerosis.

Multiple sclerosis (MS) is a severe debilitating disorder characterized by progressive demyelination and axonal damage of the central nervous system (CNS). Current therapies for MS inhibit the immune response and demonstrate reasonable benefits if applied during the early phase of relapsing­remitting MS (RRMS) while there are no treatments for patients that progress neither to the chronic phase nor for the primary progressive form of the disease. In this manuscript, we have studied the therapeutic efficacy of a cell and gene therapy strategy for the treatment of a mouse model of chronic MS [myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)]. We used allogenic mesenchymal stem cells (MSCs) asa therapeutic tool and also as vehicle to deliver fully processed 3.3-kDa vasoactive intestinal peptide (VIP) to the peripheral immune organs and to the inflamed CNS. Intraperitoneal administrations of MSCs expressing VIP stopped progression and reduced symptoms when administered at peak of disease. The improvement in clinical score correlated with diminished peripheral T-cell responses against MOG as well as lower inflammation,lower demyelination, and higher neuronal integrity in the CNS. Interestingly, neither lentiviral vectors expressing VIP nor unmodified MSCs were therapeutic when administer at the peak of disease. The increased therapeutic effect of MSCs expressing VIP over unmodified MSCs requires the immunoregulatory and neuroprotective roles of both VIP and MSCs and the ability of the MSCs to migrate to peripheral lymph organs and the inflamed CNS.

Alterations in DRBD3 ribonucleoprotein complexes in response to stress in Trypanosoma brucei.

Regulation of RNA polymerase II transcription initiation is apparently absent in trypanosomes. Instead, these eukaryotes control gene expression mainly at the post-transcriptional level. Regulation is exerted through the action of numerous RNA-binding proteins that modulate mRNA processing, turnover, translation and localization. In this work we show that the RNA-binding protein DRBD3 resides in the cytoplasm, but localizes to the nucleus upon oxidative challenge and to stress granules under starvation conditions. DRBD3 associates with other proteins to form a complex, the composition of which is altered by cellular stress. Interestingly, target mRNAs remain bound to DRBD3 under stress conditions. Our results suggest that DRBD3 transports regulated mRNAs within the cell in the form of ribonucleoprotein complexes that are remodeled in response to environmental cues.

High doses of 4-aminopyridine improve functionality in chronic complete spinal cord injury patients with MRI evidence of cord continuity.

BACKGROUND AND AIMS: Many patients with complete spinal cord injury (SCI) exhibit demyelinated and poorly myelinated nerve fibers traversing the lesion site. Conventional doses of 4-aminopyridine (4-AP, 30 mg/day) have shown to provide no or minor functional improvement in these patients. We undertook this study to test the functional effect of high doses of 4-AP on patients with chronic complete SCI with cord continuity at the site of injury demonstrated by magnetic resonance imaging. METHODS: Fourteen patients were included in a double-blind, randomized, placebo-controlled trial followed by an open label long-term follow-up. Initially, patients received 4-AP or placebo orally, with 4-AP being increased gradually (5 mg/week) to reach 30 mg/day. For long-term treatment, 4-AP was increased 10 mg periodically according to negative electroencephalogram and blood test abnormalities and minor adverse reactions. Pre-treatment, 12 and 24 weeks of the controlled trial, and 6 and 12 months of open trial evaluations, or with the highest doses reached were obtained. RESULTS: Three of 12 patients were able to walk with the assistance of orthopedic devices, 1/12 became incomplete (AIS B), 7/12 improved their somatosensory evoked potentials, 5/12 had sensation and control of bladder and anal sphincters, and 4/9 male patients had psychogenic erection. CONCLUSIONS: Positive changes were seen mainly in patients with cyst (4/5) or atrophy (3/5) of the injury site. Two patients withdrew from the study: one had seizures and one had intolerant adverse reactions. We conclude that high doses of 4-AP in the studied population produced several functional benefits not observed using lower doses.

Exosomes from human lymphoblastoid B cells express enzymatically active CD38 that is associated with signaling complexes containing CD81, Hsc-70 and Lyn.

Exosome vesicles of endocytic origin are involved in communication between tumor and immune cells. In addition, membrane rafts (MR) may support the sorting of proteins associated with exosomes. CD38 is found at the plasma membrane and in recycling endosomes, which are both redistributed toward the immunological synapse (IS) upon T cell antigen receptor (TCR) engagement. The data of this study provide evidence that CD38 is expressed on the surface of secreted exosomes derived from lymphoblastoid B cells. Exosomic CD38 is associated with the signaling molecules CD81, Hsc-70 and Lyn. Likewise, in MR, CD38 is associated with CD81, CD19, Lyn, Galphai-2, Hsc-70 and actin. Therefore, a high degree of overlap in the pattern of signaling proteins associated with CD38 in exosomes and MR exists. Exosomic and MR CD38, by virtue of these interactions, have signaling potential. Indeed, CD38 is enzymatically active in both exosomes and MR, and CD38 ligation induces Akt/PKB and Erk activation, which is accompanied by increased translocation of CD38 into MR. In conclusion, the present study indicates that CD38 localizes to MR, where it promotes cell signaling, and it is exported out of the cells through the exosome-mediated exocytic pathway, where it may act as an intercellular messenger.

Antigen-induced clustering of surface CD38 and recruitment of intracellular CD38 to the immunologic synapse.

During immunologic synapse (IS) formation, human CD38 redistributes to the contact area of T cell-antigen-presenting cell (APC) conjugates in an antigen-dependent manner. Confocal microscopy showed that CD38 preferentially accumulated along the contact zone, whereas CD3-zeta redistributed toward the central zone of the IS. APC conjugates with human T cells or B cells transiently expressing CD38-green fluorescent protein revealed the presence of 2 distinct pools of CD38, one localized at the cell membrane and the other in recycling endosomes. Both pools were recruited to the T/APC contact sites and required antigen-pulsed APCs. The process appeared more efficient in T cells than in APCs. CD38 was actively recruited at the IS of T cells by means of Lck-mediated signals. Overexpression of CD38 in T cells increased the levels of antigen-induced intracellular calcium release. Opposite results were obtained by down-regulating surface CD38 expression by means of CD38 siRNA. CD38 blockade in influenza HA-specific T cells inhibited IL-2 and IFN-gamma production, PKC phosphorylation at Thr538, and PKC recruitment to the IS induced by antigen-pulsed APCs. These results reveal a new role for CD38 in modulating antigen-mediated T-cell responses during IS formation.