Extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communications and exert various biological activities via delivering unique cargos of functional molecules such as RNAs and proteins to recipient cells. Previous studies showed that EVs produced and secreted by human mesenchymal stem cells (MSCs) can substitute intact MSCs for tissue repair and regeneration. In this study, we examined properties and functions of EVs from human induced pluripotent stem cells (iPSCs) that can be cultured infinitely under a chemically defined medium free of any exogenous EVs. We collected and purified EVs secreted by human iPSCs and MSCs. Purified EVs produced by both stem cell types have similar sizes (∼150 nm in diameter), but human iPSCs produced 16-fold more EVs than MSCs. When highly purified iPSC-EVs were applied in culture to senescent MSCs that have elevated reactive oxygen species (ROS), human iPSC-EVs reduced cellular ROS levels and alleviated aging phenotypes of senescent MSCs. Our discovery reveals that EVs from human stem cells can alleviate cellular aging in culture, at least in part by delivering intracellular peroxiredoxin antioxidant enzymes. Stem Cells 2019;37:779–790

The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs.

We have previously shown that blood astrocytic-origin extracellular vesicles (AEVs) from Alzheimer’s disease (AD) patients contain high complement levels. To test the hypothesis that circulating EVs from AD patients can induce complement-mediated neurotoxicity involving Membrane Attack Complex (MAC) formation, we assessed the effects of immunocaptured AEVs (using anti-GLAST antibody), in comparison with neuronal-origin (N)EVs (using anti-L1CAM antibody), and nonspecific CD81+ EVs (using anti-CD81 antibody), from the plasma of AD, frontotemporal lobar degeneration (FTLD), and control participants. AEVs (and, less effectively, NEVs) of AD participants induced Membrane Attack Complex (MAC) expression on recipient neurons (by immunohistochemistry), membrane disruption (by EthD-1 assay), reduced neurite density (by Tuj-1 immunohistochemistry), and decreased cell viability (by MTT assay) in rat cortical neurons and human iPSC-derived neurons. Demonstration of decreased cell viability was replicated in a separate cohort of autopsy-confirmed AD patients. These effects were not produced by CD81+ EVs from AD participants or AEVs/NEVs from FTLD or control participants, and were suppressed by the MAC inhibitor CD59 and other complement inhibitors. Our results support the stated hypothesis and should motivate future studies on the roles of neuronal MAC deposition and AEV/NEV uptake, as effectors of neurodegeneration in AD.

Although the majority of Alzheimer's disease (AD) cases are sporadic, about 5% of cases are inherited in an autosomal dominant pattern as familial AD (FAD) and manifest at an early age. Mutations in the presenilin 1 (PSEN1) gene account for the majority of early-onset FAD. Here, we describe the generation of virus-free human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients harboring the FAD PSEN1 mutation A246E and fibroblasts from healthy age-matched controls using nonintegrating episomal vectors. We have differentiated these hiPSC lines to the neuronal lineage and demonstrated that hiPSC-derived neurons have mature phenotypic and physiological properties. Neurons from mutant hiPSC lines express PSEN1-A246E mutations themselves and show AD-like biochemical features, that is, amyloidogenic processing of amyloid precursor protein (APP) indicated by an increase in β-amyloid (Aβ)42/Aβ40 ratio. FAD hiPSCs harboring disease properties can be used as humanized models to test novel diagnostic methods and therapies and explore novel hypotheses for AD pathogenesis.

Blood–brain barrier dysfunction is associated with many late-stage neurodegenerative diseases. An emerging question is whether the mutations associated with neurodegenerative diseases can independently lead to blood–brain barrier (BBB) dysfunction. Studies from patient-derived induced pluripotent stem cells suggest that mutations associated with neurodegenerative disease are non-cell autonomous, resulting in gain of toxic function in derived neurons and astrocytes. Here we assess whether selected mutations associated with neurodegenerative diseases can contribute to impairment of the blood–brain barrier. We assessed barrier function of confluent monolayers of human brain microvascular endothelial cells (hBMECs) derived from induced pluripotent stem cells (iPSC) from three healthy individuals and eight individuals with neurodegenerative disease. We systematically assessed protein and gene expression of BBB biomarkers, transendothelial resistance (TEER), permeability of Lucifer yellow, permeability of d-glucose, permeability of rhodamine 123, the efflux ratio of rhodamine 123, and P-gp inhibition using Tariquidar for confluent monolayers of human brain microvascular endothelial cell (hBMECs). We provide evidence supporting the hypothesis that mutations associated with neurodegenerative disease can independently cause BBB dysfunction. These functional changes are not catastrophic since barrier breakdown would result in BBB impairment during development. Synergistic interactions between non-cell autonomous cerebrovascular dysfunction and the effects of gain-of-toxic function in neurons (e.g. toxic oligomers) are likely to increase disease burden through a positive feedback mechanism. These results suggest that the accumulation of defects in brain microvascular endothelial cells may ultimately lead to impairment of the BBB. Small changes in barrier function over time could lead to accumulated defects that result in positive feedback to unrelated central nervous system diseases.

Introduction: Diffuse axonal injury is an extremely common type of traumatic brain injury encountered in motor vehicle crashes, sports injuries, and in combat. Although many cases of diffuse axonal injury result in chronic disability, there are no current treatments for this condition. Its basic lesion, traumatic axonal injury, has been aggressively modeled in primate and rodent animal models. The inexorable axonal and perikaryal degeneration and dysmyelination often encountered in traumatic axonal injury calls for regenerative therapies, including therapies based on stem cells and precursors. Here we explore the proof of concept that treatments based on transplants of human oligodendrocyte progenitor cells can replace or remodel myelin and, eventually, contribute to axonal regeneration in traumatic axonal injury. Methods: We derived human oligodendrocyte progenitor cells from the human embryonic stem cell line H9, purified and characterized them. We then transplanted these human oligodendrocyte progenitor cells into the deep sensorimotor cortex next to the corpus callosum of nude rats subjected to traumatic axonal injury based on the impact acceleration model of Marmarou. We explored the time course and spatial distribution of differentiation and structural integration of these cells in rat forebrain. Results: At the time of transplantation, over 90 % of human oligodendrocyte progenitor cells expressed A2B5, PDGFR, NG2, O4, Olig2 and Sox10, a profile consistent with their progenitor or early oligodendrocyte status. After transplantation, these cells survived well and migrated massively via the corpus callosum in both injured and uninjured brains. Human oligodendrocyte progenitor cells displayed a striking preference for white matter tracts and were contained almost exclusively in the corpus callosum and external capsule, the striatopallidal striae, and cortical layer 6. Over 3 months, human oligodendrocyte progenitor cells progressively matured into myelin basic protein(+) and adenomatous polyposis coli protein(+) oligodendrocytes. The injured environment in the corpus callosum of impact acceleration subjects tended to favor maturation of human oligodendrocyte progenitor cells. Electron microscopy revealed that mature transplant-derived oligodendrocytes ensheathed host axons with spiral wraps intimately associated with myelin sheaths. Conclusions: Our findings suggest that, instead of differentiating locally, human oligodendrocyte progenitor cells migrate massively along white matter tracts and differentiate extensively into ensheathing oligodendrocytes. These features make them appealing candidates for cellular therapies of diffuse axonal injury aiming at myelin remodeling and axonal protection or regeneration.