Abstract
SummaryHuman in vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. We have developed a reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before. The BMPS shows 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglial function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. The BMPS provides a suitable and reliable model to investigate neuron-neuroglia function as well as pathogenic mechanisms in neurotoxicology.
Highlights
There is a lack of mechanistic understanding of processes related to neurotoxicity (Smirnova et al, 2014; Schmidt et al, 2016) and neurological disorders, partly due to limited representative models of humans
We developed a novel in vitro induced pluripotent stem cells (iPSC)-derived human 3D brain microphysiological system (BMPS), which is comprised of mature neurons and glial cells
Colonies of iPSCs were manually picked after 3-6 weeks for further expansion and characterization. iPSCs were cultured on irradiated mouse embryonic fibroblasts (MEFs) in human embryonic stem cell medium comprising D-MEM/F12 (Invitrogen), 20% KnockOutTM Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine (Invitrogen), 100 μM MEM NEAA (Invitrogen), 100 μM β-mercaptoethanol (Invitrogen), and 10 ng/ml human basic FGF
Summary
There is a lack of mechanistic understanding of processes related to (developmental) neurotoxicity (Smirnova et al, 2014; Schmidt et al, 2016) and neurological disorders, partly due to limited representative models of humans. More than 90% of all drugs fail clinical trials despite extensive animal testing (Hartung, 2013), in part because animal studies do not reflect human physiology and inter-individual differences. Simple in vitro systems do not represent complex physiology and organ function (Hartung, 2007), especially that of the brain. This illustrates a critical need for better models for drug development, the study of disease, bioengineering and toxicological testing. Some attempts to generate more complex organotypic cultures or microphysiological systems (MPS) have resulted in physiological multicellular 3D co-culture models with the ability to simulate functional parts of the brain (Lancaster et al, 2013; Kadoshima et al, 2013). The discovery of induced pluripotent stem cells (iPSC) and protocols to differentiate them into various cell types has boosted the development of new hu-
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