The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.

Neuroinflammation is a common feature in neurodegenerative diseases and strategies to modulate neuroinflammatory processes are increasingly considered as therapeutic options. In such strategies, glia cells rather than neurons represent the cellular targets. Microglia, the resident macrophages of the central nervous system, are principal players in neuroinflammation and detailed cellular biological knowledge of this particular cell type is therefore of pivotal importance. The last decade has shed new light on the origin, characteristics and functions of microglia, underlining the need for specific in vitro methodology to study these cells in detail. In this review we provide a comprehensive overview of existing methodology such as cell lines, stem cell-derived microglia and primary dissociated cell cultures, as well as discuss recent developments. As there is no in vitro method available yet that recapitulates all hallmarks of adult homeostatic microglia, we also discuss the advantages and limitations of existing models across different species.

Current protocols for differentiation of human pluripotent stem cells to astrocytes are slow and inefficient, and characterization of the generated cells is incomplete. These shortcomings severely limit research on the biology of human astrocytes and their involvement in neurological disorders.Here we capitalized on recent transcription factor-driven methods to develop a novel protocol for astrocyte differentiation. We demonstrate that overexpression of two gliogenic transcription factors, Sox9 and Nfib, in human pluripotent stem cells rapidly and efficiently gives rise to a highly homogenous population of astrocytes as early as 7 days post-transduction. After 14 days, these induced astrocytes (iAs) exhibit molecular signatures and functional properties closely resembling those of adult human astrocytes. The iAs also recapitulate disease phenotype in a genomeengineered model of Alexander disease.Our approach provides an easy, time-efficient, effective and scalable method that will provide new opportunities for studies on the role of human astrocytes in health and disease.

Microglia are increasingly being recognized as druggable targets in neurodegenerative disorders, and good in vitro models are crucial to address cell biological questions. Major challenges are to recapitulate the complex microglial morphology and their in vivo transcriptome. We have therefore exposed primary microglia from adult rhesus macaques to a variety of different culture conditions including exposure to soluble factors as M-CSF, IL-34, and TGF-β as well as serum replacement approaches, and compared their morphologies and transcriptomes to those of mature, homeostatic in vivo microglia. This enabled us to develop a new, partially serum-free, monoculture protocol, that yields high numbers of ramified cells. We also demonstrate that exposure of adult microglia to M-CSF or IL-34 induces similar transcriptomes, and that exposure to TGF-β has much less pronounced effects than it does on rodent microglia. However, regardless of culture conditions, the transcriptomes of in vitro and in vivo microglia remained substantially different. Analysis of differentially expressed genes inspired us to perform 3D-spherical coculture experiments of microglia with oligodendrocytes and radial glia. In such spheres, microglia signature genes were strongly induced, even in the absence of neurons and astrocytes. These data reveal a novel role for oligodendrocyte and radial glia-derived cues in the maintenance of microglial identity, providing new anchor points to study microglia in health and disease.

Microglia are the resident macrophages of the central nervous system and contribute to maintaining brain’s homeostasis. Current 2D “petri-dish” in vitro cell culturing platforms employed for microglia, are unrepresentative of the softness or topography of native brain tissue. This often contributes to changes in microglial morphology, exhibiting an amoeboid phenotype that considerably differs from the homeostatic ramified phenotype in healthy brain tissue. To overcome this problem, multi-scale engineered polymeric microenvironments are developed and tested for the first time with primary microglia derived from adult rhesus macaques. In particular, biomimetic 2.5D micro- and nano-pillar arrays (diameters = 0.29–1.06 µm), featuring low effective shear moduli (0.25–14.63 MPa), and 3D micro-cages (volume = 24 × 24 × 24 to 49 × 49 × 49 μm3) with and without micro- and nano-pillar decorations (pillar diameters = 0.24–1 µm) were fabricated using two-photon polymerization (2PP). Compared to microglia cultured on flat substrates, cells growing on the pillar arrays exhibit an increased expression of the ramified phenotype and a higher number of primary branches per ramified cell. The interaction between the cells and the micro-pillar-decorated cages enables a more homogenous 3D cell colonization compared to the undecorated ones. The results pave the way for the development of improved primary microglia in vitro models to study these cells in both healthy and diseased conditions.