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Directed differentiation of human pluripotent stem cells to microglia

ABSTRACT: Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the central nervous system, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell (PSC) lines.

ORGANISM(S): Homo sapiens

PROVIDER: GSE97744 | GEO | 2017/04/13

SECONDARY ACCESSION(S): PRJNA382797

REPOSITORIES: GEO

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Project description:Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC) alongside several intermediate forms. The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when they are activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors. To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection (LCM). The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis helps in identifying genes that are specific to AMC and RMC. The novel and specific molecules identified in both microglial phenotypes can be targeted for therapeutic purposes in developing and adult brain diseases. We used microarrays to identify the genes specific to amoeboid and ramified microglia. RNA was isolated from the laser-captured amoeboid and ramified microglia from the corpus callosum of 5-day and 4-week old rat brain. The RNA was hybridised onto Affymetrix Rat 230 2.0 array.

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Directed differentiation of human pluripotent stem cells to microglia

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