Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSC to microglia.

There is increasing genetic evidence for the role of microglia in neurodegenerative diseases, including Alzheimer's, Parkinson's, and motor neuron disease. Therefore, there is a need to generate authentic in vitro models to study human microglial physiology. Various methods have been developed using human induced Pluripotent Stem Cells (iPSC) to generate microglia, however, systematic approaches to identify which media components are actually essential for functional microglia are mostly lacking. Here, we systematically assess medium components, coatings, and growth factors required for iPSC differentiation to microglia. Using single-cell RNA sequencing, qPCR, and functional assays, with validation across two labs, we have identified several medium components from previous protocols that are redundant and do not contribute to microglial identity. We provide an optimised, defined medium which produces both transcriptionally and functionally relevant microglia for modelling microglial physiology in neuroinflammation and for drug discovery.

Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation.

Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.

Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.

Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)-derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD.

Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage.

Development of efficient and reproducible conditions for directed differentiation of pluripotent stem cells into specific cell types is important not only to understand early human development but also to enable more practical applications, such as in vitro disease modeling, drug discovery, and cell therapies. The differentiation of stem cells to retinal pigment epithelium (RPE) in particular holds promise as a source of cells for therapeutic replacement in age-related macular degeneration. Here we show development of an efficient method for deriving homogeneous RPE populations in a period of 45 days using an adherent, monolayer system and defined xeno-free media and matrices. The method utilizes sequential inhibition and activation of the Activin and bone morphogenetic protein signaling pathways and can be applied to both human embryonic stem cells and induced pluripotent stem cells as the starting population. In addition, we use whole genome transcript analysis to characterize cells at different stages of differentiation that provides further understanding of the developmental dynamics and fate specification of RPE. We show that with the described method, RPE develop through stages consistent with their formation during embryonic development. This characterization- together with the absence of steps involving embryoid bodies, three-dimensional culture, or manual dissections, which are common features of other protocols-makes this process very attractive for use in research as well as for clinical applications. Stem Cells Translational Medicine 2017;6:490-501.