Embryonic stem cell (ESC) differentiation can be used to model development and to produce transplantable cells of the desired phenotype. ESCs can reproducibly generate retinal cells but the derivation of photoreceptor precursors is variable and depends on an array of exogenous factors and intrinsic cell–cell interactions. In this work, we have defined the use of exogenous signaling factors, dissociation, and adherent versus 3-dimensional (3D) conditions on the derivation of retinal cells from pluripotent mouse ESCs. Differentiation protocols were chosen based on the developmental stage and cell population of interest and evaluated by expression of developmental stage- and lineage-specific marker genes. We present a relatively simple protocol that guides differentiating ESCs through stages that correspond to the sequence of in vivo developmental events and is optimized for studying the time frame between eye field formation and photoreceptor precursor development in the equivalent of embryonic retina. Step-wise exposure of adherent cultures to exogenous factors facilitated expression of eye field transcription factors and limited non-specific differentiation. Dissociation after the establishment of eye field and retinal progenitor cell gene expression did not cause substantial loss in expression of markers of mature photoreceptors. Finally, 3D organoids improved expression of photoreceptor genes and region-specific architecture but required more technical manipulation. We demonstrate the usefulness of this ESC-retinal induction protocol in screening for factors that improve photoreceptor precursor yield by evaluating response to alterations in Activin signaling.
All Science Journal Classification (ASJC) codes
- Cell Biology
- Cancer Research