METHODS FOR FORMING THREE-DIMENSIONAL HUMAN RETINAL TISSUE IN VITRO

摘要

The present invention relates to the field of stem cells. More specifically, the invention provides methods and compositions useful for forming three-dimensional human retinal tissue in vitro. In a specific embodiment, an in vitro method for differentiating hiPSCs into three-dimensional retinal tissue comprising functional photoreceptors comprises the steps of (a) culturing the hiPSCs to form aggregates; (b) transitioning the aggregates into a neural induction medium; (c) seeding the aggregates on to extracellular matrix coated cell culture substrates; (d) replacing NIM with a chemically-defined differentiation medium; (e) detaching NR domains; (f) culturing in suspension; and (g) adding animal serum or plasma component and retinoic acid.

主权项

1. An in vitro method for differentiating human induced pluripotent stem cells (hiPSCs) into three-dimensional retinal tissue comprising functional photoreceptors, the method comprising the steps of:
a. on day 0 of differentiation:
i. enzymatically detaching hiPSCs cultured on extracellular matrix-coated cell culture substrates with feeder-free cell culture medium, andii. culturing the hiPSCs in suspension to induce formation of aggregates; b. during days 1-3 of differentiation, transitioning the aggregates into neural induction medium (NIM); c. during day 6 or 7, seeding the aggregates on to extracellular matrix-coated cell culture substrates; d. at any time between day 14 and day 17, replacing NIM with a chemically-defined differentiation medium; e. during the fourth week of differentiation:
i. detaching neural retina (NR) domains, andii. culturing in suspension; f. during the fifth or sixth week of differentiation, adding animal serum or plasma component to promote cell survival; and g. at any time between week 5 and week 14, adding all-trans retinoic acid to induce photoreceptor maturation.