Optimized cell-handling of human embryonic stem cells in the differentiation of photoreceptor precursor cells

Abstract

Purpose: A pressing need in regenerative medicine focused on treating common blinding diseases such as age-related macular degeneration and retinitis pigmentosa is the efficient production of large numbers of human retinal precursor cells. We proposed to optimize retinal differentiation protocols for human embryonic stem cells (hESCs) by improving cell handling.

Methods: To improve efficiency we firstly focused on the production of just one retinal precursor cell type (photoreceptor precursor cells, PPCs) rather than the production of a range of retinal cells. Combining information from a number of previous studies, in particular the use of feeder-free culture media and taurine plus tri-iodothyronine supplements, we then assessed the values of using size-controlled embryoid bodies (EBs) and negative cell selection (to remove embryonic antigen-4 positive hESCs).

Results: Using size controlled 1000-cell EBs, significant improvements were made over previous studies in that 77% CRX+ve PPCs (validated via FACS) could be produced in just 17 days. This could be increased to 93% PPCs through the added step of negative cell selection. The PPC-phenotype of these cells was further supported by their expression of BLIMP1.

Conclusions: We have developed a rapid, efficient method for producing large numbers of PPCs for future preclinical studies in common blinding conditions.

Keywords: 721 stem cells • 687 regeneration • 688 retina