Differentiation of human embryonic stem cells using size-controlled embryoid bodies and negative cell selection in the production of photoreceptor precursor cells

Tissue Eng Part C Methods. 2013 Oct;19(10):755-64. doi: 10.1089/ten.TEC.2012.0524. Epub 2013 Mar 15.

Abstract

We proposed to optimize the retinal differentiation protocols for human embryonic stem cells (hESCs) by improving cell handling. To improve efficiency, we first 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 a feeder-free culture medium and taurine plus triiodothyronine supplements, we then assessed the values of using size-controlled embryoid bodies (EBs) and negative cell selection (to remove residual embryonic antigen-4-positive hESCs). Using size-controlled 1000 cell EBs, significant improvements were made, in that 78% CRX+ve PPCs could be produced in just 17 days. This could be increased to 93% PPCs through the added step of negative cell selection. Improved efficiency of PPC production will help in efforts to undertake shorter and larger preclinical studies as a prelude to future clinical trials.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods*
  • Cell Differentiation* / drug effects
  • Cell Differentiation* / genetics
  • Cell Line
  • Cell Lineage / drug effects
  • Cell Lineage / genetics
  • Cell Size*
  • Chromosome Banding
  • Culture Media / pharmacology
  • Embryoid Bodies / cytology*
  • Embryoid Bodies / drug effects
  • Embryoid Bodies / metabolism
  • Gene Expression Profiling
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Immunomagnetic Separation*
  • Karyotyping
  • Photoreceptor Cells, Vertebrate / cytology*
  • Photoreceptor Cells, Vertebrate / drug effects
  • Photoreceptor Cells, Vertebrate / metabolism
  • Trans-Activators / genetics
  • Trans-Activators / metabolism

Substances

  • Culture Media
  • Homeodomain Proteins
  • Trans-Activators
  • cone rod homeobox protein