Derivation of multiple cranial tissues and isolation of lens epithelium-like cells from human embryonic stem cells

Stem Cells Transl Med. 2013 Feb;2(2):94-106. doi: 10.5966/sctm.2012-0100. Epub 2013 Jan 22.

Abstract

Human embryonic stem cells (hESCs) provide a powerful tool to investigate early events occurring during human embryonic development. In the present study, we induced differentiation of hESCs in conditions that allowed formation of neural and non-neural ectoderm and to a lesser extent mesoderm. These tissues are required for correct specification of the neural plate border, an early embryonic transient structure from which neural crest cells (NCs) and cranial placodes (CPs) originate. Although isolation of CP derivatives from hESCs has not been previously reported, isolation of hESC-derived NC-like cells has been already described. We performed a more detailed analysis of fluorescence-activated cell sorting (FACS)-purified cell populations using the surface antigens previously used to select hESC-derived NC-like cells, p75 and HNK-1, and uncovered their heterogeneous nature. In addition to the NC component, we identified a neural component within these populations using known surface markers, such as CD15 and FORSE1. We have further exploited this information to facilitate the isolation and purification by FACS of a CP derivative, the lens, from differentiating hESCs. Two surface markers expressed on lens cells, c-Met/HGFR and CD44, were used for positive selection of multiple populations with a simultaneous subtraction of the neural/NC component mediated by p75, HNK-1, and CD15. In particular, the c-Met/HGFR allowed early isolation of proliferative lens epithelium-like cells capable of forming lentoid bodies. Isolation of hESC-derived lens cells represents an important step toward the understanding of human lens development and regeneration and the devising of future therapeutic applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD57 Antigens / genetics
  • CD57 Antigens / metabolism
  • Cell Differentiation / physiology
  • Cell Line
  • Cell Separation / methods
  • Coculture Techniques
  • Culture Media / pharmacology
  • Ectoderm / cytology
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / physiology
  • Epithelial Cells / cytology*
  • Fibroblasts / cytology
  • Flow Cytometry / methods
  • Humans
  • Lens, Crystalline / cytology*
  • Mesoderm / cytology
  • Mice
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Neural Crest / cytology
  • Neural Plate / cytology*
  • Neural Stem Cells / cytology*
  • Neurons / cytology
  • Polymerase Chain Reaction
  • Receptors, Nerve Growth Factor / genetics
  • Receptors, Nerve Growth Factor / metabolism
  • Transcriptome

Substances

  • CD57 Antigens
  • Culture Media
  • NGFR protein, human
  • Nerve Tissue Proteins
  • Receptors, Nerve Growth Factor