March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Optimizing Retinal Progenitor Differentiation of hESC - Effect of RPE Co-Culture
Author Affiliations & Notes
  • Magdalene J. Seiler
    Anatomy & Neurobiol/Reeve-Irvine Res Ctr, Univ of California, Irvine, Irvine, California
  • David Ferguson
    Anatomy & Neurobiol/Reeve-Irvine Res Ctr, Univ of California, Irvine, Irvine, California
  • Gabriel Nistor
    California Stem Cell Inc., Irvine, California
  • Sabhya Rana
    Anatomy & Neurobiol/Reeve-Irvine Res Ctr, Univ of California, Irvine, Irvine, California
  • Irina Turovets
    International Stem Cell Corporation, Oceanside, California
  • Joshua Almodovar
    Anatomy & Neurobiol/Reeve-Irvine Res Ctr, Univ of California, Irvine, Irvine, California
  • Jared Taylor
    Anatomy & Neurobiol/Reeve-Irvine Res Ctr, Univ of California, Irvine, Irvine, California
  • Nikolay A. Turovets
    International Stem Cell Corporation, Oceanside, California
  • Hans S. Keirstead
    Anatomy & Neurobiol/Reeve-Irvine Res Ctr, Univ of California, Irvine, Irvine, California
  • Footnotes
    Commercial Relationships  Magdalene J. Seiler, None; David Ferguson, None; Gabriel Nistor, California Stem Cell (E); Sabhya Rana, None; Irina Turovets, International Stem Cell Corporation (E); Joshua Almodovar, None; Jared Taylor, None; Nikolay A. Turovets, International Stem Cell Corporation (E); Hans S. Keirstead, California Stem Cell (C)
  • Footnotes
    Support  Johnson & Johnson; International Stem Cell Corporation
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5897. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Magdalene J. Seiler, David Ferguson, Gabriel Nistor, Sabhya Rana, Irina Turovets, Joshua Almodovar, Jared Taylor, Nikolay A. Turovets, Hans S. Keirstead; Optimizing Retinal Progenitor Differentiation of hESC - Effect of RPE Co-Culture. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5897.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: : To optimize the differentiation of human embryonic stem cells (hESC) to retinal and photoreceptor progenitors for obtaining 3D sheets of retinal progenitors with RPE for transplantation.

Methods: : hESCs were differentiated according to modifications of Nistor et al. (2010), including delayed hyaluronic acid treatment, longer time in suspension culture, and a change in concentration and treatment time with transcription factors Dkk1 and LeftyA. Additional studies focused on variations in exposure time and concentration of FGF-8, Sonic Hedgehog (shh), and Activin A. Cell cultures were analyzed by immunohistochemistry for the transcription factors Chx10, CRX, NRL and various other cell markers.

Results: : Expression of photoreceptor progenitor markers could be improved compared to the original differentiation protocol by using a higher concentration of Dkk1/LeftyA in the beginning of the culture, starting hyaluronic acid treatment after 3 weeks, and maintaining the cultures in suspension until d 28-29. FGF8 and shh treatment did not improve photoreceptor progenitor differentiation. Early Avtivin A treatment together with RPE co-culture (d 44-58 of differentiation) dramatically increased expression of the photoreceptor progenitor markers CRX and NRL in more than 90% of the cells. However, no mature photoreceptor markers such as recoverin were found at d 58 of differentiation.

Conclusions: : Short treatment with transcription factors LeftyA and Dkk1 at high concentration (40-60 ng/ml) is sufficient to induce retinal differentiation. Early Activin A treatment, longer time in suspension culture, delayed hyaluronic acid and co-culture with RPE improves expression of photoreceptor progenitor markers. The next step is to test these tissues in retinal degenerate nude rats.

Keywords: transcription factors • differentiation • retinal development 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×