June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
bFGF deprivation promotes spontaneous differentiation of Human Embryonic Stem Cells into γ- synuclein positive ganglion cells and CRALBP positive muller cells
Author Affiliations & Notes
  • K V Chalam
    Ophthalmology, Univ of Florida-Jacksonville, Jacksonville, Florida, United States
  • sandeep Grover
    Ophthalmology, Univ of Florida-Jacksonville, Jacksonville, Florida, United States
  • Bharani k Mynampati
    Ophthalmology, Univ of Florida-Jacksonville, Jacksonville, Florida, United States
  • Footnotes
    Commercial Relationships   K V Chalam, None; sandeep Grover, None; Bharani k Mynampati, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5558. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      K V Chalam, sandeep Grover, Bharani k Mynampati; bFGF deprivation promotes spontaneous differentiation of Human Embryonic Stem Cells into γ- synuclein positive ganglion cells and CRALBP positive muller cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5558.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : To evaluate cellular expression profiles of γ-synuclein and Cellular retinaldehyde-binding protein (CRALBP) markers during the spontaneous differentiation of human embryonic stem cells into Ganglion and Muller cells (hESC-GC/MC).

Methods :
Human embryonic stem cells (WA09-DL-11 hESC) were seeded onto a gelatin coated 6-well plates. Prior to hESC seeding , gelatin coated wells were confluent with a layer of inactivated mouse embryonic fibroblast.hESCs were cultured in growth media containing basic fibroblast growth factor (bFGF:2μg/ml). hESCs were maintained for one week (controls) with daily media exchange. bFGF was removed to promote differentiation and first day of deprivation was considered day '0'. During spontaneous differentiation, hESC colonies were observed under phase contrast bright field light microscopy. hESC colonies expressing pigmentation pattern appeared after 5th week mixed with nonpigmented cells. Morphological assessment and immune-cytochemical analysis (γ-synuclein and CRALBP marker expression) was performed on hESC's at different time intervals i.e (36, 43, 50, 58, 65 and 72 days) to monitor differentiation into hESC- derived muller and ganglion cells. Immunofluorescence was performed under 10X magnification on an inverted Olympus IX 51-IX2-SL microscope with Olympus U-RFL-T fluorescence lamp.

Results :
Semi pigmented cells were noted 36 days after deprivation of bFGF from growth media. Immunofluorescence demonstrated progressive up regulation of both γ-synuclein and CRALBP. Immunofluorescence of γ-synuclein was (6.25±1.07,p=0.95) on day 36, (16.57±2.11, p=0.23) on day 43, (27.73±8.02, p=0.01) on day 50, ( 74.99±16.28, p<0.0001) on day 58, (101.9±20.2, p<0.0001) on day 65, ( 67.35 ± 2.17, p<0.0001) on day 72. Further immunofluorescence of CRALBP was (11.36±1.07,p=0.18) on day 36, (44.76±6.28, p<0.0001) on day 43, (48.4±7.7, p<0.0001) on day 50, ( 75.59±7.4, p<0.0001) on day 58, (73.23±10.2, p<0.0001) on day 65, ( 77.6 ± 3.5, p<0.0001) on day 72 compared to controls.

Conclusions :
bFGF deprivation led to successful differentiation of hESC into muller cells as well as ganglion cells. Longitudinal transformative changes were confirmed with measurement of γ-synuclein and CRALBP specific markers for ganglion cells and muller cells

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

cRALBP ( Muller cells)

cRALBP ( Muller cells)

 

γ-synuclein ( Ganglion cells)

γ-synuclein ( Ganglion cells)

×
×

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.

×