title: Deprivation of bFGF Promotes Spontaneous Differentiation of Human Embryonic Stem Cells into Retinal Pigment Epithelial Cells.
abstract: BACKGROUND
  The retinal pigment epithelium (RPE), an important tissue monolayer of retina, sustains visual function and retinal homeostasis. In disease conditions such as Retinitis Pigmentosa (RP) and Age related Macular degeneration (AMD), the integrity and functional capacity of RPE monolayer is compromised. Human embryonic stem cells derived RPE (hESC-RPE) is ideal for cell based therapy because of their ability to morphologically and functionally mimic native fetal and adult RPE. However protocols for optimum culture of hESC-RPE are not well established.
  
  
  OBJECTIVE
  To describe a simplified protocol for differentiating human embryonic stem cells (hESC) into retinal pigment epithelial cells.
  
  
  METHODS
  hESC (WA09-DL-11) cell lines were grown with standard stem cell culture protocol. After cell colonies were established, basic fibroblast growth factor (bFGF) was deprived (day 0). hESC colonies expressing pigmentation were characterized for expression of RPE65 and Zonular occludens--1 (ZO-1) with immunocytochemistry on days 0, 36, 42, 56 and 70 and western blot analysis on days 0, 40, 48, 53 and 63. In addition, morphological assessment was conducted on transformed cells longitudinally.
  
  
  RESULTS
  Pigmented cells were noted 36 days after deprivation of bFGF from growth media. Immunofluorescence demonstrated progressive up regulation of RPE specific proteins (ZO-1 & RPE 65). Immunofluorescence of ZO-1 (in pixels) was (3.08 ± 0.31) on day 42, (5.33 ± 0.89, p = 0.0001) on day 56 and (4.87 ± 0.57, p = 0.0011) on day 70. Similarly expression of RPE 65 was (2.44 ± 0.31) on day 42, which continued to increase (4.23 ± 0.60, p = 0.0011) on day 56 and (5.59 ± 0.36, p < 0.0001) on day 70. Protein expression patterns using western blot confirmed the trends seen in immunofluorescence. Western blot analysis of ZO-1 expression (in optical density unit) was 272.57 ± 31.75 on day 40, 4212.20 ± 911.31 (p = 0.0004) on day 48, 5182.43 ± 1230.38 (p = 0.030) on day 53 and 5848.76 ± 241.04 (p < 0.0001) on day 63. Protein expression of RPE 65 was 1607.64 ± 247.76 on day 40, 2448.07 ± 152.66 on day 48 and (2341.15 ± 52.84) on day 63. hESC-RPE cells displayed a series of specific morphological changes (cytoplasmic, nuclear pigmentary and cell shape) over the course of time frame. By day 70, cells with hexagonal pattern, dark dense nucleus and uniform cytoplasm were noted in densely pigmented RPE colonies.
  
  
  CONCLUSIONS
  bFGF deprivation leads to successful differentiation of hESC into RPE cells. Longitudinal transformative changes were confirmed with measurement of ZO-1 and RPE 65, specific markers of RPE.
authors.forename: Lee R
authors.initials: LR
authors.lastname: Ferguson
authors.forename: Sankarathi
authors.initials: S
authors.lastname: Balaiya
authors.forename: Bharani K
authors.initials: BK
authors.lastname: Mynampati
authors.forename: Kumar
authors.initials: K
authors.lastname: Sambhav
authors.forename: Kakarla V
authors.initials: KV
authors.lastname: Chalam
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D048430:Cell Shape; D002478:Cells, Cultured; D016222:Fibroblast Growth Factor 2 / Q000172:deficiency* / Q000494:pharmacology; D000066449:Human Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D051379:Mice; D055213:Retinal Pigment Epithelium / Q000502:physiology*; D013997:Time Factors
chemical_list: D016222:Fibroblast Growth Factor 2