Home › Cell Biology › Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells
Cell Biology JoVE (Open Access) Citable · DOI

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

DOI: 10.3791/56274-v
What you'll learn
  • ✓ Direct differentiation of pluripotent stem cells to retinal pigment epithelial cells using growth factors and small molecules
  • ✓ Generate immature RPE within 14 days and mature functional RPE after 3 months
  • ✓ Passage and enrich RPE populations through sequential culture steps
  • ✓ Assess RPE morphology and confluence during maturation
Protocol

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Difficulty
advanced
Total time
~3 months (14 days to immature RPE; 3 months to mature, functional RPE)
Model organism
Human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC)
Biosafety
BSL-1

Steps

1
Passage pluripotent stem cells for differentiation initiation

Prepare and passage human embryonic or induced pluripotent stem cells on day 0 to establish the starting population for directed RPE differentiation. Culture conditions and cell density are optimized for subsequent growth factor treatment.

▶ 01:02
2
Enrich and passage retinal pigment epithelial cells

Perform enrichment of differentiating RPE population and passage cells to passage 0. Apply selective culture conditions and growth factors to promote RPE specification and maturation over the subsequent culture period.

▶ 04:43
3
Evaluate RPE morphology and confluence during maturation

Assess representative morphology and confluence of maturing RPE cells at defined timepoints. Monitor pigmentation, cell organization, and culture characteristics to confirm successful differentiation progression.

▶ 08:37
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