Triphasic developmentally guided protocol to generate retinal pigment epithelium from induced pluripotent stem cells

Abstract

RPE tissues are derived from induced pluripotent stem cells (iPSCs) to model retinal diseases and as a replacement therapy for macular degeneration. Here, we developed a robust and efficient directed differentiation protocol to generate pure RPE cells that form a polarized monolayer. This protocol describes how to set up RPE differentiation and to obtain a pure population that expresses mature RPE markers and forms strong tight junctions. For complete details on the use and execution of this protocol, please refer to Sharma et al., 2019, Sharma et al., 2021 and Miyagishima et al. (2021).

Keywords: Cell Biology; Cell Differentiation; Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Health Sciences; Stem Cells.

Graphical abstract

Figure1

Morphology of iPSC colonies for setting up RPE differentiation (A and B) iPSC colonies ready to be used for differentiation. The culture should be more homogeneous with minimum dead cell floating and compact colonies. (C and D) iPSC colonies that should not be used for RPE differentiation. Heterogenous colony size (blue arrowheads), high cell death, and spiky cells (white arrowheads) lead to poor differentiation. Scale bar, 200 μm.

Figure 2

Morphology of iPSC cells transition to RIM phase: Day 2 to day 1 (A and B) Differentiating cells at day-1 (A) and day 0 (B). The cells at day-1 are spiky because of the blebbistatin in culture media. The images represent good density and morphology of differentiating cells. (C and D) Differentiating cells at day 0. The arrowheads represent the abnormal morphology that should be avoided in day 0 cells. The probable reason for such morphologies is the excessive use of TrypLE and mechanical force during passaging. Scale bar, 200 μm.

Figure 4

Morphology of differentiating cells in RMNA phase: Day 11 to day 20 (A and B) Differentiating cells at day 16 (A) and day 20 (B). At day 20, appearance of little bumps of neuronal clusters (arrowhead marked) indicates successful run. The flat land around bumps RPE progenitor cells. (C and D) Images of bad cultures at day 16 (A) and day 20 (B). The opaque cultures lacking any islands of neuronal islands end up dying at the end of day 20, i.e., RMNA phase. Scale bar, 20 μm.

Figure 5

Morphology of differentiating cells in RPE phase from committed to immature stage (A) RPE progenitor cells at day 25. (B) Reseeded cells at day 27. (C) Immature RPE cells before MACS purification. The images in this panel represent the good differentiation run. Scale bar, 200 μm. (D) RPE progenitor cells at day 25. (E) Reseeded cells at day 27. (F) Immature RPE cells before MACS purification. The images in this panel represent the bad differentiation run with no appearance of neuronal clusters at day 25 (D) and only neurons at day 27. Scale bar, 200 μm.

Figure 3

Morphology of differentiating cells in RDM phase: Day 2 to day 10 (A and B) Differentiating cells at day 5 (A) and day 10 (B). At day 10, expect lot of cell death indicating the priming to RPE fate. The differentiation runs lacking the cell death at the end of RDM phase often brings poor efficiency. Scale bar, 200 μm.

Figure 6

Morphology of differentiating cells at day 25 (A) Differentiating cells at day 25, the big holes in the. monolayer of differentiating cells indicates the failed run and that the culture is full of neurons. Scale bar, 200 μm.

Figure 7

RPE pigmentation chart

Figure 8

Breakdown culture schematic detailing all the steps