Journal: Stem Cells Translational Medicine
Article Title: Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage
Figure Lengend Snippet: Stage 2: Generation of a mixed RPE population. (A): Two days’ postseeding, SHEF1 human embryonic stem cells were treated with Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days. At day 9, cells were replated in Dulbecco's modified Eagle's medium (DMEM) KSR‐XF alone (Control) or Induction Medium 3 (ActA) for 19 days. Quantitative polymerase chain reaction was used to measure expression of a panel of RPE markers. RPE generated by using spontaneous differentiation (Sp. RPE) were used for comparison ( n = 3, ±SD). (B): Representative images showing immunocytochemistry for CRALBP and MERTK in cells derived by directed differentiation and cultured as in panel A. Dotted squares indicate the region that is magnified in the panels below. RPE generated by spontaneous differentiation were used for comparison. Images are captured at ×10 magnification. (C): Quantification of CRALBP immunocytochemistry at day 28 in which cells during stage 2 were treated with DMEM KSR‐XF (Control) or Induction Medium 3 (ActA) for 3 days, 5 days, 10 days, or 19 days. For fewer than 19‐day ActA treatments, DMEM KSR‐XF was used for the remainder of the 19‐day period. RPE derived by using spontaneous differentiation (Sp. RPE) were used for comparison ( n = 3, ±SD). Abbreviations: ActA, Activin A; d, days; RPE, retinal pigment epithelium; Sp. RPE, spontaneous differentiation retinal pigment epithelium.
Article Snippet: On day 2, cells were switched to Induction Medium 1: Dulbecco's modified Eagle's medium (DMEM) KSR‐XF (KnockOut DMEM medium [Thermo Fisher], supplemented with 20% KnockOut Serum Replacement Xeno‐Free [Thermo Fisher], 1% β‐Mercaptoethanol [Sigma‐Aldrich], 1% GlutaMax [Thermo Fisher], and 1% non‐essential amino acid solution [Thermo Fisher]), supplemented with two inhibitors (LDN/SB): 1 μM LDN‐193189 (Stemgent, Cambridge, MA, https://www.stemgent.com ) and 10 µM SB‐431542 (Sigma‐Aldrich).
Techniques: Modification, Real-time Polymerase Chain Reaction, Expressing, Generated, Immunocytochemistry, Derivative Assay, Cell Culture