TYROSINASE-Deficient Human Retinal Pigment Epithelium Exhibits Melanosome Maturation Defects

Oculocutaneous albinism type 1A (OCA1A) is a rare recessive genetic condition caused by mutations in TYROSINASE (TYR) that results in pigmentation defects of the skin, hair and eyes. This study was performed to understand melanosome biogenesis and maturation defects in an OCA1A in vitro model using retinal pigment epithelium (RPE) derived from TYR knockout human induced pluripotent stem cells (iPSC).

CRISPR-Cas9 was used to knockout the TYR gene in iPSC to generate an isogenic pair. A developmentally guided protocol was used to differentiate the isogenic iPSC pair towards RPE monolayer tissue. Monolayer organization, melanosome formation and maturation were studied using electron microscopy. Loss of TYR protein was studied using Western blot and immuno-fluorescence staining. RPE cellular morphology and junction integrity was studied using immunofluorescence staining and transepithelial resistance measurements.

An isogenic pair comprising of untargeted control and TYR knockout iPSC were successfully differentiated towards RPE monolayer tissue with polygonal cell morphology. TYR knockout RPE exhibited significantly reduced TYR protein, increased presence of immature pre-melanosomes and a complete lack of mature melanosomes. We observed abnormal junctional localization of β-catenin staining pattern, as has been reported previously for albino mouse RPE- and OCA1A patient-derived RPE.

Differentiation of TYR-deficient iPSC toward RPE displayed pigmentation defects and absence of mature melanosomes, whereas melanosome biogenesis was not affected, because pre-melanosomes were still observed. These observations were also similar to what was observed in OCA1A patient-derived RPE monolayer tissue, independently confirming the validity of these previous findings.