| Abstract |
Purpose: Human embryonic stem (ES) cells offer significant opportunities
to gain a deeper understanding of the molecular mechanisms involved in
normal tissue development and function. Similarly, disease-specific human
ES cells enable study of the molecular mechanisms responsible for disease
initiation and progression. We describe here the first human ES cells
(GENEA069) that contain the F305S BEST1 gene mutation associated with Best
vitelliform macular dystrophy (i.e., Best disease). While mutation of the
BEST1 protein is an underlying cause of Best disease little is known of
the mechanism of disease progression, which adds to our inability to treat
this disease. Investigating the differentiation and maintenance of retinal
pigment epithelium (RPE) from both GENEA069 and normal human ES cells
offers a new approach to better understand, and potentially treat, Best
disease.
Methods: The GENEA069 cells were tested for features of human ES cells via
light microscopy, PCR, flow cytometry, and colony-forming cell ability.
Human ES cells were differentiated to RPE using a 3-stage in vitro method
that mimics aspects of embryonic RPE development: (i) generation of
ectodermal cells; (ii) generation of eye field, optic vesicle and RPE cell
types; and (iii) RPE purification via cloning rings. The progress of
differentiation was assessed using PCR for expression of genes involved in
RPE development (e.g., RAX, PAX6, MITF, RPE65, BEST1).
Results: Our data shows GENEA069 cells have characteristic features of
human ES cells, including: (i) typical morphology; (ii) expression of
OCT4, NANOG, SOX2, and FOXD3; (iii) expression of high levels of TRA-1-60,
TRA-1-81, and OCT4 antigens (>90% of cells); and (iv) an ability to give
rise to alkaline phosphatase-positive colonies. Differentiation of both
GENEA069 and normal human ES cells resulted in successive patterns of gene
expression characteristic of embryonic RPE development, ultimately
producing pigmented cells with typical RPE morphology that express
transcripts for RPE65 and BEST1.
Conclusions: These data suggest that GENEA069 cells are morphologically
indistinguishable from other human ES cell lines, and that they express
key molecular components required for pluripotency. Moreover, GENEA069
cells can be differentiated to produce mature, pigmented RPE that
expresses BEST1. The RPE generated from both GENEA069 and normal human ES
cells offers exciting new avenues for investigating the molecular
mechanisms responsible for normal and pathological RPE development,
maintenance, and function. The data obtained from these studies may aid in
the development of new or improved therapies for RPE-based blinding
disorders, in particular, Best disease.
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