Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.
Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.
R&D Center, Jeil Pharmaceutical CO, LTD Yongin 449-861, Republic of Korea. tpguy@unitel.co.kr
Myung Soo
MS
Cho
Sang Jin
SJ
Kim
Seung-Yup
SY
Ku
Jung Hyun
JH
Park
Haksup
H
Lee
Dae Hoon
DH
Yoo
Un Chul
UC
Park
Seul Ae
SA
Song
Young Min
YM
Choi
Hyeong Gon
HG
Yu
D000818:Animals; D002417:Cattle; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D005136:Eye Proteins / Q000378:metabolism; D006801:Humans; D009414:Nerve Growth Factors / Q000378:metabolism; D046569:Neuroepithelial Cells / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010587:Phagocytosis; D015203:Reproducibility of Results; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D015843:Serpins / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D009414:Nerve Growth Factors; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor