Long-Term in vitro expansion of retinal progenitor cells by culturing intact neurospheres in monolayer.
This study developed a modified monolayer culture technique for expansion of retinal progenitor cells in vitro. Using this method, spheres collected by low centrifugation from free floating cultures were re-seeded onto poly-D-lysine pre-coated flasks, and cultured in the retinal progenitor cells medium (including fibrous growth factor, epidermal growth factor and leukemia inhibitor factor) plus 5% FBS for the first 24 h, then FBS was removed. These cultured cells were proliferative, and in addition to expressing the neuroectodermal marker nestin they were multipotential. By immunocytochemistry and FACS analysis, we demonstrated that the percentage of nestin-expressing cells in passage 2 and passage 8 was 92.5 and 85.5%, respectively, which was greater than the percentage of their counterparts, 81.9 and 76.2%, in simple adhesive cultures (p < 0.05). Moreover, these cells preferentially differentiated into retinal neurons rather than glial cells.
Lai
Pinghong
P
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Tang
Shibo
S
Yi
Jinglin
J
Zhu
Xiaobo
X
Zou
Yuping
Y
Xie
Hui
H
Gao
Yi
Y
Lin
Shaofen
S
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007381:Intermediate Filament Proteins / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D011247:Pregnancy; D012160:Retina / Q000196:embryology*; D012243:Rhodopsin / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D012243:Rhodopsin