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Wei Liu, Albert Lowe, Raven Harris, Punita Bhansali, Ales Cvekl; Generation of retinal tissues from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) through self-assisted progenitor cell sorting. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3169.
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© ARVO (1962-2015); The Authors (2016-present)
This study aims to model human retinal development by employing human pluripotent stem cells such as human H1 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).
H1 hESCs or hiPSCs were cultured on Matrigel-coated plates in DMEM/F12 medium supplemented with N2 and B27, 0.5% bovine serum albumin, 1mM L-glutamine, 1% penicillin-streptomycin, 100 ng/ml of bFGF (Yao et al., 2006) or in mTeSR medium. H1 hESCs or hiPSCs were passaged every 5 to 7 days with laser-enabled analysis and processing (LEAP; Cyntellect), with Dispase, or ReLeSR™. Retinal progenitor cells (RPCs) were generated using a modified Matrigel-based method.
In retinal cell differentiation from hESCs or hiPSCs in vitro, the cell cultures are frequently a mixture of randomly differentiated many cell types, and thus cell differentiation is in a disorganized manner in contrast to highly regulated morphogenesis in vivo. A key concept in organoid culture is to isolate substantial pure progenitor cells for prolong cultures. Although genetically engineered fluorescence markers are valuable tools for monitoring and isolating specific type of cells, identification and characterization of the intrinsic progenitor cell properties that can be used for cell isolation are likely to be more relevant to clinical applications. Here we report the selection and isolation of hPSC-derived retinal progenitor cells (RPCs) based on their intrinsic properties. These RPCs were self-sorted into spheres that are readily detectable under an inverted microscope. Histological and molecular characterizations of the spheres demonstrate that these cells constituted a polarized epithelium and highly expressed RPC markers SIX3, RAX, PAX6 and VSX2. Long-term cultures of these RPCs produced laminar tissues comparable to human retina.
This novel long-term organoid culture system does not require any genome engineering and generates retina-like laminar structures comprised of differentiated retinal cells. Ongoing studies aim to evaluate this system in modeling human retinal development and disease pathogenesis, drug discovery, and cell replacement therapy.
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