Spheroid transplantable and functional retinal pigment epithelium derived from non-colony dissociated human induced pluripotent stem cells

Abstract Background: Retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSCs) exhibit great promise in treating retinal degenerative diseases. Here, we would explore the feasibility of non-colony dissociated hiPSCs to differentiate into functional RPE cells (hiPSC-RPE), and offer an alternative transplantation method based on cell spheroids.Methods: hiPSC-RPE cells were identified using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence assay, Western blotting, and flow cytometry assay. The functions of hiPSC-RPE cells in vitro and in vivo were assessed by fluorescein leakage test, transepithelial electrical resistance (TEER) assay, atomic force microscopy observation, POS phagocytosis assay, frozen tissue sections, live/dead assay, SA-β-Gal staining, and immunocytochemistry.Results: hiPSC-RPE cells positively expressed biomarkers of RPE cells but not iPSCs, such as CRALBP (97.4%), EMMPRIN (93.8%), Oct4 (2.1%), and Sox2 (2.0%). hiPSC-RPE cells displayed RPE-like characteristics including barrier function, phagocytic activity, and polarized membrane. The cells derived from hiPSC-RPE spheroids positively expressed Nestin and exhibited reduced SA-β-Gal staining. hiPSC-RPE cell spheroids could form monolayer on decellularized corneal matrixes (DCM). After one month of subretinal transplantation, hiPSC-RPE cell spheroids could survive and maintain segmental sheet growth in sodium iodate (NaIO3) induced RPE-degenerated chinchilla rabbits. Conclusion: This study suggested that non-colony dissociated hiPSCs were effectively differentiated into functional RPE cells, and hiPSC-RPE cell spheroids maintained segmental sheet growth in the subretinal of RPE degenerate chinchilla rabbits in vivo, which may lay the foundation for cell spheroid transplantation as an alternative method for RPE degenerative disease therapy in the future.

Download Full-text

Transplantable and functional retinal pigment epithelium derived from non-colony-type monolayer of human induced pluripotent stem cells

10.21203/rs.2.21874/v1 ◽

2020 ◽

Author(s):

Xiaoling Guo ◽

Deliang Zhu ◽

Ruiling Lian ◽

Qiaolang Zeng ◽

Sanjana Mathew ◽

...

Keyword(s):

Stem Cells ◽

Retinal Pigment Epithelium ◽

Pluripotent Stem Cells ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Degenerative Diseases ◽

Rpe Cells ◽

Colony Type ◽

Cell Spheroids ◽

Induced Pluripotent

Abstract Background Retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSCs) exhibit great promise in treating retinal degenerative diseases. To develop transplantable and functional hiPSC-RPE cells, here, we used a novel differentiation protocol based on a non-colony-type monolayer (NCM) culture and injectable spheroids. Methods The derived hiPSC-RPE cells were identified using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence assay, Western blotting, and flow cytometry assay. The functions of transplantable hiPSC-RPE cells in vitro and in vivo were also analyzed by fluorescein leakage test, transepithelial electrical resistance (TEER) assay, atomic force microscopy observation, POS phagocytosis assay, frozen tissue sections, live/dead assay, SA-β-Gal activity assay, and immunocytochemistry. Results The derived hiPSC-RPE cells positively expressed biomarkers of RPE cells but not iPSCs, such as CRALBP (97.4%), EMMPRIN (93.8%), Oct4 (2.1%), and Sox2 (2.0%). hiPSC-RPE cells displayed RPE-like characteristics including barrier function, phagocytic activity, and polarized membrane. hiPSC-RPE cell spheroids positively expressed Nestin and exhibited reduced SA-β-Gal staining. Injectable hiPSC-RPE cell spheroids could form monolayers on decellularized corneal matrixes (DCM). After subretinal transplantation for one month, hiPSC-RPE cell spheroids could survive and maintain segmental sheet growth in RPE-degenerated chinchilla rabbits. Conclusion This study realized that NCM dissociated hiPSCs were effectively differentiated into transplantable and functional RPE through the sequential addition of defined factors but not involving exogenous genes. This study may lay the foundation for the clinical transplantation of hiPSC-RPE cell spheroids as therapy for RPE degenerative diseases in the future.

Download Full-text