scholarly journals Multiocular organoids from human induced pluripotent stem cells displayed retinal, corneal, and retinal pigment epithelium lineages

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Helena Isla-Magrané ◽  
Anna Veiga ◽  
José García-Arumí ◽  
Anna Duarri
Keyword(s):  
Stem Cells ◽  
Retinal Pigment Epithelium ◽  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Eye Development ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Types ◽  
Induced Pluripotent ◽  
Different Cell Types

Abstract Background Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated. Methods A 2-step method was established to first obtain self-organized multizone ocular progenitor cells (mzOPCs) from 2D hiPSC cultures within three weeks. Then, after the cells were manually isolated and grown in suspension, 3D multiocular organoids were generated to model important cellular features of developing eyes. Results In the 2D culture, self-formed mzOPCs spanned the neuroectoderm, surface ectoderm, neural crest, and RPE, mimicking early stages of eye development. After lifting, mzOPCs developed into different 3D multiocular organoids composed of multiple cell lineages including RPE, retina, and cornea, and interactions between the different cell types and regions of the eye system were observed. Within these organoids, the retinal regions exhibited correct layering and contained all major retinal cell subtypes as well as retinal morphological cues, whereas the corneal regions closely resembled the transparent ocular-surface epithelium and contained of corneal, limbal, and conjunctival epithelial cells. The arrangement of RPE cells also formed organoids composed of polarized pigmented epithelial cells at the surface that were completely filled with collagen matrix. Conclusions This approach clearly demonstrated the advantages of the combined 2D-3D construction tissue model as it provided a more ocular native-like cellular environment than that of previous models. In this complex preparations, multiocular organoids may be used to model the crosstalk between different cell types in eye development and disease. Graphical abstract

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

2020 ◽  
Author(s):  
Xiaoling Guo ◽  
Deliang Zhu ◽  
Ruiling Lian ◽  
Qiaolang Zeng ◽  
Sanjana Mathew ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinal Pigment Epithelium ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Great Promise ◽  
Rpe Cells ◽  
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. 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.

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

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.

scholarly journals Possibilities for Using Pluripotent Stem Cells for Restoring Damaged Eye Retinal Pigment Epithelium

Acta Naturae ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 30-39 ◽  
Author(s):  
A. E. Kharitonov ◽  
A. V. Surdina ◽  
O. S. Lebedeva ◽  
A. N. Bogomazova ◽  
M. A. Lagarkova
Keyword(s):  
Stem Cells ◽  
Retinal Pigment Epithelium ◽  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Retinal Pigment Epithelial ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
The United States ◽  
Pigment Epithelial ◽  
Pigment Epithelial Cells

The retinal pigment epithelium is a monolayer of pigmented, hexagonal cells connected by tight junctions. These cells compose part of the outer blood-retina barrier, protect the eye from excessive light, have important secretory functions, and support the function of photoreceptors, ensuring the coordination of a variety of regulatory mechanisms. It is the degeneration of the pigment epithelium that is the root cause of many retinal degenerative diseases. The search for reliable cell sources for the transplantation of retinal pigment epithelium is of extreme urgency. Pluripotent stem cells (embryonic stem or induced pluripotent) can be differentiated with high efficiency into the pigment epithelium of the retina, which opens up possibilities for cellular therapy in macular degeneration and can slow down the development of pathology and, perhaps, restore a patient's vision. Pioneering clinical trials on transplantation of retinal pigment epithelial cells differentiated from pluripotent stem cells in the United States and Japan confirmed the need for developing and optimizing such approaches to cell therapy. For effective use, pigment epithelial cells differentiated from pluripotent stem cells should have a set of functional properties characteristic of such cells in vivo. This review summarizes the current state of preclinical and clinical studies in the field of retinal pigment epithelial transplantation therapy. We also discuss different differentiation protocols based on data in the literature and our own data, and the problems holding back the widespread therapeutic application of retinal pigment epithelium differentiated from pluripotent stem cells.

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