scholarly journals Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

10.3791/52262 ◽  
2015 ◽  
Author(s):  
Alberto Muñiz ◽  
Kaini R. Ramesh ◽  
Whitney A. Greene ◽  
Jae-Hyek Choi ◽  
Heuy-Ching Wang
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ips Cells ◽  
Embryoid Bodies ◽  
Induced Pluripotent

scholarly journals Profiling the microRNA Expression in Human iPS and iPS-derived Retinal Pigment Epithelium

2014 ◽  
Vol 13s5 ◽  
pp. CIN.S14074 ◽  
Author(s):  
Heuy-Ching Wang ◽  
Whitney A. Greene ◽  
Ramesh R. Kaini ◽  
Jane Shen-Gunther ◽  
Hung-I H Chen ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Mirna Expression ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ips Cells ◽  
Differentially Expressed ◽  
Induced Pluripotent

The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore, we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs, RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129–5p may play a role in promoting differentiation, while down-regulated miRNAs such as miR-367, miR-18b, and miR-20b are implicated in cell proliferation. Subsequent miRNA-target and network analysis revealed that these miRNAs are involved in cellular development, cell cycle progression, cell death, and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis, eye differentiation and development.

scholarly journals Automating Human Induced Pluripotent Stem Cell Culture and Differentiation of iPSC-Derived Retinal Pigment Epithelium for Personalized Drug Testing

2020 ◽  
pp. 247263032097211
Author(s):  
Vincent Truong ◽  
Kevin Viken ◽  
Zhaohui Geng ◽  
Samantha Barkan ◽  
Blake Johnson ◽  
...  
Keyword(s):  
Cell Culture ◽  
Retinal Pigment Epithelium ◽  
Cell Lines ◽  
Drug Screening ◽  
Drug Testing ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
End To End ◽  
Induced Pluripotent

Derivation and differentiation of human induced pluripotent stem cells (hiPSCs) provide the opportunity to generate medically important cell types from individual patients and patient populations for research and the development of potential cell therapies. This technology allows disease modeling and drug screening to be carried out using diverse population cohorts and with more relevant cell phenotypes than can be accommodated using traditional immortalized cell lines. However, technical complexities in the culture and differentiation of hiPSCs, including lack of scale and standardization and prolonged experimental timelines, limit the adoption of this technology for many large-scale studies, including personalized drug screening. The entry of reproducible end-to-end automated workflows for hiPSC culture and differentiation, demonstrated on commercially available platforms, provides enhanced accessibility of this technology for both research laboratories and commercial pharmaceutical testing. Here we have utilized TECAN Fluent automated cell culture workstations to perform hiPSC culture and differentiation in a reproducible and scalable process to generate patient-derived retinal pigment epithelial cells for downstream use, including drug testing. hiPSCs derived from multiple donors with age-related macular degeneration (AMD) were introduced into our automated workflow, and cell lines were cultured and differentiated into retinal pigment epithelium (RPE). Donor hiPSC-RPE lines were subsequently entered in an automated drug testing workflow to measure mitochondrial function after exposure to “mitoactive” compounds. This work demonstrates scalable, reproducible culture and differentiation of hiPSC lines from individuals on the TECAN Fluent platform and illustrates the potential for end-to-end automation of hiPSC-based personalized drug testing.

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.

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

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