Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Silverchair Information Systems full text link Silverchair Information Systems Free PMC article
Full text links

Actions

Share

Comparative Study
.2019 Jul;8(7):694-706.
doi: 10.1002/sctm.18-0267. Epub 2019 Mar 27.

Systematic Comparison of Retinal Organoid Differentiation from Human Pluripotent Stem Cells Reveals Stage Specific, Cell Line, and Methodological Differences

Affiliations
Comparative Study

Systematic Comparison of Retinal Organoid Differentiation from Human Pluripotent Stem Cells Reveals Stage Specific, Cell Line, and Methodological Differences

Carla B Mellough et al. Stem Cells Transl Med.2019 Jul.

Abstract

A major goal in the stem cell field is to generate tissues that can be utilized as a universal tool for in vitro models of development and disease, drug development, or as a resource for patients suffering from disease or injury. Great efforts are being made to differentiate human pluripotent stem cells in vitro toward retinal tissue, which is akin to native human retina in its cytoarchitecture and function, yet the numerous existing retinal induction protocols remain variable in their efficiency and do not routinely produce morphologically or functionally mature photoreceptors. Herein, we determine the impact that the method of embryoid body (EB) formation and maintenance as well as cell line background has on retinal organoid differentiation from human embryonic stem cells and human induced pluripotent stem cells. Our data indicate that cell line-specific differences dominate the variables that underline the differentiation efficiency in the early stages of differentiation. In contrast, the EB generation method and maintenance conditions determine the later differentiation and maturation of retinal organoids. Of the latter, the mechanical method of EB generation under static conditions, accompanied by media supplementation with Y27632 for the first 48 hours of differentiation, results in the most consistent formation of laminated retinal neuroepithelium containing mature and electrophysiologically responsive photoreceptors. Collectively, our data provide substantive evidence for stage-specific differences in the ability to give rise to laminated retinae, which is determined by cell line-specific differences in the early stages of differentiation and EB generation/organoid maintenance methods at later stages.

Keywords: Human embryonic stem cells; Induced pluripotent stem cells; Organoids; Retina; Tissue engineering.

© 2019 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

PubMed Disclaimer

Conflict of interest statement

The authors indicated no potential conflicts of interest.

Figures

Figure 1
Figure 1
Schematic showing the various methods of embryoid body (EB) formation tested at the onset of differentiation. EB formation was initiated using either a mechanical (M), enzymatic (E), or dissociation–reaggregation approach in V‐shaped 96 well plates seeded with 9,000 (V9) cells per well plus or minus Y‐27632 (Y) in mTeSR1 medium for the first 48 hours, under either stationary (St) or shaking (Sh) conditions. Cells were then differentiated in the presence of IGF‐1 with B27 supplementation throughout and N2 supplementation from day 37. Serum supplementation was reduced to 0% by day 37. Organoids were collected at regular intervals for analysis.
Figure 2
Figure 2
Phase micrographs showing the morphology of cultures generated by different methods during the first 48 hours of differentiation. Proliferative cultures on day 0 collected by mechanical means(A, D) were observed as square‐shaped floating fragments of colonies which, by day 2(B, C, E, F), gave rise to small EBs in cultures that still contained cell debris. (D–F): Fewer embryoid bodies (EBs) were formed under shaking conditions following mechanical EB formation.(G, J): Cultures collected by enzymatic means on day 0 consisted of larger fragments in comparison to mechanical cultures, and were more phase‐bright with softer edges.(H, I, K, L): By day 2, similarly sized enzymatically derived EBs were generated under both stationary (St) and shaking (Sh) conditions with the addition of Y‐27632 (Y) resulting in EBs of increased size. Less cellular debris was observed in enzymatically generated cultures during the first 48 hours.(M–O): Micrographs showing V9 EBs formed by dissociation–reaggregation on (M) day 0 and (N, O) after 48 hours in the absence (N) or presence (O) of Y‐27632. (O): V9&Y EBs were notably larger. All scale bars = 200 μm.
Figure 3
Figure 3
Gene expression analysis reveals that retinal organoids cluster by cell type and not by method at week 5 of differentiation.(A): Principal component analysis (PCA) analysis showing clustering by cell type. All three pluripotent cell lines, undifferentiated human embryonic stem cell and human embryonic eye, and fetal retinal samples are indicated by a different color.(B): PCA analysis showing no clustering by method of retinal organoid formation; all methods are indicated by different colors.(C): Heatmap showing differentially expressed genes between retinal organoids generated from all three pluripotent stem cell lines at week 5 of differentiation,p < .05.
Figure 4
Figure 4
Retinal organoids are more similar to human embryonic eye samples than fetal/adult neural retina at week 5 of differentiation.(A): Schematic chart showing the number of differentially expressed genes between retinal organoids generated with different methods and human embryonic eye samples (hEye).(B): Schematic chart showing the number of differentially expressed genes between retinal organoids generated with different methods and human fetal retina samples (hRet). The photoreceptor, cone, and rod markers are shown by different shape and color (A, B).(C): Heatmaps based on major retinal cell types showing differentially expressed genes between retinal organoids generated with various methods and hEye samples,p < .05.(D): Heatmaps based on major retinal cell types showing differentially expressed genes between retinal organoids generated with various methods and hRet samples,p < .05.
Figure 5
Figure 5
Immunohistochemistry on human embryonic stem cell‐derived organoids revealed the highest frequency of developing retinal tissue (78.5% of embryoid bodies) in cultures derived using the “M&Y_St” method on week 5 of differentiation.(A–E): Immunohistochemical screening of retinal organoids on week 5 of differentiation revealed developing neuroepithelial tissue containing (A) retinal progenitor cells stained with VSX2 and (B) Sox2, as well as photoreceptors stained with (B) OTX2 and (C) recoverin at the apical aspect, (D) retinal ganglion cells stained with Smi32 at the basal aspect, and (E) evidence of synaptogenesis in the developing outer nuclear layer (ONL) and outer plexiform layer as indicated by synaptophysin immunoreactivity. Neural retinal formation was observed at the highest frequency in cultures generated using a “M&Y_St” method and retinal tissue continued to develop over time in culture.(F): Retinal photoreceptors in the ONL of organoids attain morphological features of maturing cells, here shown at week 21 of differentiation, stained with Crx and Recoverin. Scale bars = 50 μm.
Figure 6
Figure 6
Characterization of human induced pluripotent stem cell‐derived organoids revealed a high frequency of developing retinal tissue in cultures derived using the “M&Y_St” method on week 5 of differentiation. Developing retinal neuroepithelium in(A, B) 12.5% of Neo1‐derived and(C–F) 94.4% of AD3‐derived organoids, demonstrating the presence of (A, F) photoreceptors (Recoverin, OTX2), (B) retinal ganglion cells (Smi32, RBPMS), (C) synaptic connections, (D) interneurons (Ap2α), and (E, F) Müller glial cells (Vimentin, CRALBP). Scale bars = 50 μm.
Figure 7
Figure 7
Retinal organoids generated under static conditions are more similar to fetal retina samples at later stages of differentiation (19–21 week of differentiation).(A): Principal component analysis showing clustering by condition, shaking, and static conditions are shown with different shapes.(B): Pearson correlation between retinal organoids and human embryonic eyes and fetal retinal (hRet) samples. The range of values within groups is shown in the boxplots.(C): Heatmap showing differentially expressed genes between retinal organoids generated under static and shaking conditions and hRet samples,p < .05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hirami Y, Osakada F, Takahashi K et al. Generation of retinal cells from mouse and human induced pluripotent stem cells. Neurosci Lett 2009;458:126–131. - PubMed
    1. Lamba DA, Karl MO, Ware CB et al. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci USA 2006;103:12769–12774. - PMC - PubMed
    1. Mellough CB, Sernagor E, Moreno‐Gimeno I et al. Efficient stage‐specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells. Stem Cells 2012;30:673–686. - PubMed
    1. Osakada F, Ikeda H, Mandai M et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol 2008;26:215–224. - PubMed
    1. Osakada F, Jin ZB, Hirami Y et al. In vitro differentiation of retinal cells from human pluripotent stem cells by small‐molecule induction. J Cell Sci 2009;122:3169–3179. - PubMed

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full text links
Silverchair Information Systems full text link Silverchair Information Systems Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp