Journal: International Journal of Molecular Sciences

Article Title: The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development

doi: 10.3390/ijms22137081

Figure Lengend Snippet: Summary of the additions used to promote retinal differentiation in organoid protocols and their frequency, concentration, and time interval of use. Analysis of the literature uncovered many supplements that are added to regulate signaling pathways throughout the development of retinal organoids. We decided to use the conditions and concentrations highlighted with an asterisk (*) and test their effect on retinal differentiation in our organoid protocol, representing the more popular time points or concentrations used for a range of conditions affecting different signaling pathways. For further explanation of the individual additions used, see text.

Article Snippet: For experiments containing supplements ( n = 1), they were added with every medium change unless stated otherwise as follows: Rock Inhibitor (10 µM—SelleckChem, Houston, TX, USA) from day 0 to 1, IGF1 (5 ng/mL—Thermofisher, Waltham, MA, USA) from day 0 onwards, IWR1e (3 µM—Merck Millipore, Burlington, MA, USA) from day 0 to 12, SB431542 (10 µM—Sigma, St. Louis, MO, USA) + LDN193189 (100 nM—SelleckChem, Houston, TX, USA) from day 0 to 7, CHIR99021 (3 µM—Merck Millipore, Burlington, MA, USA) from day 18 to 24, SU5402 (5 µM—StemCell Technologies, Vancouver, BC, Canada) from day 18 to 24, CHIR99021 + SU5402 double treatment from day 18 to 24, and DAPT (10 mM—Sigma, St. Louis, MO, USA), from day 28 onwards.

Techniques: Concentration Assay, Inhibition, Binding Assay, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development

doi: 10.3390/ijms22137081

Figure Lengend Snippet: Experimental outline of pilot. A collection of frequently used agonists and antagonists for different signaling pathways reviewed in this article were systematically tested for their efficiency at directing retinal development. Adapting our previously published protocol , stem cell clumps were embedded in 3D Matrigel drops for 4 days, transitioning from mTeSR1 medium to neural induction medium (NIM). At day 4, they were removed from the gel and plated in a 2D environment for a further 10 days to allow the embryoid bodies to enter the anterior neural fate. Once neurospheres had formed, they were scraped off and cultured 3D in a floating environment in retinal differentiation medium (RDM) to form organoids. Condition 1 was the control condition, with no supplements added. Condition 2 consisted of treatment with Rock Inhibitor from day 0 to 1 only. Condition 3 included a continuous treatment of IGF1 from day 0 to 34. Condition 4 consisted of IWR1e treatment from day 0 to 12. Condition 5 included a double treatment of SB431542 and LDN193189 from day 0 to 7. Conditions 6–8 all included treatments from day 18 to 24 consisting of CHIR99021, SU5402, and CHIR99021 + SU5402, respectively. Finally, condition 9 consisted of treatment with DAPT from day 28 to 34. All culture conditions were kept until day 34, and samples were taken at day 0, 4, 14, 24 and 34 for analysis. (Pilot study: n = 1 for all conditions).

Article Snippet: For experiments containing supplements ( n = 1), they were added with every medium change unless stated otherwise as follows: Rock Inhibitor (10 µM—SelleckChem, Houston, TX, USA) from day 0 to 1, IGF1 (5 ng/mL—Thermofisher, Waltham, MA, USA) from day 0 onwards, IWR1e (3 µM—Merck Millipore, Burlington, MA, USA) from day 0 to 12, SB431542 (10 µM—Sigma, St. Louis, MO, USA) + LDN193189 (100 nM—SelleckChem, Houston, TX, USA) from day 0 to 7, CHIR99021 (3 µM—Merck Millipore, Burlington, MA, USA) from day 18 to 24, SU5402 (5 µM—StemCell Technologies, Vancouver, BC, Canada) from day 18 to 24, CHIR99021 + SU5402 double treatment from day 18 to 24, and DAPT (10 mM—Sigma, St. Louis, MO, USA), from day 28 onwards.

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development

doi: 10.3390/ijms22137081

Figure Lengend Snippet: Changes in retinal development through microscopy and gene expression assay. ( A ) All conditions developed comparable embryoid bodies to the control up to day 4. ( B ) Throughout the next 2D stage of generating neurospheres (day 4–14), changes between conditions were observed. Rock Inhibitor-treated cells had two distinct types of epithelial outgrowth, with a border separating them, whereas IWR1e-treated cells resulted in poor epithelial outgrowth and an increase in cell death. ( C ) Only IWR1e, IGF1, CHIR99021 + SU5402, and CHIR99021 treatments showed significant changes in retinal organoid yield compared to the control. IWR1e treatment severely impact retinal organoid yield and the overall organoid size. Continuous IGF1 treatment resulted in almost double the number of retinal organoids compared to the control. CHIR99021/SU5402 double treatment increased retinal organoid yield 2.5-fold; however, individual CHIR99021 treatment gave the best yield with a 5-fold increase. ( D ) The differences between conditions were also largely reflected in the gene expression of key developmental markers such as PAX6 , RAX and VSX2 , as well as cell-specific markers such as MITF and ATOH7 . Differences of retinal development genes were observed between conditions over time (Day 0, 4, 14, 24, 34). Unfortunately, IWR1e treatment resulted in poor organoid yields and subsequently, there was less RNA available for day 24, as represented by the EEF1A reference gene sample.

Article Snippet: For experiments containing supplements ( n = 1), they were added with every medium change unless stated otherwise as follows: Rock Inhibitor (10 µM—SelleckChem, Houston, TX, USA) from day 0 to 1, IGF1 (5 ng/mL—Thermofisher, Waltham, MA, USA) from day 0 onwards, IWR1e (3 µM—Merck Millipore, Burlington, MA, USA) from day 0 to 12, SB431542 (10 µM—Sigma, St. Louis, MO, USA) + LDN193189 (100 nM—SelleckChem, Houston, TX, USA) from day 0 to 7, CHIR99021 (3 µM—Merck Millipore, Burlington, MA, USA) from day 18 to 24, SU5402 (5 µM—StemCell Technologies, Vancouver, BC, Canada) from day 18 to 24, CHIR99021 + SU5402 double treatment from day 18 to 24, and DAPT (10 mM—Sigma, St. Louis, MO, USA), from day 28 onwards.

Techniques: Microscopy, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development

doi: 10.3390/ijms22137081

Figure Lengend Snippet: Retinal organoid yields. Cultures were analyzed on day 25, and both retinal and non-retinal organoids were manually counted for each condition through brightfield microscopy images. The yield of retinal organoids compared to the total amount of organoids was then calculated for each condition either focusing on the early-stage regulation (Rock Inhibitor, IWR1e, IGF1, SB431542/LDN193189) or late-stage regulation (CHIR99021, SU5402, CHIR99021/SU5402, DAPT) of signaling pathways.

Article Snippet: For experiments containing supplements ( n = 1), they were added with every medium change unless stated otherwise as follows: Rock Inhibitor (10 µM—SelleckChem, Houston, TX, USA) from day 0 to 1, IGF1 (5 ng/mL—Thermofisher, Waltham, MA, USA) from day 0 onwards, IWR1e (3 µM—Merck Millipore, Burlington, MA, USA) from day 0 to 12, SB431542 (10 µM—Sigma, St. Louis, MO, USA) + LDN193189 (100 nM—SelleckChem, Houston, TX, USA) from day 0 to 7, CHIR99021 (3 µM—Merck Millipore, Burlington, MA, USA) from day 18 to 24, SU5402 (5 µM—StemCell Technologies, Vancouver, BC, Canada) from day 18 to 24, CHIR99021 + SU5402 double treatment from day 18 to 24, and DAPT (10 mM—Sigma, St. Louis, MO, USA), from day 28 onwards.

Techniques: Microscopy