@article{81363b7f09db42b79fa9e3e2bd367d9d,
title = "CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids",
abstract = "Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.",
keywords = "Pou4f2, SIX6, hypoxia, microenvironment, optic, organoid, retina, vesicle",
author = "Wahlin, {Karl J.} and Jie Cheng and Jurlina, {Shawna L.} and Jones, {Melissa K.} and Dash, {Nicholas R.} and Anna Ogata and Nawal Kibria and Sunayan Ray and Eldred, {Kiara C.} and Catherine Kim and Heng, {Jacob S.} and Jenny Phillips and Johnston, {Robert J.} and Gamm, {David M.} and Cynthia Berlinicke and Zack, {Donald J.}",
note = "Funding Information: This work was funded by the NIH (R01EY031318, R01EY009769, R01EY030872, R21EY031122, 5P30EY001765, K99/R00EY024648, P30EY022589, and T32EY026590), California Institute for Regenerative Medicine (CIRM) DISC1-08683, Maryland Stem Cell Research Foundation, Foundation Fighting Blindness, Research to Prevent Blindness, Knights Templar Eye Foundation (KTEF) Career-Starter Research Grant, Edward N. and Della L. Thome Memorial Foundation grant for Age-Related Macular Degeneration Research, BrightFocus Foundation, and generous gifts from the Guerrieri Family Foundation (DZ), Retina Research Foundation Emmett Humble Funding Information: This work was funded by the NIH (R01EY031318, R01EY009769, R01EY030872, R21EY031122, 5P30EY001765, K99/R00EY024648, P30EY022589, and T32EY026590), California Institute for Regenerative Medicine (CIRM) DISC1-08683, Maryland Stem Cell Research Foundation, Foundation Fighting Blindness, Research to Prevent Blindness, Knights Templar Eye Foundation (KTEF) Career-Starter Research Grant, Edward N. and Della L. Thome Memorial Foundation grant for Age-Related Macular Degeneration Research, BrightFocus Foundation, and generous gifts from the Guerrieri Family Foundation (DZ), Retina Research Foundation Emmett Humble Chair and McPherson Eye Research Institute Sandra Lemke Trout Chair in Eye Research (to DG), and the Richard C. Atkinson Laboratory for Regenerative Ophthalmology (KW). This study was supported in part by a core grant to the Waisman Center (NICHHD U54 HD090256). Publisher Copyright: Copyright {\textcopyright} 2021 Wahlin, Cheng, Jurlina, Jones, Dash, Ogata, Kibria, Ray, Eldred, Kim, Heng, Phillips, Johnston, Gamm, Berlinicke and Zack.",
year = "2021",
month = nov,
day = "16",
doi = "10.3389/fcell.2021.764725",
language = "English (US)",
volume = "9",
journal = "Frontiers in Cell and Developmental Biology",
issn = "2296-634X",
publisher = "Frontiers Media S. A.",
}