CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids.

Karl J Wahlin,Sunayan Ray,Melissa K Jones,Catherine Kim,Robert J Johnston,Nawal Kibria,Shawna L Jurlina,Jenny Phillips,David M Gamm,Cynthia Berlinicke,Kiara C Eldred,Donald J Zack,Jacob S Heng,Anna Ogata,Nicholas R Dash,Jie Cheng

doi:10.3389/fcell.2021.764725

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.

Highlights

Retinal degenerative (RD) diseases range from genetically complex age-related macular degeneration (AMD), which is the most common cause of irreversible blindness in the elderly in the Western world, to Mendelian inherited orphan diseases such as retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA)
A guide RNA targeting the stop codon of the SIX homeobox 6 (SIX6) gene was used with S. pyogenes Cas9 (SpCas9) to facilitate in-frame insertion of a nuclear localized histone 2B (H2B)-GFP just before the stop codon of SIX6 (Figures 1B,C)
To rule out the possibility of mutagenesis at the “unedited” allele, we gel extracted and sequenced the lower band (Supplementary Figure 2). We differentiated these cells toward a retinal lineage using a 3D retinal organoid approach as previously described (Wahlin et al, 2017; Figure 1D)

Summary

Introduction

Retinal degenerative (RD) diseases range from genetically complex age-related macular degeneration (AMD), which is the most common cause of irreversible blindness in the elderly in the Western world, to Mendelian inherited orphan diseases such as retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA). Promising are 3D retinal organoids derived from human pluripotent stem cells (PSCs) which produce all five major neuronal cell types in the retina [i.e., photoreceptor (PRs), bipolar (BCs), horizontal (HCs), amacrine (ACs), and ganglion cells (RGCs)] (Meyer et al, 2009; Nakano et al, 2012; Kuwahara et al, 2015; Lowe et al, 2016; Volkner et al, 2016; Browne et al, 2017; Wahlin et al, 2017; Eldred et al, 2018; Capowski et al, 2019) In limited examples, they respond to light (Zhong et al, 2014; Hallam et al, 2018). Despite the great potential of PSCs, differentiation protocols are not completely standardized (Meyer et al, 2011; Nakano et al, 2012; Zhong et al, 2014; Kuwahara et al, 2015; Singh et al, 2015; Zhou et al, 2015) and variability between organoids can exist

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Conclusion