Thyroid hormone signaling specifies cone subtypes in human retinal organoids

Abstract

The mechanisms underlying the specification of diverse neuronal subtypes within the human nervous system are largely unknown. The blue (shortwavelength/S), green (medium-wavelength/M) and red (long-wavelength/L) cone photoreceptors of the human retina enable high-acuity daytime vision and trichromatic color perception. Cone subtypes are specified in a poorly understood two-step process, with a first decision between S and L/M fates, followed by a decision between L and M fates. To determine the mechanism controlling S vs. L/M fates, we studied the differentiation of human retinal organoids. We found that human organoids and retinas have similar distributions, gene expression profiles, and morphologies of cone subtypes. We found that S cones are specified first, followed by L/M cones, and that thyroid hormone signaling is necessary and sufficient for this temporal switch. Temporally dynamic expression of thyroid hormone degrading and activating proteins supports a model in which the retina itself controls thyroid hormone levels, ensuring low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining the mechanisms of cell fate specification during human development.One sentence summaryCone specification in human organoids

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids

“…Therefore, the ratio of cones to rods in our retinal organoids is higher than the ratio found in the macula and lower than the ratio in the fovea. The regulator for cone commitment is still elusive (57), although thyroid hormone signaling directs cone subtypes in mice and human retina (16,58). In our floating culture, abundant cone photoreceptors spontaneously differentiate from VSX2 + epithelium, consistent with the default cone commitment hypothesis in the retina (57).…”

“…Although retinal organoids with differentiated cone and rod photoreceptors were previously reported by us and others (7,8,11,(13)(14)(15)(16)(17), the ratio of cones to rods has not been rigorously determined using scRNA-seq until the current study. Based on immunostaining, Li et al (13) found seven rod-rich and six conerich retinal organoids out of 14 retinal organoids, but the ratio of cones to rods was not quantified.…”

“…In contrast to our findings, Li et al's (13) retinal organoids expressed L/M opsin and rhodopsin in the cytoplasm and did not display overt inner and outer segments of photoreceptors. Eldred et al (16) determined the ratio of S cones to L/M cones but did not quantify the ratio of cones to rods. Two recent scRNA-seq studies of long-term retinal organoids did not report the ratio of cones to rods (18,19).…”

“…Regenerative medicine for the retina is particularly attractive due to its impact on improving the quality of life and the accessibility of the retina (3). A number of research groups, including our own, have generated retinal structures using pluripotent stem cells (4)(5)(6)(7)(8)(9)(10)(11)(12) and demonstrated substantially mature cone and rod photoreceptors in long-term retinal organoids (7,8,11,(13)(14)(15)(16)(17). Specifically, Zhong et al (8) generated rod-dominant retinal organoids confirmed by their recent follow-up study (14).…”

The primate model for understanding and restoring vision

“…Photoreceptor transplantation studies focusing on cones can now be tested in the genetic model of achromatopsia, taking advantage of the cone-silent state and relatively stable architecture of the macula in these NHPs (9). Cone replacement has suffered from challenges in the production of cone photoreceptors in vitro from human iPSC-derived retinal organoids, although some strategies have been reported to bias cells to adopt this fate (126,127). There is now renewed interest in investigating human cell transplantation to the retina (128), as advances in the past decade using mouse models appear to be in part secondary to material transfer between transplanted cells and endogenous cells (129,130).…”