Showing papers by "Tiansen Li published in 2019"

Improved Retinal Organoid Differentiation by Modulating Signaling Pathways Revealed by Comparative Transcriptome Analyses with Development In Vivo

Abstract: Summary Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

Abstract: Purpose: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. Methods: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. Results: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. Conclusion: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

Abstract: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability in temporal dynamics of differentiation. To assess the maturity of neural retina in vitro, we performed transcriptome analyses of developing organoids from human embryonic and induced pluripotent stem cell lines. We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrated that addition of 9-cis retinal, instead of widely-used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. Our studies thus provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids, which should facilitate disease modeling and evaluation of therapies in vitro. Summary Statement Three-dimensional organoids derived from human pluripotent stem cells have been extensively applied for investigating organogenesis, modeling diseases and development of therapies. However, substantial variations within organoids pose challenges for comparison among different cultures and studies. We generated transcriptomes of multiple distinct retinal organoids and compared these to human fetal and adult retina gene profiles for molecular staging of differentiation state of the cultures. Our analysis revealed the advantage of using 9-cis retinal, instead of the widely-used all-trans retinoic acid, in facilitating rod photoreceptor differentiation. Thus, a transcriptome-based comparison can provide an objective method to uncover the maturity of organoid cultures across different lines and in various study platforms.

Retinal disease in ciliopathies: Recent advances with a focus on stem cell-based therapies

Abstract: Ciliopathies display extensive genetic and clinical heterogeneity, varying in severity, age of onset, disease progression and organ systems affected. Retinal involvement, as demonstrated by photoreceptor dysfunction or death, is a highly penetrant phenotype among a vast majority of ciliopathies. Photoreceptor cells possess a specialized and modified sensory cilium with membrane discs where efficient photon capture and ensuing signaling cascade initiate the visual process. Disruptions of cilia biogenesis and protein transport lead to impairment of photoreceptor function and eventually degeneration. Despite advances in elucidation of ciliogenesis and photoreceptor cilia defects, we have limited understanding of pathogenic mechanisms underlying retinal phenotype(s) observed in human ciliopathies. Patient-derived induced pluripotent stem cell (iPSC)-based approaches offer a unique opportunity to complement studies with model organisms and examine cilia disease relevant to humans. Three-dimensional retinal organoids from iPSC lines feature laminated cytoarchitecture, apical-basal polarity and emergence of a ciliary structure, thereby permitting pathogenic modeling of human photoreceptors in vitro. Here, we review the biology of photoreceptor cilia and associated defects and discuss recent progress in evolving treatment modalities, especially using patient-derived iPSCs, for retinal ciliopathies.

Differential expression and subcellular localization of Copines in mouse retina.

Abstract: Combinatorial expression of Brn3 transcription factors is required for the development of cell-specific morphologies in retinal ganglion cells (RGCs). The molecular mechanisms by which Brn3s regulate RGC type specific features are largely unexplored. We previously identified several members of the Copine (Cpne) family of molecules as potential targets of Brn3 transcription factors in the retina. We now use in situ hybridization and immunohistochemistry to characterize Copine expression in the postnatal and adult mouse retina. We find that Cpne5, 6, and 9 are expressed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in both amacrine cells and RGCs. Cpne4 expression is restricted to one amacrine cell population of the INL, but is specifically expressed in RGCs in the GCL. Cpne4 expression in RGCs is regulated by Brn3b both cell autonomously (in Brn3b+ RGCs) and cell nonautonomously (in Brn3b- RGCs). Copines exhibit a variety of subcellular distributions when overexpressed in tissue culture cells (HEK293), and can induce the formation of elongated processes reminiscent of neurites in these non-neuronal cells. Our results suggest that Copines might be involved in a combinatorial fashion in Brn3b-dependent specification of RGC types. Given their expression profile and previously proven role as Ca2+ sensors, they may participate in the morphogenetic processes that shape RGC dendrite and axon formation at early postnatal ages.