Generation of Lens Progenitor Cells and Lentoid Bodies from Pluripotent Stem Cells: Novel Tools for Human Lens Development and Ocular Disease Etiology

Abstract

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids and specific ocular tissues from pluripotent stem cells has provided invaluable insights into the formation of lineage-committed progenitor cell populations, signal transduction pathways, and self-organization principles. This review provides a comprehensive summary of recent advances in generation of adenohypophyseal, olfactory, and lens placodes, lens progenitor cells and three-dimensional (3D) primitive lenses, "lentoid bodies", and "micro-lenses". These cells are produced alone or "community-grown" with other ocular tissues. Lentoid bodies/micro-lenses generated from human patients carrying mutations in crystallin genes demonstrate proof-of-principle that these cells are suitable for mechanistic studies of cataractogenesis. Taken together, current and emerging advanced in vitro differentiation methods pave the road to understand molecular mechanisms of cataract formation caused by the entire spectrum of mutations in DNA-binding regulatory genes, such as PAX6, SOX2, FOXE3, MAF, PITX3, and HSF4, individual crystallins, and other genes such as BFSP1, BFSP2, EPHA2, GJA3, GJA8, LIM2, MIP, and TDRD7 represented in human cataract patients.

Keywords: PAX6; cranial placodes; crystallins; de-nucleation; differentiation; gene expression; lens progenitor cells; lentoid bodies; optic cup; pluripotent stem cells; self-organization.

Figure 1

Formation of three anterior cranial placodes during mid-stage mouse embryogenesis. (A) Highly schematic visualization of future location of adenohypophyseal, olfactory, and lens placodes relative to the neural tube and its anterior/posterior patterning at the E8.0 mouse embryo. Regarding the eye and lens formation, note that the eye field is already formed within the anterior subregion of the neural plate [30] (not shown), and undergoes its symmetric division, later forming a symmetric pair of optic vesicles (see panel D). (B) Adenohypophyseal (pituitary) gland development from E9.5 via Rathke’s pouch [31] to E13.5, forming the infundibular stalk and the pars tuberalis [32]. (C) Olfactory placode development from E9.5 to E12.5, forming the vomeronasal organ [33]. (D) Lens placode development from E8.5 to E11.5, forming the lens vesicle [34]. The prospective corneal epithelium formed after the separation of the lens vesicle from the surface ectoderm is highlighted in gray. Note that for simplification, neural crest cells including periocular mesenchymal cells are not shown, but they are generally located in the space between the neuroectoderm and surface ectoderm. For additional details, see https://syllabus.med.unc.edu/ for ultrastructural images of mouse eye development between these stages.

Figure 2

Summary of three-stage procedure to generate human lentoid bodies. (A) Lentoid bodies were differentiated over a 35-day period on Matrigel using specific concentrations of Noggin, BMP4, BMP7, FGF2, and WNT3A at the time points shown. Additional components and their concentrations of the basal medium are also shown [125]. (B) Western blot analysis shows expression of key lens markers PAX6, αA-, αB-, β-, and γ-crystallins, filensin (BFSP1), CP49 (BFSP2), and MIP (AQP0). Expression of β-actin was used as a loading control [125]. (C) Brightfield image of lentoid bodies produced at day 35 of differentiation.

Figure 3

A summary model of the generation of anterior placodes from hPSCs using the dSMADi-based procedures. The diagram illustrates multiple cell fate decisions of the pluripotent stem cells, including those following Noggin- and SB431542-mediated phase of inhibition and TGF-β, Activin, and Nodal signaling inhibition [139]. Formation of anterior pre-placodal cell fate commitment can be subsequently induced through BMP activation. In contrast, epidermal precursors can be generated through surface ectoderm induction mediated by FGF inhibition. The anterior pre-placodal cells can generate anterior pituitary placodal cells through activation of SHH signaling to produce gonadotrops at day 30 of the cultures [139]. Note that these cells treated subsequently by FGF8, BMP2, and both factors together generate hormone-producing cells analyzed at day 60 [140]. The olfactory placodal cell fate pathway analysis was limited to identification of a few cell markers, SIX1, DLX5, and FOXG1, and is marked (?) accordingly. Addition of BMP4 between days 7 and 11 increased the expression of lens lineage-specific transcription factors PAX6 and PITX3. Lens placodal cells were enriched through inhibition of FGF signaling by SU5402 at day 7, followed by analyses of lens placode markers PAX6, SIX3, and PITX3 and subsequent formation of αB-crystallin-expressing cells analyzed at day 57 [139].