Generation of Human Corneal Endothelial Cells via In Vitro Ocular Lineage Restriction of Pluripotent Stem Cells

Abstract

Purpose: We generate a renewable supply of corneal endothelial cells (CEC) from human pluripotent stem cells (PSCs) under defined culture conditions.

Methods: Corneal endothelial cell induction was driven by small molecules in a stepwise fashion of lineage specification. During the initial phase, PSC fate was restricted to the eye field-like state and became eye field stem cells (EFSCs). In the second phase, PSC-derived EFSCs were further directed toward either neural crest lineage or retinal lineage. The CECs were directly induced from ocular neural crest stem cells (NCSCs) by suppressing TGF-β and ROCK signaling.

Results: Under chemically defined conditions, PSCs were massively converted into EFSCs and subsequently NCSCs. Eye field cell identity was characterized by the expression of key fate restriction factors for early eye field cells, such as PAX6, LHX2, and VSX2. The induction of ocular NCSCs was initiated by promoting WNT signaling in EFSCs. Within 2 weeks of induction, the majority of cells expressed the typical neural crest markers p75NTR and HNK-1. Eye field stem cell-derived NCSCs can be propagated and cryopreserved. Subsequently, a CEC monolayer was induced from adherent NCSCs in the presence of small molecular inhibitors to suppress TGF-β and ROCK signaling. The polygon-shaped CEC-like cells became visible after a week in culture. The NCSC-derived CECs expressed typical CEC markers, such as N-Cadherin and Na+/K+-ATPase.

Conclusions: A novel small molecule-based approach was developed to derive human CECs from PSCs via ocular lineage specification. Moreover, EFSC-derived NCSCs could serve as an immediate source cell for rapid CEC induction in vitro.

Figure 1

Induction of EFSC from human PSC. Confocal images of immunofluorescence staining of typical early eye field transcription factors PAX6 (A) and LHX2 (B) in EFSCs derived from PSCs after 1 week of induction. Cell nuclei were counterstained with Hoechst 33342 (C). Eye field stem cells were stained for a retinal progenitor early transcription factor CHX10 (D), a cell proliferation marker Ki67 (E), and a neuroectodermal cell marker NESTIN (F). Scale bars: (A–C) = 60 μm; and (D–F) = 30 μm. (G) Real-time quantitative PCR (qPCR) analysis of the induced gene expression of representative early eye field transcription factors.

Figure 2

Characterization of EFSC differentiation to either neuronal or nonneuronal retinal cell fates under defined culture conditions. (A) Retinal ganglion cell differentiation from EFSCs. After 2 weeks of culture under conditions for RGC induction, differentiated cells displayed long neuronal processes and expressed typical RGC markers, such as BRN3 (red) and TUJ1 (green). Cell nuclei were counterstained with Hoechst 33342 (blue). (B) Photoreceptor induction was evidenced by immunocytochemistry on the expression of an early photoreceptor marker CRX (red), and cell nuclei were counterstained with Hoechst 33342 (blue). (C) Pigmented RPE appeared after prolonged RPE induction and maturation in culture. Scale bars: 30 μm.

Figure 3

Induction of NCSCs from PSCs in vitro. The cells were stained positively for two typical NCSC markers, HNK-1 (red, [A]) and p75NTR (green, [B]). (C) Immunostaining for the nuclear translocation of β-catenin (green), an indication of increased WNT signaling. Cell nuclei were counterstained with Hoechst 33342. Scale bars: (A) 200 μm; (B, C), 50 μm.

Figure 4

Expression-pattern shifts of pluripotency genes during the lineage specification.

Figure 5

Morphology and characterization of human NCSC-derived CECs. (A) Representative phase-contrast micrograph of a confluent monolayer of human NCSC-derived CECs with light treatment with Accutase for 30 seconds. Immunofluorescent images of NCSC-derived CECs which express ZO-1 (green, [B]), Na⁺/K⁺ATPase (red, [C]), and N-cadherin (green, [D]), three indicative markers of the corneal endothelium. Cell nuclei were counterstained with Hoechst 33342. Scale bars: (A, C, D) 50 μm; (B) = 100 μm. (E) Real-time qPCR analysis of increased mRNA expression of representative CEC genes.

Figure 6

Schematic of fate restriction of major eye cell types under the small molecule-driven processes.