Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions

Abstract

The eye lens is an encapsulated avascular organ whose function is to focus light on the retina. Lens comprises a single progenitor cell lineage in multiple states of differentiation. Disruption of lens function leading to protein aggregation and opacity results in age-onset cataract. Cataract is a complex disease involving genetic and environmental factors. Here, we report the development of a new 3-stage system that differentiates human embryonic stem cells (hESCs) into large quantities of lens progenitor-like cells and differentiated 3-dimensional lentoid bodies. Inhibition of BMP signaling by noggin triggered differentiation of hESCs toward neuroectoderm. Subsequent reactivation of BMP and activation of FGF signaling stimulated formation of lens progenitor cells marked by the expression of PAX6 and alpha-crystallins. The formation of lentoid bodies was most efficient in the presence of FGF2 and Wnt-3a, yielding approximately 1000 lentoid bodies/30-mm well. Lentoid bodies expressed and accumulated lens-specific markers including alphaA-, alphaB-, beta-, and gamma-crystallins, filensin, CP49, and MIP/aquaporin 0. Collectively, these studies identify a novel procedure to generate lens cells from hESCs that can be applied for studies of lens differentiation and cataractogenesis using induced pluripotent stem (iPS) cells derived from various cataract patients.

Figure 1.

Expression of PAX6, SIX3, SOX2, αA- and αB-crystallins during initial differentiation (d 0 to 14) of human ES cells into lens progenitor-like cells. A) qRT-PCR analysis of lens-lineage specific transcription factors PAX6, SIX3, and SOX2. B) qRT-PCR analysis of “early” αA- and αB-crystallin genes. C) Immunofluorescence detection of αA- and αB-crystallin proteins at d 12.

Figure 2.

Formation of lentoid bodies from human ES cells. A) Visualization of lentoid body formation by phase contrast microscopy (×40) at d 35. B) Quantitative analysis of lentoid body formation. Analysis was performed from d 21 to 32. Results are shown as means ± sd from 3 independent experiments. Cultures using BMPs, Wnt-3a, and BMPs/Wnt-3a did not stimulate lentoid body formation (data not shown).

Figure 3.

Human ES cell differentiation into lens progenitor-like cells and lentoid bodies. A) Three-step differentiation procedure and “optimized” concentrations of growth factors used. B–E) Morphology of the cells (×100) at d 0 (B), d 6 (C), d 18 (D), and d 35 (E).

Figure 4.

Quantitative RT-PCR analysis of 10 lens differentiation markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, CRYGC, CRYBB2, BFSP1, BFSP2, and MIP) from d 0 to 35. Results were calculated relative to the average C_t value of B2M, GAPDH, and SDHA genes, as described in Materials and Methods, and are shown as means ± sd from 3 independent experiments.

Figure 5.

Western blot analysis of protein expression during the differentiation of human ES cells. Specific antibodies were used to detect expression of PAX6, αA-, αB-, β-, and γ-crystallins, filensin, CP49, and MIP/aquaporin 0. Expression of β-actin is shown as a loading control.

Figure 6.

Immunofluorescence localization of proteins in lentoid bodies (d 35). Specific antibodies were used to detect expression of PAX6, αA-, αB-, β-, and γ-crystallins, filensin, CP49, and MIP/aquaporin 0. DAPI counterstaining shows nuclei in lentoid bodies. Scale bar = 100 μm.

Figure 7.

Comparative analysis of protein expression in lentoid bodies and human lens. Western blot analysis was performed using α-, αA-, αB-, and β-crystallin and MsrA-specific antibodies and indicated amounts (μg) of total lens protein extracts.