Facile and efficient reprogramming of ciliary body epithelial cells into induced pluripotent stem cells

Abstract

Induced pluripotent stem (iPS) cells are attractive for cell replacement therapy, because they overcome ethical and immune rejection issues that are associated with embryonic stem cells. iPS cells have been derived from autonomous fibroblasts at low efficiency using multiple ectopic transcription factors. Recent evidence suggests that the epigenome of donor cell sources plays an important role in the reprogramming and differentiation characteristics of iPS cells. Thus, identification of somatic cell types that are easily accessible and are more amenable for cellular reprogramming is critical for regenerative medicine applications. Here, we identify ciliary body epithelial cells (CECs) as a new cell type for iPS cell generation that has higher reprogramming efficiency compared with fibroblasts. The ciliary body is composed of epithelial cells that are located in the anterior portion of the eye at the level of the lens and is readily surgically accessible. CECs also have a reduced reprogramming requirement, as we demonstrate that ectopic Sox2 and c-Myc are dispensable. Enhanced reprogramming efficiency may be due to increased basal levels of Sox2 in CECs. In addition, we are the first to report a cellular reprogramming haploinsufficiency observed when reprogramming with fewer factors (Oct4 and Klf4) in Sox2 hemizygous cells. Taken together, endogenous Sox2 levels are critical for the enhanced efficiency and reduced exogenous requirement that permit facile cellular reprogramming of CECs.

FIG. 1.

Mouse ciliary body epithelial cell (CEC) culture. (A) Diagram of the anatomical position of the ciliary body. (B) Brightfield (left) and fluorescent (right) images of monolayer of Sox2^EGFP CECs. The Sox2^EGFP CECs were heterogeneous and some expressed weak EGFP. Scale bar: 50 μm. (C) RT-PCR showed that Sox2^EGFP CECs expressed epithelial markers cytokeratin-18, connexin-43, E-cadherin, and palmdelphin. Color images available online at www.liebertpub.com/scd

FIG. 2.

Generation of induced pluripotent stem (iPS) cells from Sox2^EGFP CECs with four and three reprogramming factors. (A) Timeline for reprogramming Sox2^EGFP CECs into iPS cells. CECs from hemizygote Sox2 mouse were transduced with Oct4, Sox2, Klf4, and c-Myc (4F) or with Oct4, Sox2, and Klf4 (3F). Transduction mix was exchanged for fresh growth medium at day 1. At day 2, transduced CECs were subcultured onto SNL feeder cells (a mouse fibroblast STO cell line-derived, neomycin-resistant, leukemia inhibitory factor (LIF)-producing cell line) in embryonic stem (ES) cell medium minus leukemia inhibitory factor (LIF). (B) Representative 4F Sox2-GFP⁺ iPS colony. Scale bar, 50 μm. (C) Representative 3F Sox2-GFP⁺ iPS colony, plus Nanog staining. Scale bar, 50 μm. (D) Representative 4F and 3F Sox2-GFP⁺ iPS cell lines cultured in N2B27+ 2i/LIF medium on gelatin-coated plate without feeder cells, plus AP staining of 3F Sox2-GFP⁺ iPS cell line. Scale bar, 100 μm. Color images available online at www.liebertpub.com/scd

FIG. 3.

Characteristics of the mouse CEC-derived 4F and 3F Sox2-GFP⁺ iPS cells. (A) Genotyping of 4F and 3F iPS cells. Genomic PCR showed all four reprogramming factors in 4F iPS cells, and the absence of the c-Myc transgene in 3F iPS cells. Parental CECs served as negative control. (B) RT-PCR of pluripotency-associated genes and GAPDH. 4F and 3F iPS cells express all tested endogenous pluripotency genes. Parental CECs express Sox2, Klf4, and c-Myc. ES cells served as positive control. (C) Immunostaining of pluripotency markers Oct4, Sox2, Nanog, and SSEA-1 in 3F iPS cells. Sox2-GFP shows GFP expression from the Sox2 locus. Scale bar, 100 μm. (D) RT-PCR of representative lineage markers after differentiation of embryoid bodies (EBs) generated from 3F iPS clones. (E) Hematoxylin and eosin staining of teratoma sections derived from 3F iPS clones showing all three embryonic germ layers. Scale bar, 100 μm. (F) Immunostaining of teratoma sections derived from 3F iPS clones showing tubulin-β III positive neural epithelium, α-smooth muscle actin-positive muscle, and E-cadherin-positive endodermal cells. Scale bar, 100 μm. Color images available online at www.liebertpub.com/scd

FIG. 4.

Endogenous expression of reprogramming factors in wild-type and Sox2^EGFP CECs. Quantitative real-time PCR was performed on passage 2 wild-type and hemizygous Sox2^EGFP CECs. Neither of the cells expresses Oct4 at appreciable levels. Compared with the wild type, expression levels of Sox2, Klf4, and c-Myc are reduced in hemizygous Sox2^EGFP CECs. Data are normalized for GAPDH expression and presented as mean±SEM for three replicates. *P value<0.05; **P value<0.01. Color images available online at Y

FIG. 5.

Generation of iPS cells from mouse wild-type CECs with two reprogramming factors. (A) Brightfield and AP staining of representative 2F iPS colony. Scale bar, 50 μm. (B) Genotyping of 2F iPS cells. Genomic PCR showed only Oct4 and Klf4 transgenes in 2F iPS cells as expected. (C) RT-PCR of pluripotency-associated genes and GAPDH. 2F iPS cells express all tested endogenous pluripotency genes, and parental wild-type CECs express Sox2, Klf4 and c-Myc. (D) Immunostaining of pluripotency markers Oct4, Sox2, Nanog, and SSEA-1 in 2F iPS cells. Scale bar, 100 μm. (E) Representative 2F iPS cell line cultured in N2B27+ 2i/LIF medium on gelatin-coated plate without feeder cells. Scale bar, 50 μm. (F) RT-PCR of typical lineage markers after differentiation of EBs derived from 2F iPS clones. (G) Teratoma formation. Hematoxylin and eosin staining of teratoma sections derived from 2F iPS clones showed all three embryonic germ layers. Immunostaining of teratoma sections showed tubulin-β III-positive neural epithelium, α-smooth muscle actin-positive muscle, and E-cadherin-positive endodermal cells. Scale bar, 100 μm. Color images available online at www.liebertpub.com/scd