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.

Sphere Formation Permits Oct4 Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

“…To generate floating spheres, CE cells were plated at a density of 20 cells per microliter in ultra-low attachment 24-well plates (Corning) [8]. To establish monolayer cultures, cells were grown in SFM supplemented with 1% fetal bovine serum (FBS) as previously described [1]. The medium was changed every 3 days.…”

“…The procedure of iPS cells generation was essentially as previously described with minor modifications [1]. In brief, all spheres at day 7 were dissociated into single cells with Accutase (Sigma) and plated at 1 · 10 5 cells per well of gelatin-coated six-well plates in 1% FBS-containing SFM.…”

“…T he ciliary body is a region that harbors ocular cells that are easily surgically accessible, and are of interest for regenerative ophthalmology [1][2][3][4]. The surface of the ciliary body is composed of a series of ridges called ciliary processes.…”

“…We previously found that mouse CE-derived cells, grown in monolayer cultures, express Sox2, Klf4, and c-Myc, and that these monolayer CE cells can be reprogrammed into iPS cells with ectopic Oct4 and Klf4 (2F), but not with Oct4 alone [1]. Given that adult neural stem cells, capable of sphere formation, have been previously reprogrammed into iPS cells with Oct4 alone [11,12], and that CE cells have the remarkable plasticity to form spheres displaying certain characteristics of neuroepithelial progenitors [7,8], we attempted to reprogram CE sphere-derived cells with Oct4 alone.…”

End of Inevitability: Programming and Reprogramming

“…The many diverse adult tissue sources/cell types being used to generate iPS cells, including e.g., prostatic epithelial cell [14], ciliary body epithelial cells [15], and human hair follicular mesenchymal stem cells [16]. While these and other studies provide strong support for the view that most cell types are reprogrammable, it is clear that the process is very inefficient and conditions vary from cell type to cell type.…”