Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules

Abstract

Reprogramming of somatic cells into desired functional cell types by small molecules has vast potential for developing cell replacement therapy. Here, we developed a stepwise strategy to generate chemically induced neural crest cells (ciNCCs) and chemically induced corneal endothelial cells (ciCECs) from mouse fibroblasts using defined small molecules. The ciNCCs exhibited typical NCC features and could differentiate into ciCECs using another chemical combination in vitro. The resulting ciCECs showed consistent gene expression profiles and self-renewal capacity to those of primary CECs. Notably, these ciCECs could be cultured for as long as 30 passages and still retain the CEC features in defined medium. Transplantation of these ciCECs into an animal model reversed corneal opacity. Our chemical approach for direct reprogramming of mouse fibroblasts into ciNCCs and ciCECs provides an alternative cell source for regeneration of corneal endothelia and other tissues derived from neural crest.

Improvement of an Effective Protocol for Directed Differentiation of Human Adipose Tissue-Derived Adult Mesenchymal Stem Cells to Corneal Endothelial Cells

“…As differential factors, the Wagoner protocol uses CHIR99021, an inhibitor of GSK-3 activity, which leads to a nuclear accumulation of β-catenin, which, in turn, activates the Wnt canonical signaling pathway, preventing terminal cell differentiation [ 33 ]. Recent studies have achieved long-term mouse CEC differentiation from mouse embryonic fibroblasts using a similar protocol with TGF-β and GSK3 inhibitors for NCC induction and Wnt inhibitors to further induce CEC differentiation [ 34 ], showing polygonal cells that proved functional in vivo.…”

Development and In Vitro Differentiation of Schwann Cells

“…While these cell populations exhibit neural crest cell properties, it is unclear if they represent cells directly derived from embryonal neural crest that remain in a quiescent state in adult tissues, or if they assume other adult cell fates but dedifferentiate in vitro after isolation and culture. A few other cell types have also been employed, such as skin-derived precursors [ 219 , 220 , 221 , 222 ], fibroblasts [ 177 , 223 , 224 , 225 , 226 , 227 , 228 ], and keratinocytes [ 229 , 230 ]. An overview of selected studies is provided in Section 3.3.3 .…”

Cell therapy in corneal endothelial disease

“…A dual-media approach with alternating stabilisation (low-mitogenic) and proliferation (high mitogenic) conditions has been shown to be effective [25,44,45]. More recently, human platelet lysate (HPL) [46][47][48][49], small molecules [50,51] and mesenchymal stem cell derived conditioned media [52,53] have been reported to be helpful.…”