Small-Molecule Induction Promotes Corneal Endothelial Cell Differentiation From Human iPS Cells

Abstract

Purpose: Corneal endothelial cells (CECs) serve as a barrier and foothold for the corneal stroma to maintain the function and transparency of the cornea. Loss of CECs during aging or disease states leads to blindness, and cell replacement therapy using either donated or artificially differentiated CECs remains the only curative approach.Methods: Human induced pluripotent stem cells (hiPSCs) that were cultured in chemically defined medium were induced with dual-SMAD inhibition to differentiate into neural crest cells (NCCs). A small-molecule library was screened to differentiate the NCCs into corneal endothelial-like cells. The characteristics of these cells were identified with real-time PCR and immunofluorescence. Western blotting was applied to detect the signaling pathways and key factors regulated by the small molecules.Results: We developed an effective protocol to differentiate hiPSCs into CECs with defined small molecules. The hiPSC-CECs were characterized by ZO-1, AQP1, Vimentin and Na+/K+-ATPase. Based on our small-molecule screen, we identified a small-molecule combination, A769662 and AT13148, that enabled the most efficient production of CECs. The combination of A769662 and AT13148 upregulated the PKA/AKT signaling pathway, FOXO1 and PITX2 to promote the conversion of NCCs to CECs.Conclusion: We established an efficient small molecule-based method to differentiate hiPSCs into corneal endothelial-like cells, which might facilitate drug discovery and the development of cell-based therapies for corneal diseases.

Focus on seed cells: stem cells in 3D bioprinting of corneal grafts

“…ASCs, ESCs, iPSCs, umbilical cord-derived mesenchymal stem cells (UC-MSCs) and PSCs were found to be potential seed cells in recent studies ( Dai et al, 2014 ; Zhang et al, 2014 ; McCabe et al, 2015 ; Song et al, 2016 ; Zhao and Afshari, 2016 ; Chen et al, 2018 ; Wagoner et al, 2018 ; Yamashita et al, 2018b ; Li et al, 2019 ; Chen et al, 2021 ; Gong et al, 2021 ; Grönroos et al, 2021 ; Marta et al, 2021 ; Sun et al, 2021 ; So et al, 2022 ; Ye et al, 2022 ). Moreover, human bone marrow-derived endothelial progenitor cells (BEPCs), skin-derived precursors (SKPs), cornea-derived precursors (COPs), rabbit oral mucosa epithelial cells and rat neural crest cells (rNCCs) have also been successfully derived from corneal endothelial cell-like cells ( Shao et al, 2011 ; Ju et al, 2012 ; Hatou et al, 2013 ; Jiang et al, 2014 ; Inagaki et al, 2017 ; Shen et al, 2017 ).…”

Stem Cell Therapies for Sensory Organ Disorders

“…The cornea is developed from the non-neural ectoderm and is a transparent tissue that covers the front of the eye; it works with the lens to focus light to the retina [19]. There are a number of studies that show the production of corneal endothelial cells from hPSCs [43][44][45][46][47][48][49][50][51][52][53]. These are well-established protocols for 3 different regions in the eye; however, they do not mimic the natural environment as they are created separately.…”

Corneal Endothelial-like Cells Derived from Induced Pluripotent Stem Cells for Cell Therapy

“…In that study, the use of serum was replaced with a chemically defined lipid concentrate [62]. Similar approaches include the use of knock-out serum replacement in addition to inhibitors or activators of specific pathways such as DKK-2 (Wnt inhibitor), SB-431542, CHIR-99021 [58][59][60]63,82], or a combination of small molecules such as A-769662 (AMPK activator) and AT-13148 (Akt inhibitor) [82]. Hatou et al described an induction approach to directly differentiate iPSCs by regulating the gene expression levels of C-JUN, C-FOS, NR3C2, and SOX9 using a combination of optimized cytokines in a chemically defined, animal and human origin-free corneal endothelial differentiation medium (CEDM) [61].…”

“…The process of characterization should begin from the induction of iPSCs to NCCs, and all the way till the CEC differentiation is deemed complete. Some of the markers that are often tested at the pluripotency stage include OCT3/4, SSEA-3, SSEA-4, SOX2, GCTM-2, TRA-1-60, and Nanog; those tested at the neural crest stem cell (NCSCs) stage include AP2ɑ (TFAP2A) and Nestin, NCC markers (SOX9, SOX10, NGFR, HNK-1, Vimentin) and human CEC markers (Na + K + -ATPase, ZO-1, SLC4A11, AQP1, N-cadherin, COL4A1, COL8A1, COL8A2) [17,[57][58][59][60]62,63,82].…”