Directed Differentiation of Human Pluripotent Stem Cells towards Corneal Endothelial-Like Cells under Defined Conditions

Abstract

The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na+/K+)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method.

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

“…Three hPSC lines, including one hiPSC and two hESC lines, previously established in our laboratory, were used for LSC differentiations. The WT001.TAU.bB2 hiPSCs were reprogrammed from healthy donor peripheral blood monocytes (PBMCs) using the CytoTune-iPS Sendai Reprogramming kit (Thermo Fisher Scientific) and characterized as previously described [ 40 ]. Derivation and characterization of the hESCs lines Regea 08/017 and Regea 11/013 was carried out as previously described in [ 41 ].…”

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“…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.…”

Generation of Lens Progenitor Cells and Lentoid Bodies from Pluripotent Stem Cells: Novel Tools for Human Lens Development and Ocular Disease Etiology

“…These studies highlighted the concept of self-organization (see Section 1 , Section 5.2 and Section 7 for additional information) as the mechanistic foundation explaining in vitro formation of 3D biological structures [ 7 ]. For comparisons, the procedures to generate retinal and eye organoids of various complexity [ 105 , 110 , 111 , 126 , 129 , 131 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 ] are listed in Table 1 , as they are important for human eye disease modeling (see Section 6 ).…”