Transplantation of human induced pluripotent stem cell-derived neural crest cells for corneal endothelial regeneration

Abstract

Background: The corneal endothelium maintains corneal hydration through the barrier and pump function, while its dysfunction may cause corneal edema and vision reduction. Considering its development from neural crest cells (NCCs), here we investigated the efficacy of the human induced pluripotent stem cell (hiPSC)-derived NCCs for corneal endothelial regeneration in rabbits.

Methods: Directed differentiation of hiPSC-derived NCCs was achieved using the chemically defined medium containing GSK-3 inhibitor and TGF-β inhibitor. The differentiated cells were characterized by immunofluorescence staining, FACS analysis, and in vitro multi-lineage differentiation capacity. For in vivo functional evaluation, 1.0 × 10⁶ hiPSC-derived NCCs or NIH-3 T3 fibroblasts (as control) combined with 100 μM Y-27632 were intracamerally injected into the anterior chamber of rabbits following removal of corneal endothelium. Rabbit corneal thickness and phenotype changes of the transplanted cells were examined at 7 and 14 days with handy pachymeter, dual-immunofluorescence staining, and quantitative RT-PCR.

Results: The hiPSC-derived NCCs were differentiated homogenously through 7 days of induction and exhibited multi-lineage differentiation capacity into peripheral neurons, mesenchymal stem cells, and corneal keratocytes. After 7 days of intracameral injection in rabbit, the hiPSC-derived NCCs led to a gradual recovery of normal corneal thickness and clarity, when comparing to control rabbit with fibroblasts injection. However, the recovery efficacy after 14 days deteriorated and caused the reappearance of corneal edema. Mechanistically, the transplanted cells exhibited the impaired maturation, cellular senescence, and endothelial-mesenchymal transition (EnMT) after the early stage of the in vivo directional differentiation.

Conclusions: Transplantation of the hiPSC-derived NCCs rapidly restored rabbit corneal thickness and clarity. However, the long-term recovery efficacy was impaired by the improper maturation, senescence, and EnMT of the transplanted cells.

Keywords: Corneal endothelium; Neural crest cells; Transplantation; iPSC.

Fig. 1

Inductive differentiation of hiPSCs into NCCs. a Schematic diagram of inductive differentiation. The hiPSCs were maintained in the SCM and induced the differentiation into NCCs in the NDM for 7 days. b Immunofluorescence staining of the hiPSCs before induction. c Immunofluorescence staining of the hiPSC-derived NCCs after 7 days of induction. d FACS analysis of P75 and HNK-1 dual-positive cells. e qRT-PCR analysis of the hiPSC-related genes (NANOG, OCT4, SOX2) and the NCC-related genes (P75, TFAP2A, TFAP2B, SOX9, SOX10). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Multipotent characteristics of hiPSC-derived NCCs. a Schematic diagram of multipotent neural crest cells. b Morphology and immunofluorescence staining of the differentiated neurons. c Morphology of the differentiated MSCs. d FACS analysis of NCC-related marker P75 and MSC markers CD73 and CD90 in the differentiated MSCs. e Multipotent differentiation into adipocytes, osteocytes, and chondrocytes by Oil Red O staining, Alizarin Red S staining, and Alcian Blue staining, respectively. f Immunofluorescence staining of the differentiated corneal keratocytes

Fig. 3

Intracameral injection of the hiPSC-derived NCCs in rabbits. a Slit-lamp photographs of rabbit corneas injected with either NIH-3 T3 fibroblasts or the hiPSC-derived NCCs. b Changes of corneal thickness after cell injection (n = 3 per group). c Representative images of corneal endothelium captured after 7 and 14 days of NCC group. Note the full coverage of polygonal cells at day 7 and blurred image at day 14

Fig. 4

Improper maturation of the hiPSC-derived NCCs after intracameral injection. a qRT-PCR analysis of the corneal endothelial-related genes (Na⁺/K⁺-ATPase, ZO-1, SLC4A11, AQP1, N-cadherin, and COL8A2) and the NCC-related genes (P75, TFAP2A, TFAP2B, and SOX10) in the hiPSC-derived NCCs before transplantation (Pre), 7 days and 14 days after transplantation (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. b Immunofluorescence staining of the hiPSC-derived NCCs before transplantation, 7 days and 14 days after transplantation. DNA was visualized by staining with DAPI (blue) and Human Nuclei (HuNu)

Fig. 5

Fate-decision of hiPSC-derived NCCs after intracameral injection. a The qRT-PCR analysis results show the analysis of the EnMT-related genes (α-SMA and SNAIL2) and senescence-related genes (P21 and P16) before transplantation (Pre), 7 days and 14 days after transplantation (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. b Immunofluorescence staining of the hiPSC-derived NCCs before transplantation, 7 days and 14 days after transplantation. DNA was visualized by staining with DAPI (blue) and Human Nuclei (HuNu). c SA-β-gal staining of the hiPSC derived-NCCs at day 7 and 14 after transplantation