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Purpose Decreased trabecular meshwork (TM) cellularity has been implicated as a major reason for TM dysfunction and aqueous humor (AH) outflow abnormalities in primary open angle glaucoma. We previously found that transplantation of induced pluripotent stem cell (iPSC)-derived TM cells can restore TM function and stimulate endogenous TM cell division. The goal of the present study is to investigate whether signaling via gap junctions is involved in this process. Methods Differentiated iPSCs were characterized morphologically, transcriptionally, and immunohistochemically. After purification, iPSC-TM were co-cultured with mouse TM (MTM) cells to mimic the transplantation procedure. Through the pharmacological antagonists and short hairpin RNA (shRNA) technique, the gap junction function in iPSC-based therapy was determined. Results In the co-culture system, iPSC-TM increase MTM cell division as well as transfer of Ca 2+ to MTM. This effect was blocked by treatment with the gap junction inhibitors carbenoxolone (CBX) or flufenamic acid (FFA). The shRNA mediated knock down of connexin 43 (Cx43) expression in iPSC-TM also results in decreased Ca 2+ transfer and lower MTM proliferation rates. In vivo, Cx43 downregulation in transplanted iPSC-TM weakened their regenerative role in an Ad5.myocilin Y437H mouse model of glaucoma. Mice receiving these cells exhibited lower TM cellularity and higher intraocular pressure (IOP) than those receiving unmodified iPSC-TM. Conclusions Our findings reveal a crucial role of gap junction, especially Cx43, in iPSC-based TM regeneration, and provides insights to enhance the regenerative effect of iPSCs in glaucoma therapy.

Purpose Stem cell-based therapy has the potential to become one approach to regenerate the damaged trabecular meshwork (TM) in glaucoma. Co-culture of induced pluripotent stem cells (iPSCs) with human TM cells has been a successful approach to generate autologous TM resembling cells. However, the differentiated cells generated using this approach are still problematic for clinical usage. This study aimed to develop a clinically applicable strategy for generating TM-like cells from iPSCs. Methods Highly expressed receptors during iPSC differentiation were identified by AutoSOME, Gene Ontology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. The recombinant cytokines that bind to these receptors were used to generate a new differentiation protocol. The resultant TM-like cells were characterized morphologically, immunohistochemically, and transcriptionally. Results We first determined two stages of iPSC differentiation and identified highly expressed receptors associated with the differentiation at each stage. The expression of these receptors was further confirmed by RT-PCR analysis. Exposure to the recombinant cytokines that bind to these receptors, including transforming growth factor beta 1, nerve growth factor beta, erythropoietin, prostaglandin F2 alpha, and epidermal growth factor, can efficiently differentiate iPSCs into TM-like cells, which express TM biomarkers and can form dexamethasone-inducible CLANs. Conclusions We successfully generated a xeno- and feeder-free differentiation protocol with recombinant cytokines to generate the TM progenitor and TM-like cells from human iPSCs. Translational Relevance The new approach minimizes the risks from contamination and also improves the differentiation efficiency and consistency, which are particularly crucial for clinical use of stem cells in glaucoma treatment.