iPSC-Derived Trabecular Meshwork Cells Stimulate Endogenous TM Cell Division Through Gap Junction in a Mouse Model of Glaucoma.

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 Ca2+ 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 Ca2+ transfer and lower MTM proliferation rates. In vivo, Cx43 downregulation in transplanted iPSC-TM weakened their regenerative role in an Ad5.myocilinY437H 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.

Xeno- and Feeder-Free Differentiation of Human iPSCs to Trabecular Meshwork-Like Cells by Recombinant Cytokines.

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.

The role of Piezo1 in conventional aqueous humor outflow dynamics.

Controlling intraocular pressure (IOP) remains the mainstay of glaucoma therapy. The trabecular meshwork (TM), the key tissue responsible for aqueous humor (AH) outflow and IOP maintenance, is very sensitive to mechanical forces. However, it is not understood whether Piezo channels, very sensitive mechanosensors, functionally influence AH outflow. Here, we characterize the role of Piezo1 in conventional AH outflow. Immunostaining and western blot analysis showed that Piezo1 is widely expressed by TM. Patch-clamp recordings in TM cells confirmed the activation of Piezo1-derived mechanosensitive currents. Importantly, the antagonist GsMTx4 for mechanosensitive channels significantly decreased steady-state facility, yet activation of Piezo1 by the specific agonist Yoda1 did not lead to a facility change. Furthermore, GsMTx4, but not Yoda1, caused a significant increase in ocular compliance, a measure of the eye's transient response to IOP perturbation. Our findings demonstrate a potential role for Piezo1 in conventional outflow, likely under pathological and rapid transient conditions.