Matsuda JJ, Filali MS, Volk KA, Collins MM, Moreland JG, Lamb FS. Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent.

Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific neurons that are ideal for autologous cell replacement for treatment of neurodegenerative diseases. In the case of retinal degeneration and associated photoreceptor cell therapy, polymer scaffolds are critical for cellular survival and integration; however, prior attempts to materialize this concept have been unsuccessful in part due to the materials’ inability to guide cell alignment. In this work, we used two-photon polymerization to create 180 μm wide non-degradable prototype photoreceptor scaffolds with varying pore sizes, slicing distances, hatching distances and hatching types. Hatching distance and hatching type were significant factors for the error of vertical pore diameter, while slicing distance and hatching type most affected the integrity and geometry of horizontal pores. We optimized printing parameters in terms of structural integrity and printing time in order to create 1 mm wide scaffolds for cell loading studies. We fabricated these larger structures directly on a porous membrane with 3 μm diameter pores and seeded them with human iPSC-derived retinal progenitor cells. After two days in culture, cells nested in and extended neuronal processes parallel to the vertical pores of the scaffolds, with maximum cell loading occurring in 25 μm diameter pores. These results highlight the feasibility of using this technique as part of an autologous stem cell strategy for restoring vision to patients affected with retinal degenerative diseases.

ClC-3, -4, and -5 are Cl Ϫ /H ϩ antiporters that are expressed primarily in intracellular organelles. They have been proposed to provide shunt conductances for proton current generated by the vacuolar (V-type) H ϩ -ATPase (V-ATPase) 2 (1). Sufficient ClC-4 and -5 protein localizes to the plasma membrane to readily allow recording of ion currents when these proteins are heterologously expressed in Xenopus oocytes (2). Unfortunately, recombinant ClC-3 currents have been much more difficult to express (2-4) and interpretation of the currents observed (5-8) has been controversial (for review see Ref. 9). ClC-3 localizes to endosomes (10) and lysosomes (11) as well as secretory vesicles of various types (12)(13)(14) suggesting that the protein cycles through the plasma membrane. Membrane localization of ClC-3 was quantified in cultured fibroblasts using recombinant protein with both extracellular and intracellular epitope tags. It had a half-life in the membrane of ϳ9 min and about 6% of total ClC-3 protein localized to the membrane at a given time (15). A proportion of ClC-3-eGFP fusion protein clearly appears to localize to the plasma membrane of ClC-3 expressing HEK293 cells (8).We recently demonstrated that adenoviral-mediated overexpression of ClC-3 produced novel currents in HEK293 cells at neutral pH (8). These currents exhibited very steep outward rectification and time-dependent activation that was reminiscent of ClC-4 and ClC-5 (2, 16) except that the kinetics of activation were slower. Activation was also significantly slower than observed when much smaller currents were elicited by overexpression of ClC-3 plasmids in Chinese hamster ovary-K1 cells (11), although rectification properties and the effect of a specific mutation (E224A) were very similar. Changes in current amplitude and reversal potential observed in response to alterations in extracellular Cl Ϫ and H ϩ concentration were consistent with ClC-3 acting as a Cl Ϫ /H ϩ antiporter and deviated significantly from the behavior of an anion channel. These results supported predictions, based upon sequence homology, that like ClC-4 and ClC-5, ClC-3 would be a ClClC-3 is required for proper acidification of synaptic vesicles (12), insulin granules (19), lysosomes (11), and endosomes (20) by the V-ATPase. This acidification process leads to intra-organellar pH values ranging from 5.9 to 6.2 for early endosomes to 5.0 -6.0 for late endosomes, and 4.6 -5.5 for lysosomes (21,22). A Cl Ϫ channel is well suited to provide charge neutralization for a proton pump. However, the realization that the intracellular ClCs are Cl Ϫ /H ϩ antiporters (8,17,18) yielded the surprising requirement that protons move out of the endosome in exchange for Cl Ϫ to provide countercurrent for the V-ATPase. The 2Cl Ϫ to 1H ϩ stoichiometry of this process (23, 24) makes this mechanism feasible, but has led to speculation that the primary goal of this coupled transport system may be to concentrate Cl Ϫ in the compartment rather than to facilitate acidification (18,22). This adds an u...

This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs and derive autologous retinal cells via 3D differentiation. The universal methods described herein for the isolation of dermal fibroblasts, and generation of iPSCs can be employed regardless of disease, tissue or cell type of interest.