Purchase this article with an account.
ORIANE OLIVIA RABESANDRATANA, Antoine J Chaffiol, Amélie Slembrouck, Sacha Reichman, Céline Nanteau, Giuliana Gagliardi, Jens Duebel, Olivier Goureau, Gael Orieux; Promoting functional maturation of human iPS cell-derived retinal ganglion cells by a 3D/2D stepwise differentiation protocol. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1372.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Optic neuropathies have currently no effective treatment to stop the progressive loss of retinal ganglion cells (RGCs) and the degeneration of the optic nerve. Stem cell-based therapy using human induced pluripotent stem cells (hiPSCs) is one of the most promising strategy to replace lost RGCs. The present work displayed the optimized generation and characterization of purified hiPSC-derived RGCs, based on our retinal differentiation method using adherent hiPSCs.
3D retinal organoids containing RGCs were generated according to our previous protocol (Reichman et al. PNAS 2014; 111:8518). To promote RGC axonal growth, retinal organoids were dissociated to further cultured retinal cells onto poly-D-lysin/laminin coated plates. At different time points, maturation of hiPSC-derived RGCs was evaluated by immunohistochemistry with specific RGC markers and by electrophysiological recordings. For cell separation, cells were dissociated into single cells, immunostained with a CD90 antibody and Magnetic-Activated Cell Sorting (MACS) was performed by using anti-CD90 microbeads. Dissociated retinal cells (unsorted, CD90- and CD90+ fractions) were analyzed and characterized by cytofluorometry, immunohistochemistry and electrophysiological recordings.
Dissociated cells from retinal organoids cultured in 2D, expressed specific RGC markers (BRN3A, ISLET1, RBPMS, ATOH7, CD90). Axonal growth was highlighted by the expression of CD90 and βIII tubulin. Cytofluorometric analysis revealed an enrichment up to 87% of RGCs based on CD90 expression after MACS purification. On both unsorted and CD90+ cells, whole-cell patch-clamp recording revealed that hiPSC-derived RGCs displayed voltage-dependent currents (up to 1500pA at +40mV). Furthermore, action potentials were triggered by depolarizing current injections (+40pA from the resting membrane potential), and in some cells spontaneous spiking was observed.
Our results support promotion of axonal growth of hiPSC-derived RGCs in adherent culture conditions. MACS of CD90+ cells resulted in a significant enrichment of RGCs exhibiting specific RGC electrophysiological profile, which will be of a great utility for future clinical translation. For this purpose, the ability of hiPSC-derived RGCs to survive, to fully differentiate and to establish new connections with the brain has to be demonstrated.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only