Abstract
BackgroundCell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. In the current study, we aimed to assess if peripheral blood-derived monocytes which are highly abundant and accessible could be utilized as a potential candidate for phenotypic differentiation into neuron-like cells.MethodsThe peripheral blood-derived monocytes were reconditioned phenotypically using extrinsic growth factors to induce pluripotency and proliferation. The reconditioned monocytes (RM) were further incubated with a cocktail of growth factors involved in retinal development and growth to induce retinal neuron-like properties. These cells, termed as retinal neuron-like cells (RNLCs) were characterized for their morphological, molecular and functional behaviour in vitro and in vivo.ResultsThe monocytes de-differentiated in vitro and acquired pluripotency with the expression of prominent stem cell markers. Treatment of RM with retinal growth factors led to an upregulation of neuronal and retinal lineage markers and downregulation of myeloid markers. These cells show morphological alterations resembling retinal neuron-like cells and expressed photoreceptor (PR) markers. The induced RNLCs also exhibited relative membrane potential change upon light exposure suggesting that they have gained some neuronal characteristics. Further studies showed that RNLCs could also integrate in an immune-deficient retinitis pigmentosa mouse model NOD.SCID-rd1 upon sub-retinal transplantation. The RNLCs engrafted in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the RP afflicted retina. Mice transplanted with RNLCs showed improvement in depth perception, exploratory behaviour and the optokinetic response.ConclusionsThis proof-of-concept study demonstrates that reconditioned monocytes can be induced to acquire retinal neuron-like properties through differentiation using a defined growth media and can be a potential candidate for cell therapy-based interventions and disease modelling for ocular diseases.
Highlights
Cell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa
Reconditioned monocytes exhibit pluripotency and proliferation The CD14+ monocytes obtained from Peripheral blood mononuclear cell (PBMC) by enrichment on plastic tissue culture flasks and flow cytometric sorting exhibited a purity of 54.5 ± 9.39% and 75.5 ± 6.24% respectively (Fig. 1a (i, ii))
(See figure on previous page.) Fig. 1 Isolation, recondition and characterization of PBMC derived monocytes into reconditioned monocytes (RM). a (i) PBMCs were isolated by density gradient centrifugation method, wherein the buffy coat was diluted in RPMI-1640 media (1:3) which was layered over ficoll
Summary
Cell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. Retinitis pigmentosa (RP) is a progressive irreversible retinal degenerative disease which causes partial or complete blindness [1, 2]. This phenomenon predominantly affects rod photoreceptors, and subsequently, other retinal cells are affected [3]. Mutations in the gene PDE6B encoding for the β-subunits of phosphodiesterase 6 enzyme cause autosomal recessive RP. The condition is incurable, and the treatment modules offer only symptomatic relief. Cell-based therapy can benefit multiloci genetic mutations by possibly recreating the natural anatomy and circuitry of retina
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