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Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium.

Demontis GC, Aruta C, Comitato A, De Marzo A, Marigo V - PLoS ONE (2012)

Bottom Line: Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro.We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP.We also identified voltage-gated channels necessary for rod maturation and viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy.

ABSTRACT
In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells.

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Effects of differentiation media on the expression of voltage-gated currents by rod-like cells derive from RNS.(A–D) Red, green and magenta bars: time of exposure to fetal bovine serum (FBS), basic fibroblast growth factor (FGF) and sodium butyrate (NaB) and T3, respectively. Blue bars: exposure time to retinoic acid (RA) and taurine (T) for 7 (A and C) and 30 days (B and D). Time 0: beginning of treatment and corresponds to the 5th day in culture (D5). (e–h′) B/W and fluorescent images of cells cultured as shown in A (e, e′), B (f, f′), C (g, g′) and D (h, h′). (I–L) Normalized hyperpolarization-activated currents before (black – CNTR), during (red – CdCl2) and after washing out 2 mM CdCl2 (blue – WASH) by cells shown in e (I), f (J), g (K), h (L). Calibration bars in I also hold for panels J–L. (M–N) Net current densities recorded in either FBS (M, red diamonds) or FGF (N, green diamonds) with NaB/T3 plus RA/T either for 7 (red open diamonds, N = 5; green open diamonds, N = 7) or 30 (red filled diamonds, N = 4; green filled diamonds, N = 10) days. (O) Depolarization-activated fast inward currents in a D30 cells cultured in the presence of FGF and 1week treatment with RA/T/NaB/T3 (panel C). Black trace: time course of voltage steps. Color-coded numbers: voltages match noisy records. (P) Inward currents evoked by a 20 ms-long step from −140 to −20 mV were rapidly inactivated. The pre-pulse voltages are plotted by the black line above the records. (Q) Inward currents in a cell cultured as in panel A, before (black trace), during (red trace – 0 Ca) and after exposure to saline with 0-added Ca2+ (blue trace). (R) Inward currents of a cell cultured as in A before (black trace), during (red trace – CdCl2) and after exposure to 2 mM CdCl2 (blue trace - WASH).
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pone-0033338-g009: Effects of differentiation media on the expression of voltage-gated currents by rod-like cells derive from RNS.(A–D) Red, green and magenta bars: time of exposure to fetal bovine serum (FBS), basic fibroblast growth factor (FGF) and sodium butyrate (NaB) and T3, respectively. Blue bars: exposure time to retinoic acid (RA) and taurine (T) for 7 (A and C) and 30 days (B and D). Time 0: beginning of treatment and corresponds to the 5th day in culture (D5). (e–h′) B/W and fluorescent images of cells cultured as shown in A (e, e′), B (f, f′), C (g, g′) and D (h, h′). (I–L) Normalized hyperpolarization-activated currents before (black – CNTR), during (red – CdCl2) and after washing out 2 mM CdCl2 (blue – WASH) by cells shown in e (I), f (J), g (K), h (L). Calibration bars in I also hold for panels J–L. (M–N) Net current densities recorded in either FBS (M, red diamonds) or FGF (N, green diamonds) with NaB/T3 plus RA/T either for 7 (red open diamonds, N = 5; green open diamonds, N = 7) or 30 (red filled diamonds, N = 4; green filled diamonds, N = 10) days. (O) Depolarization-activated fast inward currents in a D30 cells cultured in the presence of FGF and 1week treatment with RA/T/NaB/T3 (panel C). Black trace: time course of voltage steps. Color-coded numbers: voltages match noisy records. (P) Inward currents evoked by a 20 ms-long step from −140 to −20 mV were rapidly inactivated. The pre-pulse voltages are plotted by the black line above the records. (Q) Inward currents in a cell cultured as in panel A, before (black trace), during (red trace – 0 Ca) and after exposure to saline with 0-added Ca2+ (blue trace). (R) Inward currents of a cell cultured as in A before (black trace), during (red trace – CdCl2) and after exposure to 2 mM CdCl2 (blue trace - WASH).

Mentions: Clc-2 current up-regulation required continuous exposure to RA and Taurine (Figure 9). Of note, most cells treated for only 1 week with RA and taurine initially activated Rho, as shown by EGFP expression (Figure 9 panels e′–f′, g′–h′), but failed later on to up-regulate Clc-2 currents (open diamonds in Figure 9 M–N) and instead expressed fast inward calcium currents, similar to rod bipolar [25] rather than to rod cells (Figure 9 O–R).


Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium.

Demontis GC, Aruta C, Comitato A, De Marzo A, Marigo V - PLoS ONE (2012)

Effects of differentiation media on the expression of voltage-gated currents by rod-like cells derive from RNS.(A–D) Red, green and magenta bars: time of exposure to fetal bovine serum (FBS), basic fibroblast growth factor (FGF) and sodium butyrate (NaB) and T3, respectively. Blue bars: exposure time to retinoic acid (RA) and taurine (T) for 7 (A and C) and 30 days (B and D). Time 0: beginning of treatment and corresponds to the 5th day in culture (D5). (e–h′) B/W and fluorescent images of cells cultured as shown in A (e, e′), B (f, f′), C (g, g′) and D (h, h′). (I–L) Normalized hyperpolarization-activated currents before (black – CNTR), during (red – CdCl2) and after washing out 2 mM CdCl2 (blue – WASH) by cells shown in e (I), f (J), g (K), h (L). Calibration bars in I also hold for panels J–L. (M–N) Net current densities recorded in either FBS (M, red diamonds) or FGF (N, green diamonds) with NaB/T3 plus RA/T either for 7 (red open diamonds, N = 5; green open diamonds, N = 7) or 30 (red filled diamonds, N = 4; green filled diamonds, N = 10) days. (O) Depolarization-activated fast inward currents in a D30 cells cultured in the presence of FGF and 1week treatment with RA/T/NaB/T3 (panel C). Black trace: time course of voltage steps. Color-coded numbers: voltages match noisy records. (P) Inward currents evoked by a 20 ms-long step from −140 to −20 mV were rapidly inactivated. The pre-pulse voltages are plotted by the black line above the records. (Q) Inward currents in a cell cultured as in panel A, before (black trace), during (red trace – 0 Ca) and after exposure to saline with 0-added Ca2+ (blue trace). (R) Inward currents of a cell cultured as in A before (black trace), during (red trace – CdCl2) and after exposure to 2 mM CdCl2 (blue trace - WASH).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303820&req=5

pone-0033338-g009: Effects of differentiation media on the expression of voltage-gated currents by rod-like cells derive from RNS.(A–D) Red, green and magenta bars: time of exposure to fetal bovine serum (FBS), basic fibroblast growth factor (FGF) and sodium butyrate (NaB) and T3, respectively. Blue bars: exposure time to retinoic acid (RA) and taurine (T) for 7 (A and C) and 30 days (B and D). Time 0: beginning of treatment and corresponds to the 5th day in culture (D5). (e–h′) B/W and fluorescent images of cells cultured as shown in A (e, e′), B (f, f′), C (g, g′) and D (h, h′). (I–L) Normalized hyperpolarization-activated currents before (black – CNTR), during (red – CdCl2) and after washing out 2 mM CdCl2 (blue – WASH) by cells shown in e (I), f (J), g (K), h (L). Calibration bars in I also hold for panels J–L. (M–N) Net current densities recorded in either FBS (M, red diamonds) or FGF (N, green diamonds) with NaB/T3 plus RA/T either for 7 (red open diamonds, N = 5; green open diamonds, N = 7) or 30 (red filled diamonds, N = 4; green filled diamonds, N = 10) days. (O) Depolarization-activated fast inward currents in a D30 cells cultured in the presence of FGF and 1week treatment with RA/T/NaB/T3 (panel C). Black trace: time course of voltage steps. Color-coded numbers: voltages match noisy records. (P) Inward currents evoked by a 20 ms-long step from −140 to −20 mV were rapidly inactivated. The pre-pulse voltages are plotted by the black line above the records. (Q) Inward currents in a cell cultured as in panel A, before (black trace), during (red trace – 0 Ca) and after exposure to saline with 0-added Ca2+ (blue trace). (R) Inward currents of a cell cultured as in A before (black trace), during (red trace – CdCl2) and after exposure to 2 mM CdCl2 (blue trace - WASH).
Mentions: Clc-2 current up-regulation required continuous exposure to RA and Taurine (Figure 9). Of note, most cells treated for only 1 week with RA and taurine initially activated Rho, as shown by EGFP expression (Figure 9 panels e′–f′, g′–h′), but failed later on to up-regulate Clc-2 currents (open diamonds in Figure 9 M–N) and instead expressed fast inward calcium currents, similar to rod bipolar [25] rather than to rod cells (Figure 9 O–R).

Bottom Line: Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro.We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP.We also identified voltage-gated channels necessary for rod maturation and viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy.

ABSTRACT
In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells.

Show MeSH
Related in: MedlinePlus