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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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Differentiation of cells from RNS.(A–B) Changes in cells proliferation (BrdU) (A) or cell death (B) were analyzed at different times before and after treatment with differentiation medium at day 5 (indicated by an arrow). Data are derived from 8 fields from 2 independent experiments and represented as mean +/− s.e.m. (C–D) Real-time PCR analyses of CE markers Mitf (C), Rpe65 (D) and Rlbp1 (D; also expressed in Müller glia) show lower or similar levels in retinal neurospheres (RNS) compared to the ciliary epithelium (CE) and down-regulation of these genes during differentiation. (E) Real-time PCR analysis confirms higher levels of Nestin and Nanog mRNAs in RNS (black bars) compared to CE (white bars). In C, D and E data derive from the formula 2−ΔCt. S26 was used as reference gene. (F) Real-time PCR analysis of retinal progenitors markers (Pax6, Chx10, Rax and Nestin) in RNS compared to expression in undifferentiated ES (set as 0). On the right-hand side of the graph the Real-time PCR products, after separation in an agarose gel, are shown. (G–H) Bright field images of cells at D4 (G) and D30 (H) show pigmentation reduction and changes in morphology in cells differentiated for 30 days. Scale bar is 50 µm.
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pone-0033338-g001: Differentiation of cells from RNS.(A–B) Changes in cells proliferation (BrdU) (A) or cell death (B) were analyzed at different times before and after treatment with differentiation medium at day 5 (indicated by an arrow). Data are derived from 8 fields from 2 independent experiments and represented as mean +/− s.e.m. (C–D) Real-time PCR analyses of CE markers Mitf (C), Rpe65 (D) and Rlbp1 (D; also expressed in Müller glia) show lower or similar levels in retinal neurospheres (RNS) compared to the ciliary epithelium (CE) and down-regulation of these genes during differentiation. (E) Real-time PCR analysis confirms higher levels of Nestin and Nanog mRNAs in RNS (black bars) compared to CE (white bars). In C, D and E data derive from the formula 2−ΔCt. S26 was used as reference gene. (F) Real-time PCR analysis of retinal progenitors markers (Pax6, Chx10, Rax and Nestin) in RNS compared to expression in undifferentiated ES (set as 0). On the right-hand side of the graph the Real-time PCR products, after separation in an agarose gel, are shown. (G–H) Bright field images of cells at D4 (G) and D30 (H) show pigmentation reduction and changes in morphology in cells differentiated for 30 days. Scale bar is 50 µm.

Mentions: We previously demonstrated that retinal progenitors, differentiated from RNS, acquire distinctive fates if exposed to different growth factors [8]. This underscored the importance of culture conditions for differentiation into specific cell types in vitro. We sought to enrich the population of cells acquiring a rod fate. To this purpose we seeded primary RNS onto an extracellular matrix substrate (see Methods) and allowed the cells to migrate out from the RNS and proliferate in the presence of bFGF. At the 5th day (D5), cells were treated with differentiating medium containing factors known to favor rod differentiation: retinoic acid (RA) [11], sodium butyrate [12], T3 [13] and taurine [14]. Cells treated with differentiation medium quickly exited cell cycle, as demonstrated by reduced BrdU labeling (Figure 1A). Cells were induced to maturation, as suggested by gene expression (see below), and partially selected by the differentiation medium, as suggested by cell death observed during the first week of differentiation (Figure 1B).


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)

Differentiation of cells from RNS.(A–B) Changes in cells proliferation (BrdU) (A) or cell death (B) were analyzed at different times before and after treatment with differentiation medium at day 5 (indicated by an arrow). Data are derived from 8 fields from 2 independent experiments and represented as mean +/− s.e.m. (C–D) Real-time PCR analyses of CE markers Mitf (C), Rpe65 (D) and Rlbp1 (D; also expressed in Müller glia) show lower or similar levels in retinal neurospheres (RNS) compared to the ciliary epithelium (CE) and down-regulation of these genes during differentiation. (E) Real-time PCR analysis confirms higher levels of Nestin and Nanog mRNAs in RNS (black bars) compared to CE (white bars). In C, D and E data derive from the formula 2−ΔCt. S26 was used as reference gene. (F) Real-time PCR analysis of retinal progenitors markers (Pax6, Chx10, Rax and Nestin) in RNS compared to expression in undifferentiated ES (set as 0). On the right-hand side of the graph the Real-time PCR products, after separation in an agarose gel, are shown. (G–H) Bright field images of cells at D4 (G) and D30 (H) show pigmentation reduction and changes in morphology in cells differentiated for 30 days. Scale bar is 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303820&req=5

pone-0033338-g001: Differentiation of cells from RNS.(A–B) Changes in cells proliferation (BrdU) (A) or cell death (B) were analyzed at different times before and after treatment with differentiation medium at day 5 (indicated by an arrow). Data are derived from 8 fields from 2 independent experiments and represented as mean +/− s.e.m. (C–D) Real-time PCR analyses of CE markers Mitf (C), Rpe65 (D) and Rlbp1 (D; also expressed in Müller glia) show lower or similar levels in retinal neurospheres (RNS) compared to the ciliary epithelium (CE) and down-regulation of these genes during differentiation. (E) Real-time PCR analysis confirms higher levels of Nestin and Nanog mRNAs in RNS (black bars) compared to CE (white bars). In C, D and E data derive from the formula 2−ΔCt. S26 was used as reference gene. (F) Real-time PCR analysis of retinal progenitors markers (Pax6, Chx10, Rax and Nestin) in RNS compared to expression in undifferentiated ES (set as 0). On the right-hand side of the graph the Real-time PCR products, after separation in an agarose gel, are shown. (G–H) Bright field images of cells at D4 (G) and D30 (H) show pigmentation reduction and changes in morphology in cells differentiated for 30 days. Scale bar is 50 µm.
Mentions: We previously demonstrated that retinal progenitors, differentiated from RNS, acquire distinctive fates if exposed to different growth factors [8]. This underscored the importance of culture conditions for differentiation into specific cell types in vitro. We sought to enrich the population of cells acquiring a rod fate. To this purpose we seeded primary RNS onto an extracellular matrix substrate (see Methods) and allowed the cells to migrate out from the RNS and proliferate in the presence of bFGF. At the 5th day (D5), cells were treated with differentiating medium containing factors known to favor rod differentiation: retinoic acid (RA) [11], sodium butyrate [12], T3 [13] and taurine [14]. Cells treated with differentiation medium quickly exited cell cycle, as demonstrated by reduced BrdU labeling (Figure 1A). Cells were induced to maturation, as suggested by gene expression (see below), and partially selected by the differentiation medium, as suggested by cell death observed during the first week of differentiation (Figure 1B).

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