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A Hyaluronan-Based Injectable Hydrogel Improves the Survival and Integration of Stem Cell Progeny following Transplantation.

Ballios BG, Cooke MJ, Donaldson L, Coles BL, Morshead CM, van der Kooy D, Shoichet MS - Stem Cell Reports (2015)

Bottom Line: The pro-survival mechanism of HAMC is ascribed to the interaction of the CD44 receptor with HA.Transient disruption of the retinal outer limiting membrane, combined with HAMC delivery, results in significantly improved rod survival and visual function.The HAMC delivery system improves cell transplantation efficacy in two CNS models, suggesting broad applicability.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.

No MeSH data available.


Related in: MedlinePlus

CD44 Mediates the Direct Survival Effect of HAMC on RSC-Derived Rods In Vitro(A) Percentage survival of mature RSC-derived rods (assayed with ethidium homodimer-1) cultured in HAMC and its individual components (HA and MC) demonstrates that only those mixtures containing HA show a significant pro-survival effect compared to non-HA-containing mixtures (two-way ANOVA, interaction effect of culture time and mixture composition on rod survival, F(12,102) = 10.33, p < 0.05; Tukey-Kramer post hoc, p < 0.05).(B) CD44 expression by qRT-PCR remains constant regardless of culture in HA- or MC-containing mixtures.(C) Mature RSC-derived rods express CD44 by immunocytochemistry, which co-localizes with GFP consistent with surface localization.(D) Staining for activated caspase III suggests cell death occurs primarily through apoptosis without HA-CD44 interaction in non-HA-containing mixtures (interaction of culture time and mixture on rod numbers, F(12,96) = 37.78, p < 0.05).(E) When mature CD44−/− RSC-derived rods are cultured for 7 days, cell survival is decreased by the same proportion compared to day 0, independent of mixture composition. Two-way ANOVA showed a main effect of culture time (F(1,40) = 258.92, p < 0.05), but no effect of mixture composition (F(3,40) = 0.71, p > 0.05) and no interaction effect (F(3,40) = 2.12, p > 0.05) on rod survival.(F) CD44−/− RSC-derived rods maintain Rhodopsin expression, suggesting that the absence of CD44 does not affect cell phenotype (no interaction effect, F(3,40) = 0.58, p > 0.05; no main effects of time or mixture composition) Scale bar represents 100 μm. Mean± SEM of n = 3 independent transplants are plotted; ∗p < 0.05.
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fig4: CD44 Mediates the Direct Survival Effect of HAMC on RSC-Derived Rods In Vitro(A) Percentage survival of mature RSC-derived rods (assayed with ethidium homodimer-1) cultured in HAMC and its individual components (HA and MC) demonstrates that only those mixtures containing HA show a significant pro-survival effect compared to non-HA-containing mixtures (two-way ANOVA, interaction effect of culture time and mixture composition on rod survival, F(12,102) = 10.33, p < 0.05; Tukey-Kramer post hoc, p < 0.05).(B) CD44 expression by qRT-PCR remains constant regardless of culture in HA- or MC-containing mixtures.(C) Mature RSC-derived rods express CD44 by immunocytochemistry, which co-localizes with GFP consistent with surface localization.(D) Staining for activated caspase III suggests cell death occurs primarily through apoptosis without HA-CD44 interaction in non-HA-containing mixtures (interaction of culture time and mixture on rod numbers, F(12,96) = 37.78, p < 0.05).(E) When mature CD44−/− RSC-derived rods are cultured for 7 days, cell survival is decreased by the same proportion compared to day 0, independent of mixture composition. Two-way ANOVA showed a main effect of culture time (F(1,40) = 258.92, p < 0.05), but no effect of mixture composition (F(3,40) = 0.71, p > 0.05) and no interaction effect (F(3,40) = 2.12, p > 0.05) on rod survival.(F) CD44−/− RSC-derived rods maintain Rhodopsin expression, suggesting that the absence of CD44 does not affect cell phenotype (no interaction effect, F(3,40) = 0.58, p > 0.05; no main effects of time or mixture composition) Scale bar represents 100 μm. Mean± SEM of n = 3 independent transplants are plotted; ∗p < 0.05.

Mentions: In order to determine the component of HAMC responsible for the survival effect noted in the post-mitotic rods, cells were cultured in HA and MC separately. HA and HAMC had pro-survival effects, whereas cells cultured in MC and SFM showed similar cell death rates (Figure 4A). We hypothesized that HA supports cell survival through a direct CD44-mediated interaction with donor cells. CD44 is the putative HA receptor, and its activation is important in cell survival pathways in other tissues. Mature RSC-derived rods were found to express CD44 both by immunocytochemistry (ICC) and qRT-PCR (Figures 4B and 4C). Just as RSC-derived rods maintain progenitor levels of P-cad expression during differentiation in vitro, they continue to express high expression of CD44, unlike mature rod photoreceptors in adult retina (Sarthy et al., 2007; Chaitin et al., 1994). The levels of CD44 gene expression were maintained over more than 7 days in HA or MC-containing mixtures, demonstrating that the medium itself did not affect gene expression. Additional evidence for the pro-survival effect of HA in HAMC was achieved by quantification of activated caspase-3 levels in these cultures. Significantly greater levels of apoptosis were observed with RSC-derived rods cultured in non-HA-containing, compared to HA-containing, hydrogels (p < 0.05) (Figure 4D).


A Hyaluronan-Based Injectable Hydrogel Improves the Survival and Integration of Stem Cell Progeny following Transplantation.

Ballios BG, Cooke MJ, Donaldson L, Coles BL, Morshead CM, van der Kooy D, Shoichet MS - Stem Cell Reports (2015)

CD44 Mediates the Direct Survival Effect of HAMC on RSC-Derived Rods In Vitro(A) Percentage survival of mature RSC-derived rods (assayed with ethidium homodimer-1) cultured in HAMC and its individual components (HA and MC) demonstrates that only those mixtures containing HA show a significant pro-survival effect compared to non-HA-containing mixtures (two-way ANOVA, interaction effect of culture time and mixture composition on rod survival, F(12,102) = 10.33, p < 0.05; Tukey-Kramer post hoc, p < 0.05).(B) CD44 expression by qRT-PCR remains constant regardless of culture in HA- or MC-containing mixtures.(C) Mature RSC-derived rods express CD44 by immunocytochemistry, which co-localizes with GFP consistent with surface localization.(D) Staining for activated caspase III suggests cell death occurs primarily through apoptosis without HA-CD44 interaction in non-HA-containing mixtures (interaction of culture time and mixture on rod numbers, F(12,96) = 37.78, p < 0.05).(E) When mature CD44−/− RSC-derived rods are cultured for 7 days, cell survival is decreased by the same proportion compared to day 0, independent of mixture composition. Two-way ANOVA showed a main effect of culture time (F(1,40) = 258.92, p < 0.05), but no effect of mixture composition (F(3,40) = 0.71, p > 0.05) and no interaction effect (F(3,40) = 2.12, p > 0.05) on rod survival.(F) CD44−/− RSC-derived rods maintain Rhodopsin expression, suggesting that the absence of CD44 does not affect cell phenotype (no interaction effect, F(3,40) = 0.58, p > 0.05; no main effects of time or mixture composition) Scale bar represents 100 μm. Mean± SEM of n = 3 independent transplants are plotted; ∗p < 0.05.
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fig4: CD44 Mediates the Direct Survival Effect of HAMC on RSC-Derived Rods In Vitro(A) Percentage survival of mature RSC-derived rods (assayed with ethidium homodimer-1) cultured in HAMC and its individual components (HA and MC) demonstrates that only those mixtures containing HA show a significant pro-survival effect compared to non-HA-containing mixtures (two-way ANOVA, interaction effect of culture time and mixture composition on rod survival, F(12,102) = 10.33, p < 0.05; Tukey-Kramer post hoc, p < 0.05).(B) CD44 expression by qRT-PCR remains constant regardless of culture in HA- or MC-containing mixtures.(C) Mature RSC-derived rods express CD44 by immunocytochemistry, which co-localizes with GFP consistent with surface localization.(D) Staining for activated caspase III suggests cell death occurs primarily through apoptosis without HA-CD44 interaction in non-HA-containing mixtures (interaction of culture time and mixture on rod numbers, F(12,96) = 37.78, p < 0.05).(E) When mature CD44−/− RSC-derived rods are cultured for 7 days, cell survival is decreased by the same proportion compared to day 0, independent of mixture composition. Two-way ANOVA showed a main effect of culture time (F(1,40) = 258.92, p < 0.05), but no effect of mixture composition (F(3,40) = 0.71, p > 0.05) and no interaction effect (F(3,40) = 2.12, p > 0.05) on rod survival.(F) CD44−/− RSC-derived rods maintain Rhodopsin expression, suggesting that the absence of CD44 does not affect cell phenotype (no interaction effect, F(3,40) = 0.58, p > 0.05; no main effects of time or mixture composition) Scale bar represents 100 μm. Mean± SEM of n = 3 independent transplants are plotted; ∗p < 0.05.
Mentions: In order to determine the component of HAMC responsible for the survival effect noted in the post-mitotic rods, cells were cultured in HA and MC separately. HA and HAMC had pro-survival effects, whereas cells cultured in MC and SFM showed similar cell death rates (Figure 4A). We hypothesized that HA supports cell survival through a direct CD44-mediated interaction with donor cells. CD44 is the putative HA receptor, and its activation is important in cell survival pathways in other tissues. Mature RSC-derived rods were found to express CD44 both by immunocytochemistry (ICC) and qRT-PCR (Figures 4B and 4C). Just as RSC-derived rods maintain progenitor levels of P-cad expression during differentiation in vitro, they continue to express high expression of CD44, unlike mature rod photoreceptors in adult retina (Sarthy et al., 2007; Chaitin et al., 1994). The levels of CD44 gene expression were maintained over more than 7 days in HA or MC-containing mixtures, demonstrating that the medium itself did not affect gene expression. Additional evidence for the pro-survival effect of HA in HAMC was achieved by quantification of activated caspase-3 levels in these cultures. Significantly greater levels of apoptosis were observed with RSC-derived rods cultured in non-HA-containing, compared to HA-containing, hydrogels (p < 0.05) (Figure 4D).

Bottom Line: The pro-survival mechanism of HAMC is ascribed to the interaction of the CD44 receptor with HA.Transient disruption of the retinal outer limiting membrane, combined with HAMC delivery, results in significantly improved rod survival and visual function.The HAMC delivery system improves cell transplantation efficacy in two CNS models, suggesting broad applicability.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.

No MeSH data available.


Related in: MedlinePlus