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Purification and ex vivo expansion of fully functional salivary gland stem cells.

Nanduri LS, Baanstra M, Faber H, Rocchi C, Zwart E, de Haan G, van Os R, Coppes RP - Stem Cell Reports (2014)

Bottom Line: Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages.Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function.Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, 9713AV Groningen, the Netherlands.

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Potency of Expanded Salisphere-Derived CD24hi/CD29hi Cells(A) Percentage sphere formation related to the number of passages (x axis) of CD24hi/CD29hi cells from n = 4 independent experiments. Error bars are SEM.(B) Increase (p < 0.05) in percentage of CD24hi/CD29hi cells determined by flow cytometry in self-renewal passage 1 (P1) and P9 from n = 4 independent experiments. Error bars are SEM.(C) Expansion of CD24hi/CD29hi and unselected cells at different passages in self-renewal conditions (two-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). Data from n = 4 independent experiments. Error bars are SEM.(D) Photomicrograph showing single-cell-derived ductal organoids from expanded cells at various passages (P1, P4, P6, and P11); scale bar, 50 μm.(E) Graph showing saliva (% of preirradiation; y axis) at different days postirradiation from the irradiated, nontransplanted mice and mice transplanted with different cell populations.(F) The box-whisker plot shows recovery of saliva (as % of preirradiation values) in irradiated (15 Gy) mice 90 days posttransplantation with primary-sphere-derived CD24hi/CD29hi cells, passage 2, or passage 13 cells in comparison to irradiated, nontransplanted controls (n = 6–10 mice/group). Note the uniform response in all mice transplanted with passage 13 cells. (∗p < 0.01; ∗∗p < 0.001; ∗∗∗p < 0.001; one-way ANOVA). All error bars are SEM.See also Figures S1–S3 and Tables S3 and S4.
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fig4: Potency of Expanded Salisphere-Derived CD24hi/CD29hi Cells(A) Percentage sphere formation related to the number of passages (x axis) of CD24hi/CD29hi cells from n = 4 independent experiments. Error bars are SEM.(B) Increase (p < 0.05) in percentage of CD24hi/CD29hi cells determined by flow cytometry in self-renewal passage 1 (P1) and P9 from n = 4 independent experiments. Error bars are SEM.(C) Expansion of CD24hi/CD29hi and unselected cells at different passages in self-renewal conditions (two-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). Data from n = 4 independent experiments. Error bars are SEM.(D) Photomicrograph showing single-cell-derived ductal organoids from expanded cells at various passages (P1, P4, P6, and P11); scale bar, 50 μm.(E) Graph showing saliva (% of preirradiation; y axis) at different days postirradiation from the irradiated, nontransplanted mice and mice transplanted with different cell populations.(F) The box-whisker plot shows recovery of saliva (as % of preirradiation values) in irradiated (15 Gy) mice 90 days posttransplantation with primary-sphere-derived CD24hi/CD29hi cells, passage 2, or passage 13 cells in comparison to irradiated, nontransplanted controls (n = 6–10 mice/group). Note the uniform response in all mice transplanted with passage 13 cells. (∗p < 0.01; ∗∗p < 0.001; ∗∗∗p < 0.001; one-way ANOVA). All error bars are SEM.See also Figures S1–S3 and Tables S3 and S4.

Mentions: To assess their self-renewal potential, cells from all populations were cultured under MM conditions. Most cells from CD24hi/CD29lo and CD24lo/CD29hi subsets do not proliferate or die during culture as observed with trypan-blue-based cell counting. Very few form secondary spheres that did not yield enough cells to allow passaging. CD24med/CD29hi- and CD24hi/CD29hi-derived cells, however, were able to self-renew for four passages and greater than five passages, respectively (Figure S1A), indicating the higher potential of these populations. During self-renewal, spheres are dissociated to single cells at every passage. To minimize dissociation-induced stress, the Rho-inhibitor Y-27632, known to protect against dissociation-induced cell stress, was added (Zhang et al., 2011; enriched medium [EM]). It enhanced the initial secondary sphere-formation percentage of CD24hi/CD29hi cells (Figure S1B) from 2.5% ± 0.68% (MM) to 10.9% ± 3.9% (EM). Next, CD24hi/CD29hi cells were cultured under EM conditions, which resulted in a more-rapid and pronounced expansion of cells capable of forming secondary spheres. In general, the percentage of cells capable of forming spheres was increased with increasing passages (Figure 4A) from 11.54% ± 5.02% at passage 1 (P1) to 25.51% ± 1.86% at P12 (p < 0.05; Figure S1C), indicative of enrichment for sphere-forming cells. This is further emphasized by the significant increase (Figure 4B) in cells expressing CD24hi/CD29hi from P1 to P9 (Figure S1D).


Purification and ex vivo expansion of fully functional salivary gland stem cells.

Nanduri LS, Baanstra M, Faber H, Rocchi C, Zwart E, de Haan G, van Os R, Coppes RP - Stem Cell Reports (2014)

Potency of Expanded Salisphere-Derived CD24hi/CD29hi Cells(A) Percentage sphere formation related to the number of passages (x axis) of CD24hi/CD29hi cells from n = 4 independent experiments. Error bars are SEM.(B) Increase (p < 0.05) in percentage of CD24hi/CD29hi cells determined by flow cytometry in self-renewal passage 1 (P1) and P9 from n = 4 independent experiments. Error bars are SEM.(C) Expansion of CD24hi/CD29hi and unselected cells at different passages in self-renewal conditions (two-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). Data from n = 4 independent experiments. Error bars are SEM.(D) Photomicrograph showing single-cell-derived ductal organoids from expanded cells at various passages (P1, P4, P6, and P11); scale bar, 50 μm.(E) Graph showing saliva (% of preirradiation; y axis) at different days postirradiation from the irradiated, nontransplanted mice and mice transplanted with different cell populations.(F) The box-whisker plot shows recovery of saliva (as % of preirradiation values) in irradiated (15 Gy) mice 90 days posttransplantation with primary-sphere-derived CD24hi/CD29hi cells, passage 2, or passage 13 cells in comparison to irradiated, nontransplanted controls (n = 6–10 mice/group). Note the uniform response in all mice transplanted with passage 13 cells. (∗p < 0.01; ∗∗p < 0.001; ∗∗∗p < 0.001; one-way ANOVA). All error bars are SEM.See also Figures S1–S3 and Tables S3 and S4.
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fig4: Potency of Expanded Salisphere-Derived CD24hi/CD29hi Cells(A) Percentage sphere formation related to the number of passages (x axis) of CD24hi/CD29hi cells from n = 4 independent experiments. Error bars are SEM.(B) Increase (p < 0.05) in percentage of CD24hi/CD29hi cells determined by flow cytometry in self-renewal passage 1 (P1) and P9 from n = 4 independent experiments. Error bars are SEM.(C) Expansion of CD24hi/CD29hi and unselected cells at different passages in self-renewal conditions (two-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). Data from n = 4 independent experiments. Error bars are SEM.(D) Photomicrograph showing single-cell-derived ductal organoids from expanded cells at various passages (P1, P4, P6, and P11); scale bar, 50 μm.(E) Graph showing saliva (% of preirradiation; y axis) at different days postirradiation from the irradiated, nontransplanted mice and mice transplanted with different cell populations.(F) The box-whisker plot shows recovery of saliva (as % of preirradiation values) in irradiated (15 Gy) mice 90 days posttransplantation with primary-sphere-derived CD24hi/CD29hi cells, passage 2, or passage 13 cells in comparison to irradiated, nontransplanted controls (n = 6–10 mice/group). Note the uniform response in all mice transplanted with passage 13 cells. (∗p < 0.01; ∗∗p < 0.001; ∗∗∗p < 0.001; one-way ANOVA). All error bars are SEM.See also Figures S1–S3 and Tables S3 and S4.
Mentions: To assess their self-renewal potential, cells from all populations were cultured under MM conditions. Most cells from CD24hi/CD29lo and CD24lo/CD29hi subsets do not proliferate or die during culture as observed with trypan-blue-based cell counting. Very few form secondary spheres that did not yield enough cells to allow passaging. CD24med/CD29hi- and CD24hi/CD29hi-derived cells, however, were able to self-renew for four passages and greater than five passages, respectively (Figure S1A), indicating the higher potential of these populations. During self-renewal, spheres are dissociated to single cells at every passage. To minimize dissociation-induced stress, the Rho-inhibitor Y-27632, known to protect against dissociation-induced cell stress, was added (Zhang et al., 2011; enriched medium [EM]). It enhanced the initial secondary sphere-formation percentage of CD24hi/CD29hi cells (Figure S1B) from 2.5% ± 0.68% (MM) to 10.9% ± 3.9% (EM). Next, CD24hi/CD29hi cells were cultured under EM conditions, which resulted in a more-rapid and pronounced expansion of cells capable of forming secondary spheres. In general, the percentage of cells capable of forming spheres was increased with increasing passages (Figure 4A) from 11.54% ± 5.02% at passage 1 (P1) to 25.51% ± 1.86% at P12 (p < 0.05; Figure S1C), indicative of enrichment for sphere-forming cells. This is further emphasized by the significant increase (Figure 4B) in cells expressing CD24hi/CD29hi from P1 to P9 (Figure S1D).

Bottom Line: Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages.Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function.Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, 9713AV Groningen, the Netherlands.

Show MeSH
Related in: MedlinePlus