Purification and ex vivo expansion of fully functional salivary gland stem cells.
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.
Affiliation: Department of Cell Biology, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, 9713AV Groningen, the Netherlands.Show MeSH
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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).
Affiliation: Department of Cell Biology, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, 9713AV Groningen, the Netherlands.