A novel aldehyde dehydrogenase-3 activator leads to adult salivary stem cell enrichment in vivo

Abstract

Purpose: To assess aldehyde dehydrogenase (ALDH) expression in adult human and murine submandibular gland (SMG) stem cells and to determine the effect of ALDH3 activation in SMG stem cell enrichment.

Experimental design: Adult human and murine SMG stem cells were selected by cell surface markers (CD34 for human and c-Kit for mouse) and characterized for various other stem cell surface markers by flow cytometry and ALDH isozymes expression by quantitative reverse transcriptase PCR. Sphere formation and bromodeoxyuridine (BrdUrd) incorporation assays were used on selected cells to confirm their renewal capacity and three-dimensional (3D) collagen matrix culture was applied to observe differentiation. To determine whether ALDH3 activation would increase stem cell yield, adult mice were infused with a novel ALDH3 activator (Alda-89) or with vehicle followed by quantification of c-Kit(+)/CD90(+) SMG stem cells and BrdUrd(+) salispheres.

Results: More than 99% of CD34(+) huSMG stem cells stained positive for c-Kit, CD90 and 70% colocalized with CD44, Nestin. Similarly, 73.8% c-Kit(+) mSMG stem cells colocalized with Sca-1, whereas 80.7% with CD90. Functionally, these cells formed BrdUrd(+) salispheres, which differentiated into acinar- and ductal-like structures when cultured in 3D collagen. Both adult human and murine SMG stem cells showed higher expression of ALDH3 than in their non-stem cells and 84% of these cells have measurable ALDH1 activity. Alda-89 infusion in adult mice significantly increased c-Kit(+)/CD90(+) SMG population and BrdUrd(+) sphere formation compared with control.

Conclusion: This is the first study to characterize expression of different ALDH isozymes in SMG stem cells. In vivo activation of ALDH3 can increase SMG stem cell yield, thus providing a novel means for SMG stem cell enrichment for future stem cell therapy.

Figure 1

Isolation of human salivary stem cells. A, flow cytometric analysis of CD34-positive cells isolated from human SMGs shows colocalization of CD34 (94.7%), CD90 (99.4%), c-Kit (99.5%), and CD44/Nestin (70%). B, the cultured salispheres show positive staining for BrdUrd when compared with negative staining control. C, the salispheres showed positive staining for CD34 and c-Kit when compared with negative staining control. D, salispheres formed ductal-like structures (indicated by arrow) when cultured in 3D collagen matrix. The acinar structure is shown by periodic acid—Schiff (PAS) staining.

Figure 2

ALDH expression in salivary stem cells. A, quantitative PCR analysis of expression of different ALDHs in mouse SMG c-Kit⁺ cells showed that there is a significant (P < 0.05) increase in the expression of ALDH3A2 (1.70 ± 0.44) and ALDH6A (1.76 ± 0.22) isozymes when compared with the normalized c-Kit⁻ cells (1.00 ± 0.00). ALDH1A3 (1.04 ± 0.02) and ALDH3A1 (1.10 ± 0.05) expression did not differ from the c-Kit⁻ cells. In contrast, ALDH2 (0.49 ± 0.03, P < 0.05) expression was significantly lower in the c-Kit⁺ cells. Analysis using the ALDEFLUOR assay showed that 69% of the mouse SMG stem cells have elevated ALDH1 activity. B, quantitative PCR analysis of human SMG CD34⁺ cells shows a significant (P < 0.05) increase in the expression of ALDH3A2 (7.94 ± 3.91), ALDH1A1 (1.90 ± 0.33), and ALDH1A3 (4.55 ± 1.31) when normalized to their respective expressions in CD34⁻ cells. The increased expression of ALDH2 (2.00 ± 1.09, P > 0.05) in the CD34⁺ cells was not significant. Analysis using the ALDEFLUOR assay showed that 84% of the human SMG stem cells have elevated ALDH1 activity.

Figure 3

In vivo ALDH3 activation enriches salivary stem cells. A, Alda-89 (5-allyl-1,3-benzodioxol; molecular weight = 162) is an ALDH3 isozyme-selective activator. At 50 μmol/L, Alda-89 significantly increases the catalytic activity of human ALDH3 recombinant enzyme by 304.4% ± 7.1% of control (n = 3, P < 0.01), but has no effect on ALDH1, ALDH2, or ALDH5 isozymes (left). In vivo, infusion of Alda-89 into mice by an osmotic pump at 34 mg/kg/d enhances ALDH3 enzymatic activity by 29% in esophageal homogenate as compared with vehicle control (n = 5, P < 0.05). B, flow cytometric analysis of mouse SMGs collected after 7 days of Alda-89 treatment showed a signigicant increase of c-Kit⁺/CD90⁺ cells when compared with the vehicle control (17.8% ± 0.95% vs. 7.37% ± 0.45%, P < 0.05). In contrast, there was a slightly lower increase of c-Kit⁻ cells (right). C, unselected SMG cells were collected and cultured to examine the effects of Alda-89 on BrdUrd⁺ salisphere formation. The total number of salispheres were significantly greater in the Alda-89–treated group than in vehicle control (121 ± 0.83 spheres/1 × 10⁵ cells vs. 86 ± 0.58; P < 0.05, left). The number of BrdUrd⁺ spheres were also significantly higher in the Alda-89–treated group than in the vehicle control (91 ± 0.83 spheres/1 × 10⁵ cells vs. 66 ± 1.42, P < 0.05, right).