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. 2021 May 6;12(1):276.
doi: 10.1186/s13287-021-02355-0.

Umbilical mesenchymal stem cell-derived extracellular vesicles as enzyme delivery vehicle to treat Morquio A fibroblasts

Michael Flanagan  1 , Isha Pathak  2 , Qi Gan  1 , Linda Winter  1 , Ryan Emnet  3 , Salem Akel  3 , Adriana M Montaño  4   5
Affiliations

Umbilical mesenchymal stem cell-derived extracellular vesicles as enzyme delivery vehicle to treat Morquio A fibroblasts

Michael Flanagan et al. Stem Cell Res Ther. .

Abstract

Background: Mucopolysaccharidosis IVA (Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which results in the accumulation of the glycosaminoglycans (GAGs), keratan sulfate, and chondroitin-6-sulfate in the lysosomes of all tissues causing systemic dysfunction. Current treatments include enzyme replacement therapy (ERT) which can treat only certain aspects of the disease such as endurance-related biological endpoints. A key challenge in ERT is ineffective enzyme uptake in avascular tissues, which makes the treatment of the corneal, cartilage, and heart valvular tissue difficult. The aim of this study was to culture human umbilical mesenchymal stem cells (UMSC), demonstrate presence of GALNS enzyme activity within the extracellular vesicles (EVs) derived from these UMSC, and study how these secreted EVs are taken up by GALNS-deficient cells and used by the deficient cell's lysosomes.

Methods: We obtained and cultured UMSC from the umbilical cord tissue from anonymous donors from the Saint Louis Cord Blood Bank. We characterized UMSC cell surface markers to confirm phenotype by cell sorting analyses. In addition, we confirmed that UMSC secrete GALNS enzyme creating conditioned media for co-culture experiments with GALNS deficient cells. Lastly, we isolated EVs derived from UMSC by ultracentrifugation to confirm source of GALNS enzyme.

Results: Co-culture and confocal microscopy experiments indicated that the lysosomal content from UMSC migrated to deficient cells as evidenced by the peak signal intensity occurring at 15 min. EVs released by UMSC were characterized indicating that the EVs contained the active GALNS enzyme. Uptake of GALNS within EVs by deficient fibroblasts was not affected by mannose-6-phosphate (M6P) inhibition, suggesting that EV uptake by these fibroblasts is gradual and might be mediated by a different means than the M6P receptor.

Conclusions: UMSC can deliver EVs containing functional GALNS enzyme to deficient cells. This enzyme delivery method, which was unaffected by M6P inhibition, can function as a novel technique for reducing GAG accumulation in cells in avascular tissues, thereby providing a potential treatment option for Morquio A syndrome.

Keywords: Extracellular vesicles; Morquio A; Mucopolysaccharidosis IVA; Umbilical mesenchymal stem cell.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Surface marker expression confirms the mesenchymal phenotype of UMSC. UMSC were labeled with fluorescent-tagged antibodies (red) and compared to unlabeled controls (blue). A rightward shift in peaks indicates the presence of the stained markers. UMSC expressed the markers CD90 (a), CD73 (b), and CD105 (c). The lack of the rightward shift in (d) indicated that UMSC do not display the markers CD14, CD20, CD34, and CD45
Fig. 2
Fig. 2
UMSC are capable of uptake and release GALNS enzyme. a Specific activity of UMSC and deficient fibroblast lysate when cultured in rhGALNS-supplemented media. Both cells showed a strong increase in GALNS activity from 5h to 24h. After the addition of mannose-6-phosphate, both cells showed a decrease in activity, with UMSC showing an 80–90% reduction, and the fibroblasts having a 65–85% reduction after 24 h. b Specific activity of fibroblasts co-cultured with UMSC, normalized to fibroblasts cultured without UMSC. After an initial delay, the fibroblasts displayed a 25% increase in GALNS activity on days 3 and 5, then returning to baseline on day 7. Error bars: mean ± SD. (*P < 0.05, ***P < 0.001, two-tailed unpaired t test with Welch’s correction)
Fig. 3
Fig. 3
Co-culture of UMSC reveals transfer of material to deficient cells. Lysosomes (red) originating from Hoechst labeled UMSC feeder cells (green) were fluorescently labeled with Lysotracker Red prior to co-culture with Hoechst labeled fibroblasts (blue). This signal gradually increases within the fibroblasts during co-culture with the stained feeder cells, reaching a maximum at 15 min, before gradually declining at 20 min
Fig. 4
Fig. 4
Isolated EVs contain latent enzyme. After isolating at pelleting EVs from the host UMSC, the pellet was resuspended, and the suspension was measured for GALNS (a) or GUSB (b) activity. The EV-free supernatant of the isolation process contained no detectable GALNS or GUSB activities. Suspending the EVs in PBS contained a small amount of available GALNS and GUSB. Mechanical disruption by sonication increased the amount of available active GALNS enzyme. Finally, detergent-based lysis of EVs showed a significant increase in available active enzyme for both GALNS and GUSB. Error bars: mean ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired t test with Welch’s correction)
Fig. 5
Fig. 5
FACS scanning of EVs reveals a heterogeneous population of vesicles. UMSC-derived EVs were screened for microvesicle and exosome markers by FACS sorting. In the case of all vesicles, the EVs were separated into two populations, with the first population indicating higher side scatter than the second. Rightward peak shift indicates the presence of the stained markers. Population 1 showed a small increase in CD53+ (a), CD 151+ (b), CD14+ (c), and PECAM1+ vesicles (d). Population 2 showed a slight increase in CD151+ and CD14+ vesicles. Neither population showed an increase in CD11a+ vesicles (e). Finally, both populations had a strong presence in CD9+ vesicles (f)
Fig. 6
Fig. 6
Uptake of GALNS within EVs is more gradual and unaffected by M6P inhibition. Deficient fibroblasts were cultured in 2000 U GALNS (+GALNS) in the presence or absence of M6P (blue bars). This showed a strong increase in GALNS enzyme activity through the initial 24 h before declining, and this GALNS activity was inhibited by M6P. Deficient fibroblasts cultured in GALNS with an activity equal to those found in EVs (+LowGALNS) (purple bars) as well as UMSC-derived EVs (+EVs) (red bars) had a GALNS activity increase, though not significant at 24 h. Treatment of Morquio A deficient fibroblasts with UMSC-derived EVs or small amounts of GALNS showed steady slight increase up to 48 h, and this increase was not inhibited by M6P, suggesting a more gradual uptake. Deficient Morquio A fibroblasts without addition of exogenous GALNS (black bars). Error bars: mean ± SD. (*P < 0.05, ***P < 0.001, two-tailed unpaired t test with Welch’s correction)
Fig. 7
Fig. 7
Isolation of EVs from higher-passage UMSC does not yield reduced GALNS activity. EVs were isolated from p6 and p10 UMSC, lysed, and measured for protein-normalized activity. The change in activity from p6 to p10 was not statistically significant (P=0.33) (a). Additionally, western blotting of lysates showed the GALNS enzyme present in both early and late passages (b). Signal normalized with actin showed equal intensity (c). Error bars: mean ± SD
Fig. 8
Fig. 8
Constitutive expression of GALNS enzyme from UMSC. UMSC were transfected to constitutively express GALNS enzyme. After performing three rounds of clone selection, two clones [1–8 and 2–6] were found to express GALNS over 200% of non-transfected controls (a). Western blotting of these clones confirms the presence of the GALNS enzyme within these clones, as compared to non-transfected cells (b). Error bars: mean ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired t test with Welch’s correction)
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