Salivary gland stem cell-derived exosomes produced by a Wnt-loaded microwell culture accelerates the recovery from salivary gland dysfunction in murine salivary gland damage models

Abstract

Background and objectives: The three-dimensional (3D) spheroid culture has been found to enhance stem cell properties such as differentiation potential and paracrine function. However, the potential benefits of harnessing the 3D spheroid culture for exosome production and regenerative application have not been fully investigated. This study aims to develop a microwell scaffold incorporating a biochemical niche to boost stemness and exosome production from salivary gland stem cells. Methods: We fabricated a WNT3A protein (WNT)-loaded PLGA electrospun nanofiber that can enable sustained WNT release, mimicking the microenvironment of niche molecule release. This WNT-loaded nanofiber was integrated with a PCL microwell scaffold using an insert. Human salivary gland-derived epithelial stem cells (SG-EpSCs) were then cultured on either 2D plastic dishes or 3D bare PLGA nanofiber-PCL microwells (3DPLGA) and WNT-loaded PLGA nanofiber-PCL microwells (3DWNT). We then investigate the regenerative effects of SG-EpSCderived exosomes produced in these microwells on salivary dysfunction using an irradiated murine salivary gland in mice and a human salivary organoid model. Results: Fabricated PCL microwells and WNT3A-loaded PLGA nanofibers, which were subsequently micro-patterned onto PCL electrospun nanofibers. This resulted in a WNT3Abiosubstrate composite culture system featuring microwell arrays that secrete WNT3A into the wells. Our findings suggest that this novel WNT3A-secreting microwell culture system enhances stem cell characteristics, including increased marker expression, cell proliferation, and differentiation potential, as well as improved exosome production and secretion. Moreover, the v data indicate that canonical WNT signaling regulates the functional enhancement of SG-EpSCderived exosomes in 3D spheroids cultured within WNT3A-secreting microwells. Recovery proceeded more rapidly in the 3D culture compared to 2D, with a significant increase in the number of proliferating cells observed in the 3DWNT condition. These results suggest that YWAHAZ, highly expressed in SG-EpSC-derived exosomes, significantly enhances the proliferation rate of salivary gland progenitor cells and increases phosphorylated PI3K-AKT expression, indicating its potential to improve salivary gland function.

본 논문은 지속적인 WNT3A 방출이 있는 3차원 마이크로웰 스캐폴드에서 생산된 타액선 줄기세포 유래 엑소좀의 재생 효과를 조사하기 위한 연구를 진행했습니다. 이 연구는 방사선에 노출된 쥐의 타액선의 기능에 대한 조사를 중점적으로 다루었습니다. 새로운 배양방법이 유전자 표지자 발현, 측분비 기능, 그리고 분화 잠재력 측면에서 줄기세포를 촉진하는지 여부를 결정하고, 3차원 스페로이드 배양에서 줄기세포 촉진의 역할을 조사했습니다. 이를 위해 WNT가 로드된 나노섬유를 사용하여 PCL 마이크로웰 스캐폴드와 결합되었고, 지속적인 WNT 방출을 가능하게 하기 위해 WNT3A 단백질이 로드된 PLGA 나노섬유가 제조되었습니다. 인간 타액선에서 유래된 상피 줄기세포는 2차원(단일세포) 또는 3차원 PLGA 나노섬유-PCL 마이크로웰 (3DPLGA) 및 WNT로드된 PLGA 나노섬유-PCL 마이크로웰 (3DWNT)에 배양되었습니다. 우리는 배양 매체에서 초원심분리기를 이용하여 분리한 기능성 엑소좀을 역행적으로 쥐의 타액관에 주입하고, 쥐의 타액선 손상 모델에서 타액선 기능 회복에 대한 타액선 줄기세포 유래 엑소좀의 재생 잠재력을 조사했습니다. 결과적으로, 마이크로웰 스캐폴드에 배양된 줄기세포는 모두 3차원 스페로이드를 형성했으며, 3DWNT의 스페로이드는 3DPLGA에 비해 줄기세포 관련 유전자 및 단백질 발현이 현저하게 더 높았으며, 더 많은 양의 엑소좀을 생산하고 측분비 활동이 향상되었습니다.