Tiwari Shubha, Nair Rohini M, Vamadevan Praseeda, Ali Mohammad Javed, Naik Milind N, Honavar Santosh G, Vemuganti Geeta K
Sudhakar and Sreekant Ravi Stem Cell Biology Laboratory, L V Prasad Eye Institute, Hyderabad, India.
School of Medical Sciences, University of Hyderabad, Hyderabad, 500046, India.
Graefes Arch Clin Exp Ophthalmol. 2018 Apr;256(4):717-727. doi: 10.1007/s00417-018-3926-8. Epub 2018 Feb 17.
Lacrimal gland (LG) dysfunction leading to dry eye syndrome (DES) is an important cause of ocular morbidity. One of the potential and promising long-term management therapies for restoration of LG function could be transplantation of autologous ex vivo expanded stem cells. The present study was aimed at exploring the 2D and 3D cultures of human LG, identifying inherent stem cells and evaluating their secretory potential.
Fresh human lacrimal gland (HuLG) (n = 5) from patients undergoing therapeutic exenteration was harvested after ethical approval and informed consent. The gland was enzymatically digested and the isolated cells plated in Hepato-STIM media supplemented with l-glutamine, epidermal growth factor, fibroblast growth factor, and N-2 supplement. The native HuLG and the cultured spheres (DIV14-16) were evaluated for presence of stem cells (CD117 expression, quiescence, BrdU label retention, cell cycle, colony forming efficiency) and differentiation (secretion of tear proteins).
Under the established culture conditions, suspension 3D cultures of human "lacrispheres" could be maintained and propagated for 3-4 weeks. The spheres consist of both acinar as well as ductal cells with evidence of stem cells (0.8 ± 0.05% CD117 cells), BrdU label retention (9.31 ± 0.41%), G0/G1 profile similar to native lacrimal cells at isolation (76.9 versus 79.9%) and colony forming units (3.1%). The lacrispheres also secreted quantifiable levels of tear proteins (lysozyme, lactoferrin, scIgA) into the conditioned media.
The study provides promising, first-of-its-kind evidence for the generation of lacrispheres from fresh HuLG, with enriched population of stem cells and secretory competent differentiated cells. The dual properties of these spheres make them a highly suitable source of transplantable cells for restoring the structure and function of damaged lacrimal gland.
泪腺(LG)功能障碍导致干眼症(DES)是眼部发病的一个重要原因。恢复LG功能的一种潜在且有前景的长期治疗方法可能是自体体外扩增干细胞移植。本研究旨在探索人LG的二维和三维培养,鉴定固有干细胞并评估其分泌潜能。
在获得伦理批准和知情同意后,从接受治疗性眼球摘除术的患者中获取新鲜人泪腺(HuLG)(n = 5)。将腺体进行酶消化,分离出的细胞接种于添加了L-谷氨酰胺、表皮生长因子、成纤维细胞生长因子和N-2补充剂的Hepato-STIM培养基中。对天然HuLG和培养的球体(第14 - 16天)进行干细胞存在情况(CD117表达、静止状态、BrdU标记保留、细胞周期、集落形成效率)和分化情况(泪液蛋白分泌)的评估。
在既定培养条件下,人“泪球体”的悬浮三维培养物可维持并传代3 - 4周。球体由腺泡细胞和导管细胞组成,有干细胞证据(0.8±0.05% CD117细胞)、BrdU标记保留(9.31±0.41%)、与分离时天然泪腺细胞相似的G0/G1期分布(76.9%对79.9%)以及集落形成单位(3.1%)。泪球体还向条件培养基中分泌了可定量的泪液蛋白(溶菌酶、乳铁蛋白、分泌型免疫球蛋白A)。
本研究为从新鲜HuLG生成泪球体提供了有前景的、同类研究中的首个证据,其具有富集的干细胞群体和具有分泌能力的分化细胞。这些球体具有的双重特性使其成为恢复受损泪腺结构和功能的高度合适的可移植细胞来源。