Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, 15213, USA.
Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, 15213, USA; Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, China.
Exp Eye Res. 2019 Dec;189:107860. doi: 10.1016/j.exer.2019.107860. Epub 2019 Oct 23.
Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to β-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.
脂肪来源的干细胞（ADSCs）在再生医学中具有重要贡献，范围从临床试验前模型中测试用于疾病治疗的干细胞疗法到临床试验。为了立即将干细胞用于治疗，需要在不同时间点使用高剂量的干细胞，这可以通过冷冻保存来满足。在这项研究中，我们评估了长期冷冻保存的 ADSCs 的特性及其在平均 12 年冷冻保存后的再生潜力。通过台盼蓝、钙黄绿素/ Hoechst 和 MTT 测定法检查复苏的 ADSCs 的细胞活力和增殖。通过流式细胞术、免疫染色和 qPCR 检查干细胞标志物的表达。还评估了集落形成效率和球体形成能力。通过诱导成骨细胞、脂肪细胞、神经细胞、角膜成纤维细胞和小梁网（TM）细胞来评估多能分化潜力。解冻后，ADSCs 保持干细胞标志物 CD90、CD73、CD105、CD166、NOTCH1、STRO-1、ABCG2、OCT4、KLF4 的表达。ADSCs 保持集落和球体形成能力。这些细胞能够分化为成骨细胞，通过茜素红 S 染色和骨钙蛋白和骨桥蛋白表达升高来证实；通过油红 O 染色和 PPARγ2 表达升高分化为脂肪细胞。ADSCs 可分化为神经细胞，β-III 微管蛋白、神经丝、GFAP 染色阳性，巢蛋白和神经丝 mRNA 表达升高。ADSCs 还可以产生表达角膜蛋白聚糖、角膜硫酸聚糖、ALDH 和胶原 V 的角膜成纤维细胞，以及表达 CHI3L1 和 AQP1 的 TM 细胞。分化的 TM 细胞对皮质醇治疗的反应是肌球蛋白表达增加，这可作为进一步研究的体外青光眼模型。发现 ADSCs 的条件培养基对原代 TM 细胞具有再生作用。总之，ADSCs 在长期冷冻保存后保持其干性和多能性，不同供体之间存在差异。这项研究在再生医学中具有重要意义，并为使用冷冻保存的 ADSCs 进行未来的临床试验铺平了道路。
