1 Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, China.
3 Department of Neurosurgery, Affiliated Haikou Hospital of Xiangya School of Central South University, Haikou, China.
Cell Transplant. 2017 Aug;26(8):1452-1461. doi: 10.1177/0963689717720291.
Olfactory mucosa mesenchymal stem cells (OM-MSCs) display significant clonogenic activity and may be easily propagated for Parkinson's disease therapies. Methods of inducing OM-MSCs to differentiate into dopaminergic (DAergic) neurons using olfactory ensheathing cells (OECs) are thus an attractive topic of research. We designed a hypoxic induction protocol to generate DAergic neurons from OM-MSCs using a physiological oxygen (O) level of 3% and OEC-conditioned medium (OCM; HI group). The normal induction (NI) group was cultured in O at ambient air level (21%). The role of hypoxia-inducible factor-1α (HIF-1α) in the differentiation of OM-MSCs under hypoxia was investigated by treating cells with an HIF-1α inhibitor before induction (HIR group). The proportions of β-tubulin- and tyrosine hydroxylase (TH)-positive cells were significantly increased in the HI group compared with the NI and HIR groups, as shown by immunocytochemistry and Western blotting. Furthermore, the level of dopamine was significantly increased in the HI group. A slow outward potassium current was recorded in differentiated cells after 21 d of induction using whole-cell voltage-clamp tests. A hypoxic environment thus promotes OM-MSCs to differentiate into DAergic neurons by increasing the expression of HIF-1α and by activating downstream target gene TH. This study indicated that OCM under hypoxic conditions could significantly upregulate key transcriptional factors involved in the development of DAergic neurons from OM-MSCs, mediated by HIF-1α. Hypoxia promotes DAergic neuronal differentiation of OM-MSCs, and HIF-1α may play an important role in hypoxia-inducible pathways during DAergic lineage specification and differentiation in vitro.
嗅黏膜间充质干细胞（OM-MSCs）表现出显著的克隆形成活性，并且易于增殖，可用于帕金森病的治疗。因此，使用嗅鞘细胞（OEC）诱导 OM-MSCs 分化为多巴胺能（DAergic）神经元的方法是一个有吸引力的研究课题。我们设计了一种低氧诱导方案，使用 3%的生理氧（O）水平和 OEC 条件培养基（OCM；HI 组）从 OM-MSCs 中产生 DAergic 神经元。正常诱导（NI）组在大气氧（21%）水平下培养。通过在诱导前用缺氧诱导因子-1α（HIF-1α）抑制剂处理细胞（HIR 组），研究了缺氧对 OM-MSCs 分化中 HIF-1α的作用。免疫细胞化学和 Western blot 显示，与 NI 和 HIR 组相比，HI 组中 β-微管蛋白和酪氨酸羟化酶（TH）阳性细胞的比例明显增加。此外，HI 组中多巴胺水平明显增加。使用全细胞膜片钳测试在诱导 21 天后记录到分化细胞中的外向钾电流。因此，低氧环境通过增加 HIF-1α的表达并激活下游靶基因 TH，促进 OM-MSCs 分化为 DAergic 神经元。本研究表明，在低氧条件下的 OCM 通过 HIF-1α介导，可显著上调 OM-MSCs 向 DAergic 神经元分化过程中关键转录因子的表达。低氧促进 OM-MSCs 向 DAergic 神经元分化，HIF-1α 可能在体外 DAergic 谱系特化和分化的缺氧诱导途径中发挥重要作用。
