Drela Katarzyna, Sarnowska Anna, Siedlecka Patrycja, Szablowska-Gadomska Ilona, Wielgos Miroslaw, Jurga Marcin, Lukomska Barbara, Domanska-Janik Krystyna
NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
First Department of Obstetrics and Gynecology, Medical University of Warsaw, Warsaw, Poland.
Cytotherapy. 2014 Jul;16(7):881-92. doi: 10.1016/j.jcyt.2014.02.009. Epub 2014 Apr 13.
As we approach the era of mesenchymal stem cell (MSC) application in the medical clinic, the standarization of their culture conditions are of the particular importance. We re-evaluated the influences of oxygens concentration on proliferation, stemness and differentiation of human umbilical cord Wharton Jelly-derived MSCs (WJ-MSCs).
Primary cultures growing in 21% oxygen were either transferred into 5% O2 or continued to grow under standard 21% oxygen conditions. Cell expansion was estimated by WST1/enzyme-linked immunosorbent assay or cell counting. After 2 or 4 weeks of culture, cell phenotypes were evaluated using microscopic, immunocytochemical, fluorescence-activated cell-sorting and molecular methods. Genes and proteins typical of mesenchymal cells, committed neural cells or more primitive stem/progenitors (Oct4A, Nanog, Rex1, Sox2) and hypoxia inducible factor (HIF)-1α-3α were evaluated.
Lowering O2 concentration from 21% to the physiologically relevant 5% level substantially affected cell characteristics, with induction of stemness-related-transcription-factor and stimulation of cell proliferative capacity, with increased colony-forming unit fibroblasts (CFU-F) centers exerting OCT4A, NANOG and HIF-1α and HIF-2α immunoreactivity. Moreover, the spontaneous and time-dependent ability of WJ-MSCs to differentiate into neural lineage under 21% O2 culture was blocked in the reduced oxygen condition. Importantly, treatment with trichostatin A (TSA, a histone deacetylase inhibitor) suppressed HIF-1α and HIF-2α expression, in addition to blockading the cellular effects of reduced oxygen concentration.
A physiologically relevant microenvironment of 5% O2 rejuvenates WJ-MSC culture toward less-differentiated, more primitive and faster-growing phenotypes with involvement of HIF-1α and HIF-2α-mediated and TSA-sensitive chromatin modification mechanisms. These observations add to the understanding of MSC responses to defined culture conditions, which is the most critical issue for adult stem cells translational applications.
随着我们迈入间充质干细胞（MSC）应用于临床医疗的时代，其培养条件的标准化尤为重要。我们重新评估了氧浓度对人脐带华通氏胶来源的间充质干细胞（WJ-MSC）增殖、干性及分化的影响。
将在21%氧气环境中进行原代培养的细胞，要么转移至5%氧气环境中，要么继续在标准的21%氧气条件下培养。通过WST1/酶联免疫吸附测定法或细胞计数来评估细胞扩增情况。培养2周或4周后，使用显微镜、免疫细胞化学、荧光激活细胞分选及分子方法评估细胞表型。评估间充质细胞、定向神经细胞或更原始的干细胞/祖细胞（Oct4A、Nanog、Rex1、Sox2）以及缺氧诱导因子（HIF）-1α至3α的典型基因和蛋白质。
将氧浓度从21%降至生理相关水平5%会显著影响细胞特性，诱导干性相关转录因子并刺激细胞增殖能力，增加的成纤维细胞集落形成单位（CFU-F）中心表现出OCT4A、NANOG以及HIF-1α和HIF-2α免疫反应性。此外，在21%氧气培养条件下WJ-MSC向神经谱系自发且随时间的分化能力在低氧条件下受到阻断。重要的是，曲古抑菌素A（TSA，一种组蛋白脱乙酰酶抑制剂）处理除了阻断低氧浓度的细胞效应外，还抑制HIF-1α和HIF-2α表达。
5%氧气的生理相关微环境使WJ-MSC培养恢复到分化程度更低、更原始且生长更快的表型，涉及HIF-1α和HIF-2α介导以及TSA敏感的染色质修饰机制。这些观察结果有助于加深对MSC对特定培养条件反应的理解，这是成体干细胞转化应用中最关键的问题。
