With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.

中文翻译：

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低氧和常氧条件下培养的早期和晚期传代脂肪间充质干细胞的分化潜能

随着越来越多地关注用于治疗关节和骨骼疾病（如骨关节炎）的临床应用所需的间充质干细胞（MSC）的大规模扩增，体外条件的优化MSC扩展需要仔细考虑以保持本地MSC特性。诸如氧气浓度，培养基成分和通过次数等生理参数会影响MSC的特性，并可能对其治疗潜力产生重大影响。在缺氧条件下生长的细胞已在临床上广泛记录。大规模培养MSC需要生物反应器培养。然而，在大型生物反应器容器的多个通道中维持低氧气和其他生理参数是一项挑战。扩大体外细胞生产的必要性在常氧状态下可能影响MSC的重要属性。由于这些原因，我们的研究调查了常氧和低氧培养条件对早期和晚期传代脂肪来源的MSC的影响。我们检查了每种条件对关键干细胞标记基因POU5F1，NANOG和KLF4以及分化基因RUNX2，COL1A1，SOX9，COL2A1和PPARG的表达的影响。我们发现，在正常氧状态下，干细胞标记基因，成骨和软骨形成基因的表达水平高于缺氧状态。此外，这些基因的表达随传代次数减少，但PPARG除外，脂肪分化标记，可能是由于MSC的脂肪来源所致。我们通过流式细胞仪证实了在所有条件下细胞表面标志物CD105，CD73和CD90的存在以及CD45，CD34，CD14和CD19的缺乏表达。此外，体外分化证实，在低氧或常氧状态下生长的早期和晚期脂肪来源的MSC均可分化为成软骨和成骨细胞类型。我们的结果表明，在低氧或常氧条件下培养的早期或晚期传代MSC中，可以维持定义MSC适用于基于实验室和临床前研究的最低标准标准。因此，当将生物反应器中的MSC放大以用于同种异体临床应用或组织工程以治疗关节和骨骼疾病（例如骨关节炎）时，可以使用任何这些培养条件。