Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia

Abstract

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.

Figure 1

Relative gene expression levels by qRT-PCR for cells cultured under hypoxia and normoxia. (a) qRT-PCR for pluripotency genes KLF4, NANOG, and POU5F1. (b) qRT-PCR for MSC differentiation genes; osteogenic markers COL1A1 and RUNX2, chondrogenic markers SOX9 and COL2A1, and adipogenic marker PPARG. Relative expression levels are expressed using the GAPDH as the reference gene, and values were normalized to P5H; error bars represent SD. Statistically significant difference, p < 0.05, shown by ^∗ for relative expression level when compared to P5H sample.

Figure 2

Flow cytometry of CD cell surface markers for cells cultured under hypoxia and normoxia. The positive CD markers for MSCs as detected by the fluorescent antibodies anti-CD73 FITC, anti-CD105 PE, and anti-CD90 PE Cy7. The negative markers of MSCs were detected using anti-CD14 FITC, anti-CD45 PerCP, anti-CD34-R-PE, and anti-CD19 PE-Cy7 antibodies. Unstained cell for each condition was used as negative controls.

Figure 3

Osteogenic differentiation of MSCs cultured under hypoxia and normoxia. Alizarin Red S staining for cells differentiated for 19 days imaged in (a–e) culture wells and (f–j) on coverslips. (a) Negative control, (b) passage 5 hypoxia (P5H), (c) passage 9 hypoxia (P9H), (d) passage 5 normoxia (P5N), and (e) passage 9 normoxia (P9H). Scale bar 100 μm.

Figure 4

Chondrogenic differentiation of MSCs cultured under hypoxia and normoxia. Alcian Blue staining for cells differentiated for 21 days imaged in (a–e) culture wells and (f–j) on coverslips. (a) Negative control, (b) passage 5 hypoxia (P5H), (c) passage 9 hypoxia (P9H), (d) passage 5 normoxia (P5N), and (e) passage 9 normoxia (P9H). Scale bar 100 μm.