Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions

Abstract

Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo. However, these cells do finally reach a senescent stage and lose their multipotent potential. Proliferation of these cells is limited up to the time of their senescence, which limits their supply, and they may accumulate chromosomal changes through ex vivo culturing. The safe, rapid expansion of hMSCs is critical for their clinical application. Chromosomal aberration is known as one of the hallmarks of human cancer, and therefore it is important to understand the chromosomal stability and variability of ex vivo expanded hMSCs before they are used widely in clinical applications. In this study, we examined the effects of culturing under ambient (20%) or physiologic (5%) O(2) concentrations on the rate of cell proliferation and on the spontaneous transformation of hMSCs in primary culture and after expansion, because it has been reported that culturing under hypoxic conditions accelerates the propagation of hMSCs. Bone marrow samples were collected from 40 patients involved in clinical research. We found that hypoxic conditions promote cell proliferation more favourably than normoxic conditions. Chromosomal aberrations, including structural instability or aneuploidy, were detected in significantly earlier passages under hypoxic conditions than under normoxic culture conditions, suggesting that amplification of hMSCs in a low-oxygen environment facilitated chromosomal instability. Furthermore, smoothed hazard-function modelling of chromosomal aberrations showed increased hazard after the fourth passage under both sets of culture conditions, and showed a tendency to increase the detection rate of primary karyotypic abnormalities among donors aged 60 years and over. In conclusion, we propose that the continuous monitoring of hMSCs will be required before they are used in therapeutic applications in the clinic, especially when cells are cultured under hypoxic conditions.

Fig 1

Karyotyping analysis of hMSCs. (A) Morphology of cells in primary culture (left) and in late (13th) passage (right). The size of hMSCs in culture was enlarged with increasing passages. Scale bars, 200 μm. (B) G-banding karyotyping of cells with chromosomal anomalies (donor #100). Karyotyping analysis was performed as described in ‘Materials and methods’. The karyotype shows an addition on chromosome 12 [46,XY,add(12)(p13)]. The chro-mosomal anomaly is indicated by an arrow.

Fig 2

Calculated cumulative cell divisions of hMSCs cultured from passages 0 to 7, under hypoxia (5% O₂) and normal conditions (20% O₂). (A) PDL curves of hMSCs under the two culture conditions. Cumulative numbers of cell divisions (shown as PDL) are shown for one example grown until passage 7 (donor #100). Each symbol represents a single time-point of subculturing. (B) Correlation between cumulative PDL and passage number under the two different oxygen conditions. Results illustrate population doublings during the expansion time in different culture conditions as described in ‘Materials and methods’. hMSCs cultured under hypoxic culture (×) showed a significantly higher rate of proliferation than hMSCs under normal conditions (○) (P = 0.0005). (C) Comparison of the estimated number of proliferative hMSCs from all samples cultured in the two oxygen conditions. hMSCs propagated rapidly under hypoxic culture, and the estimated number cultured in hypoxia was approximately 2.5 times that of cells cultured under normal conditions at passage 3 (**P < 0.01).

Fig 3

Comparison of change in chromoso-mal aberrations under two different culture conditions. (A) Kaplan–Meier plot showing the aberration-free survival of hMSCs cultured under normoxic (solid line) and hypoxic (dotted line) conditions (log-rank test; P = 0.032). The end-point was defined as the occurrence of chromosomal aberrations. ‘No. of At risk’ below the figure indicates the number of samples which had never undergone chromosomal aberrations until the passage. (B) Estimated hazard curve for an anomalous karyotype during continuous passages from passage 0 to 7.

Fig 4

Change in chromosome aberrations during ex vivo culture. (A) The proportion of aberrant karyotypes is illustrated at each culture passage during expansion. The vertical axis of figure indicates the total number of samples that were subjected to karyotyping analysis at each passage. The percentage of chromosome aberrations was as indicated below each graph. (B) Comparison of smoothed hazard modelling for anomalous karyotype by increasing donor age. Among hMSCs derived from donors in the age of over 60, the hazard risk increased under both conditions after passage 4 (P4). Especially under hypoxic conditions, the increase in hazard was observed at later passages in hMSCs derived from all age groups except donors aged 49 or under.