Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy

Abstract

Ex vivo expansion and manipulation of human mesenchymal stem cells are important approaches to immunoregulatory and regenerative cell therapies. Although these cells show great potential for use, issues relating to their overall nature emerge as problems in the field. The need for extensive cell quantity amplification in vitro to obtain sufficient cell numbers for use, poses a risk of accumulating genetic and epigenetic abnormalities that could lead to sporadic malignant cell transformation. In this study, we have examined human mesenchymal stem cells derived from bone marrow, over extended culture time, using cytogenetic analyses, mixed lymphocyte reactions, proteomics and gene expression assays to determine whether the cultures would retain their potential for use in subsequent passages. Results indicate that in vitro cultures of these cells demonstrated chromosome variability after passage 4, but their immunomodulatory functions and differentiation capacity were maintained. At the molecular level, changes were observed from passage 5 on, indicating initiation of differentiation. Together, these results lead to the hypothesis that human mesenchymal stem cells cultures can be used successfully in cell therapy up to passage 4. However, use of cells from higher passages would have to be analysed case by case.

Figure 1

hMSC capacity for differentiation. (a), (d) and (g) – undifferentiated hMSCs at passages 1, 5 and 8. (b), (e) and (h) – adipogenic differentiation at passages 1, 5 and 8. Accumulation of neutral lipid vacuoles stained with oil red O indicate differentiation. (c), (f) and (i) – osteogenic differentiation at passages 1, 5 and 8. Calcium deposition stained with alizarin red indicate differentiation. Scale bars ‐ 26 μm (100× magnification).

Figure 2

Maintenance of hMSC markers. Total RNA was isolated from hMSC cultures at different passages (1 to 9) and examined using Q‐PCR to determine changes in mRNA expression levels after normalisation to β‐actin and GAPDH expression. Analysis of fold changes in expression of SCF, CD44, c‐Kit and BMPR were performed at the different passages. Data are presented as fold induction relative to control group (passage 1). Mean ± SD (n = 9). *Statistical significance was considered at P < 0.001.

Figure 3

Morphologic changes indicating senescence. Using the same confluence stage, representative morphology of hMSCs is shown at early passage (P3) [(a) and (c)] and late passage (P8) [(b) and (d)]. (a) and (b): Scale bars ‐ 65 μm (40× magnification). (c) and (d): Scale bars ‐13 μm (200× magnification). Cells were seeded at the same density (2.000 cells/cm²), and at P3, the hMSC culture obtained confluence at 7 days, while at P8, confluence was obtained at 12 days.

Figure 4

hMSC‐mediated T cell inhibition. (a) hMSC inhibition of MLR. Lymphocyte proliferation was assessed using MLR in absence (MLR) or presence of 10% hMSCs/well, administered at early (P1 to P5) or late (P6 to P10) passage. Results expressed as mean ± sd for independent experiments using 9 hMSC cultures (P < 0.01); (b) Representative assay showing MLR in absence (MLR) or presence of 10% MSC/well at early (P1 to P5) or late (P6 to P9) passages. Black represents proliferative cells and grey represents non‐proliferative cells. R ‐ responders without stimulators.

Figure 5

hMSC proteomic profile obtained using cells at passages 1, 5 and 8 from the same donor. (a) 2‐DE gel ‐ passage 1. (b) 2‐DE gel ‐ passage 5. (c) 2‐DE gel ‐ passage 8. Values on horizontal axes represent pI, and values on vertical axes represent molecular weight.

Figure 6

Bidimensional gel analysis using Image Master 2D Platinum 6.0. Values on horizontal axes represent master gel 1 products from pool of 2‐DE gels from passages 1, 5 and 8 from the same donor. Values on vertical axes represent master gel 2 products from pool of 2‐DE gels from passages 1, 5 and 8 from the same donor, which were different from those of master gel 1. Scatter plot calculated using results from analysed gels and shows relationship between spot values (% volume), which show 96.9% similarity.