Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation

Abstract

Cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) are being tested in several clinical trials and encouraging outcomes have been observed. To determine whether in vitro expansion influences the genomic stability of hUC-MSCs, we maintained nine hUC-MSC clones in long-term culture and comparatively analyzed them at early and late passages. All of the clones senesced in culture, exhibiting decreased telomerase activity and shortened telomeres. Two clones showed no DNA copy number variations (CNVs) at passage 30 (P30). Seven clones had ≥1 CNVs at P30 compared with P3, and one of these clones appeared trisomic chromosome 10 at the late passage. No tumor developed in immunodeficient mice injected with hUC-MSCs, regardless of whether the cells had CNVs at the late passage. mRNA-Seq analysis indicated that pathways of cell cycle control and DNA damage response were downregulated during in vitro culture in hUC-MSC clones that showed genomic instability, but the same pathways were upregulated in the clones with good genomic stability. These results demonstrated that hUC-MSCs can be cultured for many passages and attain a large number of cells, but most of the cultured hUC-MSCs develop genomic alterations. Although hUC-MSCs with genomic alterations do not undergo malignant transformation, periodic genomic monitoring and donor management focusing on genomic stability are recommended before these cells are used for clinical applications.

Figure 1

Characterization of hUC-MSCs. Nestin (a) and Sox2 (b) expression in hUC-MSCs at P3 (red) and P30 (green) was analyzed by flow cytometry. Osteogenic and adipogenic differentiation were visualized using Alizarin Red and Oil Red O staining at early and late passages (c) separately. Senescence-associated β-galactosidase activity was observed in hUC-MSCs at the late passage (d). hUC-MSCs with smaller size and rapid proliferation (e, red arrow) were distinct from the senescent cells with a flat morphology (e, white arrow)

Figure 2

Telomerase and telomere analysis. (a) Analysis of hTERT expression in hUC-MSCs at early and late passages. Relative expression levels were calculated using the −ΔΔCt method in comparison with those of HeLa cells. (b) Telomere length analysis of two hUC-MSC clones at early and late passages using Southern blotting. A reduction in telomere length was observed in hUC-MSCs from early to late passages. The control DNA supplied with the TeloTAGGG Telomere Length Assay Kit was genomic DNA purified from immortalized cell lines. MWM, molecular weight marker. (c) Telomere length of nine pairs of hUC-MSC clones at early and late passages. A paired t-test was used in the analysis of telomere length and hTERT expression in hUC-MSCs at early and late passages. Statistical analysis revealed that the telomere length of hUC-MSCs at the late passage was significantly shorter than that at early passage

Figure 3

CNVs identified by aCGH in culture. (a) Amplifications and deletions were mapped onto the human genome for nine hUC-MSC clones at P30 versus P3. Trisomy for chromosome 10 in clone 6 was not considered CNV, and thus is not shown in this figure. Each individual CNV is marked with a symbol: , amplification; , deletion. CNV segment number and length for each hUC-MSC clone are presented in panels b and c, respectively. CNV segment number and length for each chromosome are shown in panels d and e, respectively

Figure 4

Genetic changes observed in long-term cultured hUC-MSCs. (a) R-banded karyotype of clone 6 P3 with a normal chromosomal number. (b) R-banded karyotype of clone 6 P30 with trisomy for chromosome 10 (red box). (c) aCGH analysis of clone 6 P30 displayed as a single-panel rainbow plot with each chromosome differentiated by color. The red arrow shows the amplifications on chromosome 10

Figure 5

Long-term cultured hUC-MSCs did not generate malignant tumors in SCID mice. In the mice that received hUC-MSCs, no tumor was found in the subcutaneous tissues of the injection site (a and c). However, ESCs gave rise to a teratoma (b). Hematoxylin and eosin staining of the teratoma was performed. (d) Intestinal crypts (endoderm); (e) muscles (mesoderm); and (f) nerve fibers (ectoderm)

Figure 6

Unsupervised clustering of genome-wide gene expression (adjusted FPKM of >1) for six samples (clone 2 P3, clone 2 P30, clone 6 P3, clone 6 P30, clone 8 P3, and clone 8 P30). Clustering on the horizontal axis demonstrates two clusters: normal cells and cells with slight CNVs (clone 2 P3, clone 2 P30, clone 6 P3, clone 8 P3, and clone 8 P30) and cells containing substantial chromosomal changes (clone 6 P30)