Preclinical Evaluation of the Tumorigenic and Immunomodulatory Properties of Human Bone Marrow Mesenchymal Stromal Cell Populations with Clonal Trisomy 5

Abstract

Cytogenetic aberrations may emerge in human mesenchymal stromal cells (MSC) during ex vivo expansion for cell therapy. We have detected clonal trisomy 5 in two distinct autologous MSC products expanded from bone marrow which, based on the current quality control criteria, could not be released for clinical use. Although a safety concern, it is still unclear to what extent recurrent aneuploidies detected in MSC products may affect the threshold for neoplastic transformation or the medicinal properties of these cells. We have carried out an exploratory preclinical study to evaluate these MSC products with clonal trisomy 5, regarding their oncogenic and immunomodulatory potential. Cell population growth in vitro was reduced in MSC cultures with clonal trisomy 5 compared with the population growth of their euploid MSC counterparts, based on a lower cumulative population doubling level, reduced cell proliferation index, and increased senescence-associated beta-galactosidase activity. Subcutaneous injection of clinically relevant amount of MSC population, either with or without clonal trisomy 5, did not generate tumors in immunodeficient mice within a follow-up period of six months. Most importantly, MSC population with clonal trisomy 5 kept immunomodulatory properties upon interferon gamma (IFNγ) licensing, displaying overexpression of IDO, CXCL9, CXCL10, and CXCL11, in a similar fashion than that of IFNγ-licensed euploid MSC. Our findings suggest that bone marrow MSC products with clonal trisomy 5 may retain their therapeutic potential, based on poor tumor initiating capability and preserved immunomodulatory potency. This preclinical evidence may further support the definition of release criteria of autologous MSC products for cell therapy under critical clinical scenarios. This trial is registered with Clinical Study registration number: RBR-29x2pr.

Figure 1

Cytogenetic characterization of distinct human MSC populations after ex vivo expansion. (a–d) Karyotyping results of the four investigated MSC cell lines. (a) MSC sample T5-50 karyotype 47,XX,+5. (b) MSC sample T5-53 karyotype 47,XX,+5. (c) MSC sample M10 karyotype 46,XX. (d) MSC sample M12 karyotype 46,XY. (e) CNV profiles of three MSC cell lines showing the results observed by CMA. Microarray probes are plotted along the X-axis according to their genomic coordinates and each color represents a chromosome (indicated below), from 1 to 22, X and Y (from the short to the long arm). The Y-axis represents the log² scale of the copy number ratio MSC cell lines/control (values close to 0 indicate regions with similar copy number between tested and control samples; positive values represent gains; negative values represent losses). The arrow shows the detected trisomy of chromosome 5 in mosaic.

Figure 2

Characterization of human MSC cell cultures. (a) Cumulative population doublings during cellular expansion ex vivo. Passage interval of samples: M10: P5 to P10; M12: P7 to P12; T5-50: P6 to P8; T5-53: P7 to P10. (b) Percentage of proliferative cells. One-Way ANOVA and Tukey's multiple comparisons test. (c) Percentage of cells with SA-β-galactosidase activity; 100 cells analyzed per well. One-Way ANOVA and Tukey's multiple comparisons test. (d) Intensity of SA-β-galactosidase activity per cell; cell fluorescence intensity (CF) calculated with ImageJ by CF = total fluorescence–(cell area × background intensity). Kruskal-Wallis and Dunn's multiple comparisons test. (e) Comparison of cellular morphology of M10 (Passage 8) and M12 (Passage 10) with T5-50 (Passage 7) and T5-53 (Passage 8) samples. T5-50 and T5-53 cells with typical flattened and enlarged senescent morphology. All pictures were captured using 100x magnification. Assays were performed in triplicate, at the same passage. Error bars represent standard deviation. ^∗∗∗P value < 0.001; ^∗∗∗∗P value < 0.0001; ns: statistically nonsignificant. M10 and M12: euploid human bone marrow MSC samples; T5-50 and T5-53: human bone marrow MSC samples with clonal trisomy 5.

Figure 3

Tumorigenic potential of ex vivo expanded MSC. Images of tumors or flank region of animals at the time of euthanasia. M10 and M12: euploid human bone marrow MSC samples; T5-50 and T5-53: human bone marrow MSC samples with clonal trisomy 5; USP7: tumorigenic human ATRT cell line.

Figure 4

Relative expression of cytokines related to MSC immunomodulatory response, before and after stimulation by IFN-γ. (a) IDO mRNA relative expression. (b) CXCL9 mRNA relative expression. (c) CXCL10 mRNA relative expression. (d) CXCL11 mRNA relative expression. Values were normalized by GAPDH expression, using M10 IFNγ licensed as the reference sample. ^∗∗∗∗P < 0.0001. M10 and M12: euploid human bone marrow MSC samples; T5-50 and T5-53: human bone marrow MSC samples with clonal trisomy 5.