Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer

Abstract

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

Fig. 1

(A) Plasmid chart of pLenti6/V5-hTERT (lentiviral expression construct). (B) hTERT mRNA was detected in hTERT-transduced hMSCs. hTERT: heterogeneous hTERT-hMSCs, SCP: single-cell-picked hTERT-hMSCs. No hTERT expression was detected in untransduced hMSC. (C-F) HTERT localization in MSCs. Immunohistochemical staining of untransduced hMSC (C, E) and hTERT transduced hMSC/SCP-1 (D, F). Staining patterns and distribution of hTERT are shown in E and F, DAPI nuclear counterstaining in C and D. Bar = 100 μm. (G) Telomerase activity assay. Untransduced hMSC show only background level of relative telomerase activity. hTERT-hMSCs have a significant higher relative telomerase activity (*P < 0.001). 293 cells: positive control.

Fig. 2

(A-C) Cell morphology at PDL 31, 121 and 224 of hMSC clone (SCP-1) ectopically expressing telomerase. (D, E) Senescence-associated β-galactosidase assay. Untransduced hMSCs show pronounced β-galactosidase activity at PDL 24. In contrast, hTERT-transduced hMSCs had no β-galactosidase activity even at higher passages. (F) Growth curve of untransduced hMSC (hMSCs), heterogeneous (hTERT-hMSCs) and single-cell-picked hMSC clones (SCP-1, -9, -11–12). (G) BrdU assay of heterogeneous hTERT-hMSCs and SCP-1 showed a significantly higher proliferation of SCP-1 (P= 0.00014 hTERT young [PDL 31]versus SCP-1 young passage [PDL 31]).

Fig. 3

(A) Expression of tumour suppressor genes. There is no relevant change in tumour suppressor gene (Rb, p53 and p21) expression at different time points in heterogeneous (hTERT-hMSCs) and single-cell-picked hMSCs (SCP) in PCR analysis. MG63 (positive control) showed a marked down-regulation of p53 and p21. (B) Expression of p53, p21, p14 and p16 in SCP-1 (PDL 17 and 322). There was no significant down-regulation of these genes even after almost 2 years in culture. (C, D) Soft agar assay. HTERT-transduced hMSCs did show contact inhibition, while the tumour cell line HT1080 formed colonies. (E–H)in vivo tumour formation. HTERT-transduced hMSCs (here shown SCP-1) did not show macroscopic (D) and microscopic (E, haematoxylin and eosin stain) after 8 weeks. In contrast, HT1080 formed macroscopically visible tumours within 1 week (F). Haematoxylin and eosin stain revealed a characteristic tumour growth.

Fig. 4

(A–C) Karyotyping revealed no change in hTERT-transduced SCP-1 clone (passage 92, PDL 175), while SCP-11 (passage 89, PDL 127) and SCP-12 (passage 80, PDL 90) showed identical deletions in the long arm of chromosome 16 (16q). (D–G) FISH analysis revealed that these deletions already existed in younger passages of untransduced and hTERT-transduced hMSCs and significantly accumulated with time (P < 0.018).

Fig. 5

Differentiation assay. HTERT-transduced hMSCs (SCP-1) maintained their stem cell character and were still capable to differentiate into the osteogenic (B), adipogenic (D) and chondrogenic (F) lineages. (A, C, E) Undifferentiated control cells.