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 chon-drogenic 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.
Highlights
Human bone marrow-derived mesenchymal stem cells are an attractive target for therapeutic cell transplantation, since they have a high proliferation capacity and maintain in vitro the ability to differentiate into a variety of mesenchymal tissues such as bone, cartilage, fat and muscle [1, 2]
All clones and the heterogeneous human telomerase reverse transcriptase (hTERT)-Human mesenchymal stem cells (hMSCs) expressed hTERT, while in untransduced hMSCs no hTERT expression was detected on mRNA level by polymerase chain reaction (PCR) (Fig. 1B)
HTERT protein was primarily located within the nucleus of the hTERT-expressing hMSCs
Summary
Human bone marrow-derived mesenchymal stem cells (hMSCs) are an attractive target for therapeutic cell transplantation, since they have a high proliferation capacity and maintain in vitro the ability to differentiate into a variety of mesenchymal tissues such as bone, cartilage, fat and muscle [1, 2]. The excitement to have an unlimited cell source of hMSCs for therapeutic cell transplantation by ectopic expression of hTERT was limited by more recent finding that these cells underwent neoplastic transformation [15, 16]. There are, dozens of normal cell types that have been immortalized with telomerase without signs of malignant transformation, without altering pre-existing genetic abnormalities, and without altering differentiation capacity [17]. All hTERT-expressing hMSCs, which showed malignant transformation so far, used gamma-retroviral vectors. We show for the first time that an MSC line generated by ectopic expressing of hTERT using lentiviral gene transfer did not cause malignant transformation and, may be a safe tool for ex vivo cell and gene therapy
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