Stabilization of cellular properties and differentiation mutilpotential of human mesenchymal stem cells transduced with hTERT gene in a long‐term culture

Abstract

Human bone marrow mesenchymal stem cells (hMSCs) are promising candidates for cell therapy and tissue engineering. The life span of hMSCs during in vitro culture is limited. Human telomerase catalytic subunit (hTERT) gene transduction can prolong the life span of hMSCs and maintain their potential of osteogenic differentiation. We established a line of hMSCs transduced with exogenous hTERT (hTERT-hMSCs) and investigated its sustaining cellular properties in a long-term culture. This line of hTERT-hMSCs was cultured for 290 population doublings (PDs) without loss of contact inhibition. Under adipogenic, chondrogenic and osteogenic induction, hTERT-hMSCs at PD 95 and PD 275 could differentiate respectively into adipocytes, chondrocytes, and osteocytes. hTERT-hMSCs at these PDs showed no transforming activity through both in vitro assay of cell growth in soft agar and in vivo assay of tumorigenicity in NOD-SCID mice. Karyotype analyses showed no significant chromosomal abnormalities in hTERT-hMSCs at these PDs. These results suggested that the hTERT-hMSCs at lower population doubling levels (PDLs) should be considered as a cell model for studies of cellular senescence, differentiation and in vitro tissue engineering experiment because of its prolonged life span and normal cellular properties.

Inducible immortality in hTERT‐human mesenchymal stem cells

“…22,23 However, enhanced telomerase expression is a characteristic of several tumors, 20,21 and while some studies have not found malignant changes in hTERT-immortalized hMSCs, 22,23,27,28 others have proven otherwise. Spontaneous alterations in c-myc proto-oncogene expression 29 and other genetic/epigenetic changes [30][31][32] have been shown to occur in long-term in vitro hTERTimmortalized hMSC and fibroblast monolayer cultures.…”

“…20,21 The potential tumorigenicity of hTERT hMSCs remains controversial. [21][22][23][24] Malignant transformation has been demonstrated in cells with ectopic expression of hTERT. 20,21,24 Because of this, tight control of hTERT expression in hMSCs would be a prerequisite for clinical use.…”

Studies on Culture and Osteogenic Induction of Human Mesenchymal Stem Cells under CO2-Independent Conditions

“…The conventional osteogenic medium was prepared by adding a given concentration of these three osteogenic factors to the conventional culture medium. This conventional osteogenic medium has been used for osteogenic induction experiments of hMSCs (Huang et al, 2008;Yang et al, 2010). Here, the experimental osteogenic medium should have the same concentration of cation as the experimental culture medium.…”

“…The conventional culture medium was modified by adding 10% FBS, 0.01 lM dexamethasone, 50 lg/mL ascorbic acid, 10 mM sodium b-glycerophosphate (Gibco BRL), 10,000 U/ mL penicillin, and 10,000 U/mL streptomycin (Life Technologies) to prepare the conventional osteogenic medium (Huang et al, 2008;Yang et al, 2010).…”

Cell vehicle targeting strategies

“…[119][120][121][122] Similarly, ectopic expression of the catalytic domain of human telomerase reverse transcriptase has been evaluated as a strategy to limit proliferative senescence in primary cell populations. 123,124 Despite their potential for enhancing therapeutic cell survival and proliferation, many of these gene products are also associated with malignant transformation. Therefore, nonconstitutive or pharmacologically regulated expression strategies may be required to ensure safety.…”