Generation and Characterization of an Immortalized Human Mesenchymal Stromal Cell Line

Abstract

Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use due to their capacity to self-renew and differentiate into multiple mesenchymal lineages. However, disadvantages of primary cultures of hMSCs are the limited in vitro lifespan, and the variable properties of cells from different donors and over time in culture. In this article, we describe the generation of a telomerase-immortalized nontumorigenic human bone marrow-derived stromal mesenchymal cell line, and its detailed characterization after long-term culturing (up to 155 population doublings). The resulting cell line, iMSC#3, maintained a fibroblast-like phenotype comparable to early passages of primary hMSCs, and showed no major differences from hMSCs regarding surface marker expression. Furthermore, iMSC#3 had a normal karyotype, and highresolution array comparative genomic hybridization confirmed normal copy numbers. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding DNA methylation profiles were more diverse. The cells also had proliferation characteristics comparable to primary hMSCs and maintained the capacity to differentiate into osteoblasts and adipocytes. A detailed characterization of the mRNA and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. In summary, the immortalized mesenchymal cells represent a valuable model system that can be used for studies of candidate genes and their role in differentiation or oncogenic transformation, and basic studies of mesenchymal biology.

Molecular Phenotyping of Telomerized Human Bone Marrow Skeletal Stem Cells Reveals a Genetic Program of Enhanced Proliferation and Maintenance of Differentiation Responses

“…Increase in cancer antigen families in telomerized hMSC have been reported, including previous work from our group, which studied the tumorigenic hMSC‐TERT20 . In another study of telomerized hMSC, similar changes were observed. These cancer antigen gene functions are poorly understood, although they are frequently expressed in tumor or embryonic tissues but not in somatic tissues (as reviewed in Simpson and colleagues).…”

“…We have previously reported the generation and characterization of telomerized hMSC, which we termed hMSC‐TERT, and reported that telomerization abolished replicative senescence and not only maintained but enhanced bone formation capacity of the cells when implanted subcutaneously in immune‐deficient mice . Other investigators corroborated these findings (see summary of the telomerized cell lines in Table ). Telomerized hMSC are also good cellular models for primary hMSC because of their stable phenotype and are thus suitable for proteomic and genomic studies requiring the use of a large number of cells .…”

“…hMSC isolated using an anti‐stro‐1 antibody, which is known to enrich for multipotent hMSC, were compared to hMSC‐TERT and reported that among 35 CD markers examined, 31 showed no significant quantitative change in expression. Similarly, Skarn and colleagues showed that the hMSC line (iMSC#3) showed no difference in cell surface markers compared with the primary hMSC.…”

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research

“…Positive for ARS, APS, and Runx2 upregulation [71] Positive for ABS, TBS, and GAG assay in pellet culture [72]; low chondrogenic potential but stimulation of chondrocyte differentiation Positive for OROS, adipogenesis-related genes upregulation [71,73], and NRS [73] BMA13H Positive for ARS (reduced compared with primary cells) [74] Positive for ABS and GAG assay in 2D culture [74]; also positive for TBS, PSR and aggrecan and ColII immunostaining in 3D culture [75]; chondrogenic potential reduced compared with primary cells [74] Positive for OROS (reduced compared with primary cells) [74] SCP-1…”

“…Transduction of hTERT alone has been employed to generate iMSC lines, but, since hTERT has no effect over non-replicative senescence, it has also been reported to fail to immortalize MSCs derived from bone marrow [13,18,58,74,104] and adipose tissue [85]. Skårn et al (2014) described that only one out of nine hTERT-transduced bone marrow-derived MSC clones was able to proliferate over 40 PDs, and even this clone showed a slow proliferation rate similar to that of primary MSCs [71]. Other authors have confirmed that hTERT-transduced MSCs displayed a lifespan similar to that of primary MSCs (about 30-40 PDs) [18,81].…”