From cytogenomic to epigenomic profiles: monitoring the biologic behavior of in vitro cultured human bone marrow mesenchymal stem cells

Abstract

IntroductionBone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles.MethodsIn this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors.ResultsOur data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages.ConclusionsBecause we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on hBM-MSC malignant transformation in the literature.

Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation

“…hMSC genomic stability during the culture period was assessed by monitoring the chromosomal status at several passages in vitro. No abnormalities were observed [14]. The hMSC immunophenotype remained unchanged in early and late culture passages.…”

Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation

“…[12][13][14] By analyzing the amount of CNV segments at the P30, different genomic instability was observed among hUC-MSCs derived from different donors in our study. To analyze the relationship between the transcriptome and genomic instability during prolonged culture of hUC-MSCs derived from different donors, we studied the genome-wide changes in gene expression, molecular networks, and signaling pathways for three pairs of hUC-MSC clones (at early and late passages) using the high-throughput sequencing technology.…”

“…10,11,37 However, whether MSCs with genomic anomalies gained the ability of forming tumor in vivo was unclear, even though chromosomal aberrations were found in cultured ADMSCs and BMMSCs. High-throughput methods were also used to investigate the genomic mutation of cultured ADMSCs and BMMSCs, 12,14 whereas, these studies failed to find significant genomic changes and stated that MSCs expanded in vitro confirmed an overall stability of genomic profile. This might be because of the shorter in vitro life span of ADMSCs and BMMSCs, and thus the effect of prolonged culture on the genomic stability of MSCs could not be revealed sufficiently.…”

“…Nonetheless, for the relatively shorter in vitro life span of BMMSCs and ADMSCs, few in vitro culture-related CNVs were found in these studies. [12][13][14] Whether MSCs with genomic instability undergo malignant transformation in culture or could form a tumor in an animal model was not clear.…”

Telomerase-mediated telomere elongation from human blastocysts to embryonic stem cells

“…Telomere maintenance is principally associated with telomerase activity, which protects telomere integrity through the addition of single stranded TTAGGG repeats onto the 39-ends of chromosomes. Telomerase activity is generally silenced or found at very low levels in somatic cells, and telomerase expression is insufficient to maintain telomere length in most adult stem cells, which limits cell division (Redaelli et al, 2012). In contrast, most tumor cells, germline cells and embryonic stem cells (ESC) exhibit high levels of telomerase expression.…”