Immortalizing Mesenchymal Stromal Cells from Aged Donors While Keeping Their Essential Features

Abstract

Human bone marrow-derived mesenchymal stromal cells (MSCs) obtained from aged patients are prone to senesce and diminish their differentiation potential, therefore limiting their usefulness for osteochondral regenerative medicine approaches or to study age-related diseases, such as osteoarthiritis (OA). MSCs can be transduced with immortalizing genes to overcome this limitation, but transduction of primary slow-dividing cells has proven to be challenging. Methods for enhancing transduction efficiency (such as spinoculation, chemical adjuvants, or transgene expression inductors) can be used, but several parameters must be adapted for each transduction system. In order to develop a transduction method suitable for the immortalization of MSCs from aged donors, we used a spinoculation method. Incubation parameters of packaging cells, speed and time of centrifugation, and valproic acid concentration to induce transgene expression have been adjusted. In this way, four immortalized MSC lines (iMSC#6, iMSC#8, iMSC#9, and iMSC#10) were generated. These immortalized MSCs (iMSCs) were capable of bypassing senescence and proliferating at a higher rate than primary MSCs. Characterization of iMSCs showed that these cells kept the expression of mesenchymal surface markers and were able to differentiate towards osteoblasts, adipocytes, and chondrocytes. Nevertheless, alterations in the CD105 expression and a switch of cell fate-commitment towards the osteogenic lineage have been noticed. In conclusion, the developed transduction method is suitable for the immortalization of MSCs derived from aged donors. The generated iMSC lines maintain essential mesenchymal features and are expected to be useful tools for the bone and cartilage regenerative medicine research.

Generation of Mesenchymal Cell Lines Derived from Aged Donors

“…In this study, primary MSCs derived from aged donors were immortalized by sequential spinoculation of SV40LT and hTERT, as previously described [ 30 ]. Immortalized MSCs overcame senescence and acquired an unlimited proliferation potential while maintaining most of the characteristics of MSCs.…”

“…Moreover, the expression of CD105 was higher in iMSCs and T-MSCs than in primary MSCs. We have previously observed that CD105 can either reduce or increase its expression from primary to immortalized cells [ 30 ], and its expression can also be reduced with passaging [ 29 , 33 , 34 , 35 , 36 ]. The role that CD105 expression plays in MSCs is still unclear [ 37 ].…”

“…We previously developed a method suitable for immortalizing MSCs derived from aged donors by spinoculation of SV40LT and hTERT. In this way, we previously generated four immortalized mesenchymal cell (iMSC) lines: three derived from three hip OA patients, and one derived from a hip fracture patient without OA [ 30 ]. In this study, we generated two iMSC lines by immortalization of primary MSCs derived from two elderly patients with hip fractures and without OA.…”

Mesenchymal Stem Cells as a Cornerstone in a Galaxy of Intercellular Signals: Basis for a New Era of Medicine

“…MSC immortalization can be achieved by the combination of transduction of immortalizing genes such as simian virus 40 large T antigen (SV40LT), which promote cell cycle progression and human telomerase reverse transcriptase (hTERT) and prevent telomeres shortening [ 286 ]. In addition, both morphology and functionality of MSC were found to not change after transduction of immortalization genes [ 285 , 287 ].…”

Versatility of Induced Pluripotent Stem Cells (iPSCs) for Improving the Knowledge on Musculoskeletal Diseases

“…In these lines, cells can be produced in large amounts and grown indefinitely, offering a good tool to explore molecular and biochemical processes [ 9 ]. Numerous immortalized cell lines have been generated from MSCs, bone cells, muscle cells and chondrocytes [ 22 , 23 , 24 ], which have provided valuable information about the processes involved in skeletal development. However, these cell lines may contain genetic and metabolic abnormalities due to their derivation using mainly integrative methods.…”