Adipose-Derived Mesenchymal Stem Cell (MSC) Immortalization by Modulation of hTERT and TP53 Expression Levels

Abstract

Mesenchymal stem cells (MSCs) are pivotal players in tissue repair and hold great promise as cell therapeutic agents for regenerative medicine. Additionally, they play a significant role in the development of various human diseases. Studies on MSC biology have encountered a limiting property of these cells, which includes a low number of passages and a decrease in differentiation potential during in vitro culture. Although common methods of immortalization through gene manipulations of cells are well established, the resulting MSCs vary in differentiation potential compared to primary cells and eventually undergo senescence. This study aimed to immortalize primary adipose-derived MSCs by overexpressing human telomerase reverse transcriptase (hTERT) gene combined with a knockdown of TP53. The research demonstrated that immortalized MSCs maintained a stable level of differentiation into osteogenic and chondrogenic lineages during 30 passages, while also exhibiting an increase in cell proliferation rate and differentiation potential towards the adipogenic lineage. Long-term culture of immortalized cells did not alter cell morphology and self-renewal potential. Consequently, a genetically stable line of immortalized adipose-derived MSCs (iMSCs) was established.

Keywords: MSC; MSC differentiation; immortalization; mesenchymal stem cell; tumor stroma.

Figure 1

Phenotypic characterization of adMSCs: (A) Immunophenotypic analysis of adipose-derived cells from flow cytometry-generated histograms. Light blue curves represent isotypic control and blue curves represent fluorescence peaks. (B) Flow cytometry histogram of GFP-FITC fluorescence signal. The blue curve represents negative control—non-immortalized cells; red curve represents iMSCs. (C) Cell morphology of iMSC cells at 50th passage did not change compared to control cells at 5th passage.

Figure 2

Functional characterization of iMSCs in comparison to control cells: (A) Diagram representing the MTT-assay for cell proliferation rate of iMSCs and control cells. Three replicates were obtained for each cell line. Cells were collected at the 5th passage for control cells and at the 5th and 50th passages for iMSCs. Error bars show mean ± SD; *-p < 0.05, **-p < 0.01. (B) Representative pictures of adipogenesis with Oil red O dye staining neutral lipids in cells; osteogenesis with Alizarin red S dye staining calcium deposits in cells; chondrogenesis with Alcian dye staining sulfated proteoglycan in cells. (C) The histogram of differentiated cell count of iMSCs at the 5th and 30th passages compared to control. The data is presented as a mean value ± SD; ***-p < 0.001.

Figure 3

Sphere formation efficacy evaluation. (A) Sphere formation of iMSC-c2 after cultivation in serum-free cell culture medium at 200× (left) and 400× (right) magnification. (B) Histogram of sphere count when initially 1 × 10⁴ cells were seeded. Bars show the mean ± SD of three biological replies per microscopic field at 100× magnification. (C) Histogram displaying cell number in iMSC spheres. Bars show the mean ± SD of three biological replicates.