Novel Immortalized Human Multipotent Mesenchymal Stromal Cell Line for Studying Hormonal Signaling

Abstract

Multipotent mesenchymal stromal cells (MSCs) integrate hormone and neuromediator signaling to coordinate tissue homeostasis, tissue renewal and regeneration. To facilitate the investigation of MSC biology, stable immortalized cell lines are created (e.g., commercially available ASC52telo). However, the ASC52telo cell line has an impaired adipogenic ability and a depressed response to hormones, including 5-HT, GABA, glutamate, noradrenaline, PTH and insulin compared to primary cells. This markedly reduces the potential of the ASC52telo cell line in studying the mechanisms of hormonal control of MSC's physiology. Here, we have established a novel immortalized culture of adipose tissue-derived MSCs via forced telomerase expression after lentiviral transduction. These immortalized cell cultures demonstrate high proliferative potential (up to 40 passages), delayed senescence, as well as preserved primary culture-like functional activity (sensitivity to hormones, ability to hormonal sensitization and differentiation) and immunophenotype up to 17-26 passages. Meanwhile, primary adipose tissue-derived MSCs usually irreversibly lose their properties by 8-10 passages. Observed characteristics of reported immortalized human MSC cultures make them a feasible model for studying molecular mechanisms, which regulate the functional activities of these cells, especially when primary cultures or commercially available cell lines are not appropriate.

Keywords: ASC52telo; differentiation; hormone sensitivity; immortalization; multipotent mesenchymal stromal cells (MSCs); proliferative potential; telomerase reverse transcriptase (TERT).

Figure 1

Telomerase expression in immortalized MSCs. (a) TERT mRNA expression level before (pMSCs) and after the immortalization procedure (iMSC-1 and iMSC-2) established using qPCR; (b) Western blot analysis of TERT protein content; (c) telomerase activity assessment by qPCR assay; (d) relative telomere length. *—p < 0.05 (ANOVA multiple pairwise comparison, Tukey test), n = 3. Data are presented as mean ± standard deviation.

Figure 2

iMSC proliferation. Proliferation of pMSC and iMSC cultures was assessed via measuring the dynamics of cell density (upper panels) and calculating the population doubling time (PDT, lower panels). #N—passage number, *—p < 0.05—pMSCs PDT vs. iMSCs PDT at the corresponding passage, n ≥ 4. Data are presented as mean ± standard deviation.

Figure 3

iMSCs senescence. Beta-galactosidase activity (blue color) was analyzed in pMSCs and ASC52telo cell line (upper panel’s row) as well as iMSCs-1 and iMSCs-2 cell lines. Passage number is indicated for each cell line.

Figure 4

Flow cytometry analysis of MSC-specific markers in pMSC, iMSC and ASC52telo cell lines. #N—passage number.

Figure 5

iMSC-2 differentiation potential. (a) MSCs cultured in appropriate differentiation medium for 2 weeks. Adipogenic differentiation was assessed by Nile red staining of accumulated lipids (green fluorescence), osteogenic differentiation—by Alizarin red staining of calcium deposits (red color) and chondrogenic differentiation—by Alcian blue staining of cartilage acidic glycosaminoglycans (light blue color); (b) relative expression level of marker genes of osteogenic (osteocalcin, Runx) and adipogenic (PPARgamma, adiponectin) differentiation in pMSCs and iMSCs cultured for 15 days in ordinary complete growth medium or appropriate differentiation medium.

Figure 6

iMSC response to hormones. (a) The portion of cells, responded to glutamate (10⁻⁵ M), GABA (2 × 10⁻⁵ M), dopamine (10⁻⁵ M), noradrenaline (10⁻⁶ M), angiotensin II (10⁻⁸ M), histamine (10⁻⁶ M), 5-HT (10⁻⁵ M) and PTH (10⁻⁸ M) by increase in cytoplasmic Ca²⁺ influx. (b,c) AKT and ERK phosphorylation in response to insulin. Western blot of MSC samples with P-AKT and P-ERK specific antibodies. (c) Vinculin content was used as a reference for densitometry of P-AKT and P-ERK bands.