K.Y. Smazhilo1, V.A. Usachev2, M.A. Kulebyakina3, M.V. Shchebetina4, E.S. Chechekhina5, E. Bakhchinyan6, O. I. Klychnikov7, M.V. Vorontsova8, K.Y. Kulebyakin9

1–9 Lomonosov Moscow State University (Moscow, Russia)

1 k.y.komashko@gmail.com, 2 usachjov-vova@mail.ru, 3 m.a.kulebyakina@gmail.com, 4 mshchebetina@mail.ru, 5 voynovaes.pharm@gmail.com, 6 is5492@mail.ru, 7 ollie.klychnikov@mail.ru, 8 maria.v.vorontsova@mail.ru, 9 konstantin-kuleb@mail.ru

Immortalized lines of multipotent mesenchymal stem cells (MSCs) have many advantages for use in regenerative medicine compared to primary MSC cultures. However, it has been repeatedly demonstrated that some properties of immortalized cell lines including response to various stimuli and differentiation potential may not be equivalent to those of original primary cultures. Here, we assessed these properties of immortalized MSCs in the context of bone tissue regeneration.

The aim of the study was to compare signaling and differentiation responses to parathyroid hormone (PTH), the main pro-osteogenic stimulus and regulator of bone tissue renewal in humans, given by immortalized adipose tissue MSCs line ASC52Telo and by primary adipose tissue MSC cultures. Patterns of cell signaling responses, expressed in changes in cytoplasmic calcium level, were detected by fluorescent single-cell microscopy using the Fluo-8 probe. Effect of PTH on osteogenic and adipogenic differentiation was assessed in vitro using adipogenic and osteogenic differentiation media, respectively. Visualization of differentiation results was performed by alizarin red (for osteogenic differentiation) or NileRed (for adipogenic differentiation) staining.

We demonstrated that the effect of PTH on immortalized ASC52Telo cells and on primary MSCs differs significantly. Compared to primary adipose tissue MSCs, the proportion of cells responding to PTH with cAMP-dependent calcium responses (smooth increases in intracellular calcium concentration) in the ASC52Telo line was almost twofold reduced – 17.5% versus 33.1%. At the same time, ASC52Telo line has a higher proportion of cells in which PTH causes phosphoinositide-dependent calcium responses (calcium oscillations or single calcium peaks). We also found that PTH suppresses osteogenic differentiation of ASC52Telo cells and increases their differentiation efficiency towards adipogenic lineage. These effects are opposite to the effect of PTH on cells of primary adipose tissue MSCs, on which PTH demonstrates pro-osteogenic and anti-adipogenic effects. Taken together, observed differences in responses to PTH point out possible limitations in the applicability of ASC52Telo cells for various bone defects treatment approaches as well as for fundamental studies of bone tissue regeneration.

Smazhilo K.Y., Usachev V.A., Kulebyakina M.A., Shchebetina M.V., Chechekhina E.S., Bakhchinyan E., Klychnikov O.I., Vorontsova M.V., Kulebyakin K.Y. Altered sensitivity of immortalized mesenchymal stromal cells to the homeostatic regulator of bone renewal. Technologies of Living Systems. 2026. V. 23. № 1. Р. 20-30. DOI: https://doi.org/10.18127/j20700997-202601-02 (In Russian).

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