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Comparison of dedifferentiation of primary adipocytes with adipocytes obtained by induction of mesenchymal stromal cells of rats’ fatty tissue

Keywords:

N.V. Kostiuk - Ph.D. (Biol.), Associate Professor, Department of Biology, Tver State Medical University E-mail: p001637@mail.ru
M.B. Belyakova - Ph.D. (Biol.), Senior Lecturer, Department of Biochemistry with the Course of Clinical Laboratory Diagnostics, Tver State Medical University
M.V. Miniaev - Ph.D. (Biol.), Specialist of Department of the Planning and Organization, Research Institute in Tver State Medical University
D.V. Leshchenko - Ph.D. (Biol.), Associate Professor, Department of Biochemistry with the Course of Clinical La-boratory Diagnostics, Tver State Medical University
E.N. Egorova - Dr.Sc. (Med.), Associate Professor, Head of the Department of Biochemistry with the Course of Clin-ical Laboratory Diagnostics, Tver State Medical University
M.B. Petrova - Dr.Sc. (Biol.), Professor, Head of the Department of Biology, Tver State Medical University
M.V. Chernorutsky - Student, Therapy Faculty, Tver State Medical University
A.V. Panova - Student, Pediatric Faculty, Tver State Medical University


Dedifferentiated fat cells (DFAT cells), obtained with the help of ceiling cultivation, are an interesting model for the study of the reversibility of adipogenic differentiation. Although the phenomenon of dedifferentiation was discovered about 40 years ago, the return to less differentiated state of cells, which passed their initial differentiation in the culture in vitro, has not been experimentally shown yet. In this regard, the purpose of this study was to compare the ability of dedifferentiation of primary adipocytes and adipocytes obtained as a result of in vitro differentiation of mesenchymal stromal cells (MSCs).
Primary adipocytes and MSCs were isolated from the subcutaneous fat of female rats. The fat cells after multistage purification were maintained in the form of a ceiling culture on the coverslips. MSCs were grown in a conventional monolayer culture and induced to adipogenous differentiation. Maturing pop-up adipocytes were captured with cover slips. Their population consisted entirely of mature adipocytes and did not contain cells of other morphology.
Primary adipocytes and adipocytes induced in vitro were dedifferentiated in a ceiling culture in DMEM / F12 + 10% FBS. During dedifferentiation, adipocytes passed the stages of cytoplasmic outgrowths, multi-drop adipocytes, the proliferation of fat-containing cells, and a confluent monolayer. After mitosis, fat drops could be distributed between the daughter cells symmetrically or asymmetrically. Characteristically, adipocytes induced in vitro were involved in dedifferentiation earlier, and the efficiency of transformation was 1.6 times higher than that of primary adipocytes.
In a conventional monolayer culture, both populations of the DFAT cells had a high proliferative potential. The viability of the cells was 95-97% for 5 passages. In DMEM + 10% FBS, spontaneous redifferentiation of cells was observed for 2 and 3 passages. Lipid accumulation occured in 60-70% of cells, that was several times higher than the ability to adipogenous dif-ferentiation of the MSC population. The quantity of synthesized lipids was also greater in the populations of the DFAT cells than in the MSCs, as evidenced by the colorimetrical analysis of lipophilic dye Oil Red O accumulated by cells.
Floated after redifferentiation, mature adipocytes were captured with the help of cover slips and again subjected to dediffe-rentiation. The effectiveness of re-dedifferentiation was approximately twice higher than of primary one, and not dependent on the origin of the adipocytes.
Thus, dedifferentiation is shown to be characteristic not only for adipocytes isolated from tissue material, but also for cells obtained as a result of previous differentiation of MSCs in vitro. Regardless of the origin of the culture, the DFAT show a high proliferative potential and the ability for spontaneous redifferentiation. Novel adipocytes are easily involved into a cycle of repeated dedifferentiation. This approach can be used for selection of cells aimed at creating an adequate model of the reversibility of adipogenic differentiation.

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