Impact of the Stimulation and Inhibition of NAD +  Biosynthesis on the Maintenance of Pluripotency in Mouse Embryonic Stem Cells

M. V. Antipova; Антипова М. В.; V. A. Kulikova; Куликова В. А.; L. V. Solovjeva; Соловьева Л. В.; A. V. Kropotov; Кропотов А. В.; M. P. Svetlova; Светлова М. П.; A. P. Yakimov; Якимов А. П.; K. B. Nerinovski; Нериновский К. Б.; E. I. Bakhmet; Бахмет Е. И.; A. A. Nikiforov; Никифоров А. А.

doi:10.31857/S0041377123030033

Impact of the Stimulation and Inhibition of NAD⁺ Biosynthesis on the Maintenance of Pluripotency in Mouse Embryonic Stem Cells

Authors: Antipova M.V.¹, Kulikova V.A.¹^,2, Solovjeva L.V.¹, Kropotov A.V.¹, Svetlova M.P.¹, Yakimov A.P.¹^,3, Nerinovski K.B.⁴, Bakhmet E.I.¹, Nikiforov A.A.¹
Affiliations:
1. Institute of Cytology, Russian Academy of Sciences
2. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences
3. Peter the Great St. Petersburg Polytechnic University
4. St. Petersburg State University
Issue: Vol 65, No 3 (2023)
Pages: 273-282
Section: Articles
URL: https://rjmseer.com/0041-3771/article/view/669647
DOI: https://doi.org/10.31857/S0041377123030033
EDN: https://elibrary.ru/VBPSEK
ID: 669647

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Abstract

Nicotinamide adenine dinucleotide (NAD⁺) plays a key role in cellular metabolism and signaling. In recent years, evidence has accumulated that NAD⁺-dependent processes are involved in the regulation of pluripotency and differentiation of mammalian embryonic stem cells. The major means to maintain NAD⁺ levels in mammalian cells is through its biosynthesis from various forms of vitamin B3. In this study, we examined how stimulation and inhibition of NAD⁺ biosynthesis affect the maintenance of the pluripotency of mouse embryonic stem cells E14 Tg2a (E14 cells). The pluripotency status of E14 cells was assessed by immunocytochemical and immunoblotting analysis using antibodies to the pluripotency factor Oct4, as well as by staining for alkaline phosphatase. Using NMR spectroscopy, we have found that the concentration of NAD⁺ in pluripotent E14 cells cultured in the presence of LIF is about 4 nmol/mg, and it remains unchanged after induction of differentiation with retinoic acid. We have also demonstrated that pharmacological stimulation of NAD⁺ biosynthesis by nicotinamide riboside increases the level of intracellular NAD⁺ by 20%, but it does not affect the maintenance of pluripotency in E14 cells. Moreover, under conditions of critical depletion of NAD⁺ pool by Nampt inhibition with FK866 E14 cells maintained pluripotency, though the expression level of Oct4 was decreased.

Keywords

NAD⁺, NMR spectroscopy, mouse embryonic stem cells Tg2α E14, pluripotency, differentiation, Oct4

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