TY - JOUR

T1 - The Impact of Stimulation and Suppression of NAD+ Biosynthesis on the Maintenance of Pluripotency of Mouse Embryonic Stem Cells

AU - Antipova, M.V.

AU - Kulikova, V.A.

AU - Solovjeva, L.V.

AU - Kropotov, A.V.

AU - Svetlova, M.P.

AU - Yakimov, A.P.

AU - Nerinovski, K.B.

AU - Bakhmet, E.I.

AU - Nikiforov, A.A

PY - 2023/9/22

Y1 - 2023/9/22

N2 - Abstract: Nicotinamide adenine dinucleotide (NAD+) plays a key role in cell 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 elucidated how stimulation and suppression of NAD+ biosynthesis affect the maintenance of the pluripotency of E14 Tg2a mouse embryonic stem cells (E14 cells). The pluripotency status of E14 cells was assessed by immunocytochemical analysis and immunoblotting 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 the leukemia inhibitory factor is about 4 nmol/mg and it remains unchanged after the induction of differentiation with retinoic acid. We have also shown that pharmacological stimulation of NAD+ biosynthesis with nicotinamide riboside increases the level of intracellular NAD+ by 20%, but it does not affect the maintenance of pluripotency of E14 cells. Moreover, under conditions of critical depletion of the intracellular NAD+ pool, when its synthesis from nicotinamide was suppressed by nicotinamide phosphoribosyltransferase inhibitor (FK866), E14 cells retained pluripotency, while the Oct4 protein level was reduced.

AB - Abstract: Nicotinamide adenine dinucleotide (NAD+) plays a key role in cell 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 elucidated how stimulation and suppression of NAD+ biosynthesis affect the maintenance of the pluripotency of E14 Tg2a mouse embryonic stem cells (E14 cells). The pluripotency status of E14 cells was assessed by immunocytochemical analysis and immunoblotting 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 the leukemia inhibitory factor is about 4 nmol/mg and it remains unchanged after the induction of differentiation with retinoic acid. We have also shown that pharmacological stimulation of NAD+ biosynthesis with nicotinamide riboside increases the level of intracellular NAD+ by 20%, but it does not affect the maintenance of pluripotency of E14 cells. Moreover, under conditions of critical depletion of the intracellular NAD+ pool, when its synthesis from nicotinamide was suppressed by nicotinamide phosphoribosyltransferase inhibitor (FK866), E14 cells retained pluripotency, while the Oct4 protein level was reduced.

KW - E14 Tg2a mouse embryonic stem cells

KW - NAD+

KW - NMR spectroscopy

KW - Oct4

KW - differentiation

KW - pluripotency

UR - https://www.mendeley.com/catalogue/2ccfc59e-6d84-34e1-a9ba-8adca01ba171/

U2 - 10.1134/S1990519X23050024

DO - 10.1134/S1990519X23050024

M3 - Article

VL - 17

SP - 522

EP - 530

JO - Cell and Tissue Biology

JF - Cell and Tissue Biology

SN - 1990-519X

IS - 5

ER -