Dichloroacetate is a reprogramming factor

Dichloroacetate is a reprogramming factor Sainz de la Maza D, De Miguel MP, Moratilla A, Lorca C, Martín-Morales R. Cell Engineering Laboratory, La Paz Hospital Research Institute, Madrid, Spain. Despite being unipotent cells, primordial germ cells (PGC) have the latent capability to produce a complete individual, which confers them combined characteristics of both differentiated and pluripotent cells. Previous results from our laboratory achieved mouse PGC partial reprogramming by just using hypoxia, highlighting the importance the glycolytic metabolism has in the process of cellular reprogramming (López-Iglesias et al, 2015). We hypothesize that dichloroacetate (DCA), an inhibitor of the enzyme Pyruvate Dehydrogenase Kinase (PDK) which enhances oxidative phosphorylation (OXPHOS) in detriment of glycolysis and has been reported to impair cell reprogramming (Folmes et al, 2011), would inhibit PGCs hypoxia-induced reprogramming. PGCs were isolated form 8.5 dpc mouse embryos and seeded onto a confluent STO cells monolayer. Identification of PGCs and reprogrammed cells was performed by alkaline phosphatase staining and SSEA1 expression. Groups of 8 or more cells were considered reprogrammed colnies. In vitro pluripotency was assayed by embryoid body formation. To assess mitochondrial activation PGCs were also cultured in the presence of fluorescent probe JC-1. Finally, confocal microscopy was performed to study pyruvate dehydrogenase (PDH) phosphorylation and HIF1α expression.

DCA-Reprogrammed cells maintained glycolysis predominance, as shown by phosphorylated PDH in hypoxic cultures and in DCA cultures, proving that the DCA concentration used does not shift metabolism towards OXPHOS. In addition, PGCs cultured with DCA showed a higher percentage of inactive mitochondria:

DAPI

SSEA1

p-PDH

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Norm

Hip

Norm+DCA We found that impairment of glycolysis by DCA under hypoxia inhibits PGCs reprogramming and had no effect in normoxia (A,B). However, we also observed that a ten-fold lower DCA dose added to PGCs cultured in hypoxia showed no effect in hypoxia (C,D) and resulted in PGCs reprogramming in normoxia (E,F):

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B

C

D

E

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Hip+DCA

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*

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F

Both hypoxia and DCA-induced reprogramming share HIF1α expression as a common feature:

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SSEA1

HIF1α

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Norm

Reprogrammed cells with DCA were capable of differentiating into the three germ layers: endoderm, ectoderm and mesoderm:

Hip

DCA

DCA at low concentration induces PGC reprogramming in normoxia. Under these conditions, PGCs undergo HIF1α- induced metabolic reprogramming towards glycolisis. We propose a mechanism where DCA would promote a transient increase in ROS, which would lead to HIF1α stabilization. This event would be determinant to induce a metabolic switch in PGCs towards a glycolitic phenotype and would cause the acquisition of pluripotency.

Folmes CD, Nelson TJ, Martinez-Fernandez A, Arrell DK, Lindor JZ, Dzeja PP, Ikeda Y, Perez-Terzic C, Terzic A. (2011) Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metab, 14(2):264-271. López-Iglesias P, Alcaina Y, Tapia N, Sabour D, Arauzo-Bravo MJ, Sainz de la Maza D, Berra E, O'Mara AN, Nistal M, Ortega S, Donovan PJ, Schöler HR, De Miguel MP. (2015) Hypoxia induces pluripotency in primordial germ cells by HIF1α stabilization and Oct4 deregulation. Antioxid Redox Signal, 22(3):205-223.