Lactate Accelerates Mouse ES Cell Differentiation Towards the XEN Lineage
Posted on: 7 April 2021
Preprint posted on 26 December 2020
Enhance the in vitro conversion of embryonic stem cells to the XEN lineage with a single metabolite, lactate.
Selected by Nozomu Takata
Categories: cell biology, developmental biology, molecular biology
Background
The yolk sac, a membranous structure situated outside of the embryo, has a variety of functions1. For instance, the yolk sac aids in nutrition and gas exchange between the mother and the developing embryo as well as the formation of the umbilical cord. Another important role is the production of embryonic blood cells, primitive macrophages and germ cells.
In amniotes, a major constituent of the yolk sac is created from cell derivatives of a particular cell lineage called the primitive endoderm (PrE), that appears as an epithelial monolayer visible shortly before implantation. It is one of the first three cell lineages, defined by complex mechanisms that remain elusive2 (Schematic the authors provided).
Due to the lack of accessibility to the PrE lineage on demand, there has been a plethora of well-established in vitro model systems allowing researchers to derive PrE cells and maintain them indefinitely in vitro as XEN cells3. XEN cells can be established from embryonic stem cells chemically4 or when the PrE-specific genes Gata4/65 or Sox176 are expressed in vitro. DAB2 is used as another XEN cell marker (Fig. S1), but it is likely other additional factors assist lineage specification toward the PrE.
Cells between preimplantation and post-implantation stages display distinct metabolic profiles7. The authors reported previously that F9 embryonal carcinoma cells, which are capable of adopting a XEN-like lineage, exhibited a metabolic phenotype reliant exclusively on glycolysis8. In addition, evidence indicates that the metabolic state between embryonic and extraembryonic lineages differs, suggesting that metabolism may be sufficient to influence lineage commitment. These phenomena have brought the authors to question why and how important glycolysis is for lineage specification.
In this preprint, the authors conducted metabolomic analysis on two distinct embryonic cell lineages and demonstrated that 1) XEN cells possess altered glycolytic enzyme levels involved in pyruvate and lactate homeostasis, 2) the cells are sensitive to glycolytic inhibition when compared with oxidative phosphorylation (OXPHOS) inhibition, 3) lactate is a key metabolite to enhance the XEN lineage.
Key findings
-
Feeder-free XEN cells exhibit high sensitivity to glycolytic inhibition
The morphology of the ES cell colony was insensitive to treatment with the glucose blocker 2-DG or with oligomycin, an inhibitor of OXPHOS, while in XEN cells, 2-DG had a more pronounced effect not only on morphology but also on cell viability compared to oligomycin. It suggests that XEN cells were more sensitive to glycolytic inhibition than OXPHOS inhibition (Fig. 1)
-
Metabolomic profiling between feeder-free ES and XEN cells identified lactate as a candidate metabolite
Next, the authors performed intracellular metabolomic analysis to identify changes in intracellular metabolite composition between ES and XEN cells. They revealed that 18 intracellular metabolites were significantly enriched in ES cells and 68 in XEN cells (Fig. 2), specifically, intracellular lactate levels were significantly higher in XEN cells. The data provided here indicate that XEN cells exhibit a unique metabolite profile distinct from the ES population.
-
Elevated intracellular and extracellular lactate is observed in feeder-free XEN cells
LDHA converts the metabolite pyruvate into lactate while the reverse is mediated by LDHB. Therefore, the relative ratio of LDHA/LDHB could explain why lactate production was higher in XEN cells. Consistent with the finding from the metabolome analysis, LDHA expression was indeed notably higher in XEN cells than in ES cells in quantitative PCR and western blot analysis (Fig. 4).
How about the alternative possibility that pyruvate in XEN cells travels into mitochondria and facilitates OXPHOS to generate energy? Expression analysis of the mitochondrial pyruvate carrier Mpc1 showed significantly reduced expression in XEN cells. Therefore, pyruvate in XEN cells would likely fail to enter the mitochondria and instead, is preferentially converted into lactate by the enhanced LDHA activity.
The authors conducted functional tests to prove the hypothesis that lactate can facilitate cell fate. To promote intracellular lactate accumulation, the cells were incubated with L-lactate or AZD3965, a potent inhibitor to block lactate shuttling to the extracellular space. Intriguingly, both treatments enhanced Gata6 and Dab2 expression. Therefore, promoting intracellular lactate accumulation enhanced XEN induction in vitro, highlighting a novel role for lactate as a signaling molecule involved in the differentiation of XEN cells. These results suggest that lactate participates in specifying ES cells towards the XEN lineage.
In summary, the findings strongly suggest that feeder-free XEN cells are sensitive to glycolytic inhibition, produce high levels of lactate, which enhances XEN induction in vitro. This study revealed a functional association between metabolism and cell lineage specification.
Why I like to highlight this preprint
I like the questions the authors raised and how they were challenged in this study.
There are a growing number of reports suggesting that metabolites play unexpected roles in transcriptional changes following epigenetic modifications. In multicellular systems, it would not be so surprising that layers of multiple signalling cascades secure and regulate a variety of cellular activities that lead to changes in gene expression. However, the contribution of metabolites and key mechanisms remain less understood.
Embryonic development is a beautiful system that exists before birth and one of the best platforms to witness cell lineage specification. The earlier the developmental stage in vivo, the more difficult it generally is to get access to a small number of cell types. Therefore, this study uses an in vitro culture system that clearly distinguishes two key cell types and overcomes a quantitative hurdle when metabolomics is performed.
The discovery of glycolysis and its product lactate in the XEN cells may open the avenue to not only better understand the new aspect of metabolism in mammalian cell specification, but perhaps give us better treatments for complications during pregnancy in the future 9,10.
Questions to the authors
- Is this mechanism happening in real embryos? If so, what could be the trigger for the sensitivity towards glycolysis? It would be nice if the authors could validate the findings in vivo in the future.
- What happens when LDHA is inhibited or functionally KO? I assume lactate production will be reduced. Would this treatment stop the maintenance of XEN cell fate?
- Lactate accelerates XEN cell markers. Is there any clear change in epigenetic modification? And if so, how would that be caused?
- It wouldn’t be surprising if other embryonic cell types also prefer to consume lactate. Testing other lineages would be of great interest to reveal a general mechanism during embryonic development.
- I am curious about the dynamics of glycolysis activity, which could be analyzed by visualizing the final product lactate in live by a fluorescent reporter system11.
References
1 Donovan, M. F. & Bordoni, B. in StatPearls (2021).
2 Hermitte, S. & Chazaud, C. Primitive endoderm differentiation: from specification to epithelium formation. Philos Trans R Soc Lond B Biol Sci 369, doi:10.1098/rstb.2013.0537 (2014).
3 Niakan, K. K., Schrode, N., Cho, L. T. & Hadjantonakis, A. K. Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells. Nat Protoc 8, 1028-1041, doi:10.1038/nprot.2013.049 (2013).
4 Anderson, K. G. V. et al. Insulin fine-tunes self-renewal pathways governing naive pluripotency and extra-embryonic endoderm. Nat Cell Biol 19, 1164-1177, doi:10.1038/ncb3617 (2017).
5 Fujikura, J. et al. Differentiation of embryonic stem cells is induced by GATA factors. Gene Dev 16, 784-789, doi:DOI 10.1101/gad.968802 (2002).
6 McDonald, A. C., Biechele, S., Rossant, J. & Stanford, W. L. Sox17-mediated XEN cell conversion identifies dynamic networks controlling cell-fate decisions in embryo-derived stem cells. Cell Rep 9, 780-793, doi:10.1016/j.celrep.2014.09.026 (2014).
7 Zhou, W. et al. HIF1alpha induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition. EMBO J 31, 2103-2116, doi:10.1038/emboj.2012.71 (2012).
8 Gatie, M. I. & Kelly, G. M. Metabolic profile and differentiation potential of extraembryonic endoderm-like cells. cell Death Discov 4, 42, doi:10.1038/s41420-018-0102-1 (2018).
9 Moradan, S. & Forouzeshfar, M. Are abnormal yolk sac characteristics important factors in abortion rates? Int J Fertil Steril 6, 127-130 (2012).
10 Cho, F. N., Chen, S. N., Tai, M. H. & Yang, T. L. The quality and size of yolk sac in early pregnancy loss. Aust N Z J Obstet Gynaecol 46, 413-418, doi:10.1111/j.1479-828X.2006.00627.x (2006).
11 San Martin, A. et al. A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells. PLoS One 8, e57712, doi:10.1371/journal.pone.0057712 (2013).
doi: https://doi.org/10.1242/prelights.28089
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