How to make up a new body: capturing totipotent cells in the dish…

In the quest of making cells ‘in the dish’ that could form any cell type in our bodies, a team of Helmholtz researchers led by Dr. Ane Iturbide and Prof. Maria-Elena Torres-Padilla from the Institute for Epigenetics and Stem Cells and the Faculty of Biology of the Ludwig Maximilians University have carried out a large-scale small molecule screening to identify factors that can change cell identity ‘in the dish’. They identified a compound, Retinoic acid, as a molecule that can change cells in the dish into cells resembling totipotent cells that are able to give rise to a completely new organism. Their results were just published in Nature Structural and Molecular Biology.

Retinoic acid increases the number of totipotent-like cells in culture.

Cell differentiation makes it possible that from just a single cell, the zygote - which is the result of fertilization of the egg by the sperm - all the different cell types of an organism can be generated. This capacity is called totipotency. This contrasts to Embryonic stem cells (ESCs), which are not totipotent but pluripotent. This means that ESCs are not capable of forming a full organism.

Totipotent cells hold an enormous promise towards regenerative medicine, since they have the potential to generate all the tissues and cell types of an organism. To fulfil the need of regenerative and replacement approaches as disease therapy, it is of paramount importance to understand how totipotency can be maintained. Moreover, there is an urgent need for a cell culture model, which is stable and robust. To address this, and with the aim of generating a robust, easy to handle ‘totipotent-like cells’ in the petri dish, the researchers of the Institute for Epigenetics and Stem Cells collaborated with the Assay Development & Screening platform to screen ~31,000 compounds. This led to the discovery of specific compounds with the capacity to induce robust reprogramming in cell culture and regulate cell identity. Thus, this work highlights a new, robust pathway to induce reprogramming.

Drug-based screening to identify small molecules able to generate totipotent-like cells in the dish

The group of researchers was looking for novel compounds that robustly reprogram stem cells into 2-cell-like cells (2CLCs). 2CLCs provide the unique possibility to establish a cell culture model for totipotency because of their similarity to the cells in the early mouse embryo at the 2-cell-stage, which are totipotent. “2CLCs arise spontaneously in a culture of mouse ESCs, but they are extremely rare as they only constitute less than 1% of the cell population.”, explained Dr. Ane Iturbide. The team performed the first drug screening to generate totipotent-like cells in culture and their findings will ease their use for the field and enable more robust manipulation of cells in the dish. 

From the 30,000 compounds, the researchers ended up with three major molecules. Interestingly, all three were different retinoids, among them also retinoic acid, which was the only naturally-occurring compound. “The results are very surprising, and unexpected, because retinoic acid has long been known to cause the complete opposite effect: to generate differentiated cells” says Dr. Ane Inturbide, first author of the work. Over decades, it has been assumed that RA is only important for cell differentiation. The recently published findings of Dr. Ane Iturbide and Prof. Maria-Elena Torres-Padilla from the Institute for Epigenetics and Stem Cells at the Helmholtz Zentrum München have elucidated that the role of RA during embryogenesis goes far beyond.

Already at very low doses, retinoic acid leads to a 10-fold increase of 2CLCs in the cell population. We found an outstanding conversion efficiency of reprogramming of up to 50-60% as opposed to the much lower 4-10%, which has been the current standard in the field.”, said Prof. Torres-Padilla. “Our next step was to see whether the RA signaling pathway is physiologically relevant in the early embryo.” This was indeed the case. “To investigate whether retinoic acid is important for early embryogenesis, we inhibited this pathway using another small molecule” said Ane. “We observed that these embryos simply did not develop at all!”.” This means that retinoic acid signaling is key already during the earliest stages of life, and not only for initiating the differentiation of pluripotent stem cells as it was so far believed. 

Reprogramming dynamics at the single cell level.

But, why can retinoic acid lead to totipotency in some cases and to the opposite outcome of differentiation in others? “This was a puzzle: how and why can cells in the same culture respond differently – this is an amazing sign of cellular plasticity too, and analyzing single-cell RNAseq datasets gave us the opportunity to further understand this process” said Dr. Scialdone, co-author of the manuscript and expert in single cell approaches. To investigate this apparent conundrum, the researchers analyzed thousands of cells at the single cell level. The results were very clear, showing that a population of embryonic stem cells differentiate in response to retinoic acid, but another population reverts ‘back’ in time, to a more embryonic-like phenotype resembling totipotency. 

Altogether, the multidisciplinary approach undertaken is a great proof-of-principle for how molecular mechanisms and drug screening can synergize to identify new molecular pathways for cellular reprogramming. The findings pave the way for validating 2CLCs as model system for understanding the regulatory network of totipotency and shed light into one of the most fascinating phenomena of mammalian life: How can a single cell give rise to a full organism?

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