Direct reprogramming in the context of cells, involves the conversion of one cell type into another. For example, a skin cell to a neuron. Only a handful of the large number of cell-type transitions have been tested so far, but what seems apparent is that the processis not that efficient. To improve efficiency, an understanding of the molecular steps made throughout the transition by the cell are needed. To decipher these molecular steps, read-outs of single cells are required to exclude the reprogramming variation between cells, in addition to tracking the cell’s daughters and predecessors (the cell’s lineage). A new technique published in Nature by Biddy et al. (1) may provide this solution. The group used their technique, called ‘CellTagging’, to follow the direct reprogramming of fibroblasts to induced endoderm progenitors. Two main fates were seen for the cell population – they were either successfully reprogrammed or they weren’t. Importantly though, from the data collected using CellTagging, they identified a key methyltransferase protein that aids successful reprogramming.
What makes reprogramming direct?
All the cells in your body originate from one fertilised egg that then divides. Early during development, a population of stem cells have the potential to turn into any type of cell inthe body. These unspecialised cells become specialised as they take on a certain cell-fate. This is the process of differentiation. Once a cell differentiates there is typically no going back. A landmark was reached when Yamanaka et al.2 showed that differentiated structural cells (fibroblasts) could be converted back to stem cells under certain conditions. The advantage of these stem cells is that they could then be induced to form a different type of cell. Direct reprogramming skips this stem cell state and attempts instead to change cells in one step from cell type to another (Figure 1).
However, under the conditions used to induce reprogramming, not all cells will successfully make this transition. But why? And how can the success rate be improved?
During reprogramming, it is like the cells are going on a journey…well it’s not ‘like’, they are going on a journey. And to give this some context, let’s imagine we want the cells to travel from Birmingham to London. All the cells start at Birmingham, but only a handful end up in London (Figure2). What happened to the rest? Obviously, some of the cells took a wrong turn somewhere. But did they all take the same wrong turn, or were there multiple stages along the route that tripped the cells up?
Analogy aside, these are actually really important questions to understand. If you can determine at which stages during the journey the cells start heading in the wrong direction, you can find a rational solution to prevent it from happening. In the case of the Birmingham-London trip, it may involve blocking one of the roads, forcing the cells to go a certain way. For cellular transformation, this could involve increasing or decreasing the levels of a protein and doing this at a specific time during reprogramming.
Biddy’s team’s method to track individual cells during direct reprogramming involves CellTags – heritable random barcodes. Sequential delivery of tags throughout the reprogramming process enables each cell to uptake and express a unique subset of barcodes. This enables individual cells to be distinguished during sequencing of the tags. Key to the method though is that the tags are heritable – the tag is passed onto the two new cells after cell division. Therefore, by sequencing the tags present in a cell, the cells lineage can be traced.
To test the technique, the group applied CellTagging to the direct reprogramming of mouse embryonic fibroblasts to induced endoderm progenitors (iEP). The reprogramming process was induced by the overexpression of two proteins, FOXA1 and HNFα, that both regulate the expression of many other genes. The CellTags were added to the cells before the reprogramming, and at 3 days and 13 days after the reprogramming was initiated. Cells were collected with their RNA extracted and sequenced at multiple time points throughout a 28-day time course. The RNA transcriptome enables identification of CellTags and the fate of the cell – whether it had converted to an iEP or was still a fibroblast.
We are family
Depending on which RNAs were expressed in each cell, the team could group the cells into four phases depending on how ‘far’ through the reprogramming they were; fibroblast, early transition, transition and reprogrammed. Then based on which CellTags were present, the cells could then be compared to their relatives. Related cells tended to show similar reprogramming outcomes – this suggested that whether a cell would be successfully reprogrammed was determined early on during the process.
More importantly though, as the cells transcriptome is also known, analysing differences between cells that reprogrammed versus those that didn’t highlights gene signatures that may be critical for reprogramming. One gene significantly upregulated for a short period during successful reprogramming was Mettl7a1, a potential methyltransferase. When the team added Mettl7a1 in addition to the other transcription factors, they achieved a 3-fold increase in successful reprogramming.
On the road to success?
CellTagging was successfully used to make direct reprogramming more successful. It therefore seems like this technique can now be used to uncover other factors that can increase direct reprogramming efficiency between different cell types. It has also raised more questions as to how the potential methyltransferase helps reprogramming. But, the technique has wider applications than just direct reprogramming, for example following cancer development. More processes could be followed depending on the temporal resolution that can be resolved using the CellTags. It will be interesting to see how CellTagging can be further used.
- BA Biddy. Single-cell mapping of lineage and identity in direct reprogramming, Nature, (2018) https://doi.org/10.1038/s41586-018-0744-4
2. Takahashi, K. & Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126, 663–676 (2006).