A cell’s differentiation to its terminal state isn’t always as direct as, say, a direct flight. The cell may have to make the developmental equivalent of a connecting flight—or several connecting flights. And if the cell should miss any of its connections, it may become stranded instead of landing at its hoped-for destination. Unfortunately, most of the cells that are supposed to follow deliberately modified developmental itineraries end up losing their way.

“Deliberately modified” itineraries might be called reprogrammed itineraries. They may be built by developmental biologists, stem cell scientists, disease modelers, drug screeners, or specialists in regenerative medicine. Although these specialists have different goals, they all act like cell-development travel agents. They try to keep cells moving from developmental state to developmental state as reliably as people travel from country to country.

To help scientists design cell-development itineraries, researchers at the Washington University School of Medicine have developed a form of passport control, a methodology for tracking individual cells across the developmental landscape. The methodology is called CellTagging. In place of an inked passport stamp, it deploys a viral vector loaded with DNA barcodes, or CellTags.

Collecting passport stamps

As cells subjected to reprogramming traverse the developmental landscape, they are given CellTags at intervals. Subsequently, cell samples collected at each of the intervals are directed to a passport inspector, that is, an RNA sequencer. Finally, all the information is analyzed, enabling the construction of multilevel lineage trees.

CellTagging has been demonstrated by its developers, a group of researchers led by Samantha A. Morris, PhD, a developmental biologist at Washington University. In a paper (“Single-cell mapping of lineage and identity in direct reprogramming”) that appeared recently in Nature, these researchers described how they used CellTagging to reconstruct reprogramming trajectories followed by mouse embryonic fibroblasts that were meant to become induced endoderm progenitors (iEPs), self-renewing cells of interest in regenerative medicine.

“CellTagging,” the article indicated, “reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a ‘dead end’ state, paths determined in the earliest stages of lineage conversion.” CellTagging also allowed the researchers to identify a pro-reprogramming factor, a methyltransferase called Mettl7a1. Adding Mettl7a1 to the reprogramming cocktail increased the yield of iEPs threefold.

These findings suggest that nonrandom factors may explain the divergent paths followed by reprogrammed cells. Once mapped, these factors could be circumvented, allowing cellular reprogramming to attain much higher efficiencies. Moreover, points at which healthy cells take a wrong turn, becoming dysfunctional and contributing to diseases such as cancer, may be identified.

“We can introduce unique genetic labels into each individual cell of interest,” Morris tells GEN. “But to analyze each cell, we must destroy it, losing valuable spatial information. Coupling high-throughput lineage-tracing strategies with cell imaging represents an important next step in this field.”

Improving travel services

When GEN asked Morris to explain how CellTagging improves on earlier lineage tracing approaches, she suggested that the evolution of linage tracing could be outlined as follows:

  • At least as far back as the 1970s, lineage tracing techniques have involved labeling cells with a heritable marker such as green fluorescent protein, followed by imaging to trace all the progeny of the originally labeled cell. (These are low-throughput approaches. They fail to capture many features of cell identity.)
  • More sophisticated lineage-tracing techniques emerged with the introduction of random DNA barcodes. DNA barcodes made it possible to label single cells, which could then pass the DNA barcodes to their progeny. (This technique increased the numbers of cells that could be analyzed, but again, it was limited in the number of phenotypic features that could be analyzed from each cell.)
  • More recently, technologies have been developed that allow cell lineage and identity to be measured in parallel, via single-cell RNA-sequencing. (This technique provides a more comprehensive and unbiased view of cell identity and retains information on how individual cells are related to each other.)

“Some of these approaches are based on CRISPR-Cas9 genome editing to introduce heritable labels into the genome of cells to be tracked,” Morris notes. “Although these approaches are elegant, they do require genetic manipulation of cells that is restrictive in some circumstances.” According to Morris, a less-restrictive approach is possible with CellTagging, which aims for something simpler, something that can be easily deployed across diverse cell types.

How CellTagging works

“CellTagging uses lentivirus to insert random, heritable DNA barcodes into the genomes of individual cells,” details Morris. “These DNA barcodes are transcribed into many RNA copies that are readily detected in standard single-cell library preparation protocols.

“Thus, we can simultaneously track the identity and lineage of many thousands of cells, simply by adding CellTag lentivirus to the cells of interest. Moreover, by including successive rounds of CellTagging, we can build lineage trees to understand where cell fate decisions are made in the reprogramming process—this is a very flexible lineage tracing system that can easily be applied to many biological questions.”

Whither CellTagged cells?

In the Nature article contributed by Morris and colleagues, CellTagging identified successful reprogramming trajectories and the molecular changes associated with them. CellTagging also provided information that was used to increase the yield of successfully reprogrammed cells. Significantly, Morris and colleagues observed that clonally related cells tended to follow the same reprogramming trajectories.

Currently, the Morris team is working to reveal the very early molecular changes that place a cell on the successful reprogramming trajectory. “Eventually, this information has the potential to enable the efficient and faithful reprogramming to any desired cell identity,” she emphasizes. “This will be valuable in terms of producing cells with the defined potential to develop new cell transplant therapies. It also has value for the production of cells ‘in the dish’ that recapitulate the properties of disease-relevant tissues and cell types, enabling the study of disease of otherwise inaccessible cell types.”

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