Color-Coding Technology Reveals New Insights About Stem Cell Biology

Rainbow iPSCs three days after inducing the rainbow labeling. Clonally expanding iPSCs from the same parental origin are apparent as clusters of same-colored cells.

Several years ago, Danny El-Nachef was given a task. His mission: create a tool to observe and track  how stem cells behaved during the earliest stages of human heart development. At the time, El-Nachef was a postdoc in the Davis and Sniadecki Labs in the Institute for Stem Cell and Regenerative Medicine (ISCRM) and a member of an interdisciplinary research team assembled to engineer a human heart tube. While the focus of the challenge was human biology, the x-factor in the solution came from the sea.

To create the surveillance system needed to track the heart cells, El-Nachef turned to rainbow reporter technology. He engineered human stem cells embedded with color-coding genes found naturally in jellyfish, sea anemones, and coral. When activated, those genes turned on proteins that become fluorescent tracers, which in turn, allowed the ISCRM researchers to follow individual cells as they moved and divided in laboratory environments. Just as importantly, the first generation of color-coded cells passed their unique fluorescence barcode on to their progeny, enabling lineage tracing of individual cells.

“We knew if we could track the individual cells and see how they were proliferating and moving, in terms of migration and contraction, we would know what we were doing right in terms of mimicking heart development,” says El Nachef.  “And the heart tube was an exciting context to test our system out.”

Now, El-Nachef is the lead author of a paper published recently in the journal Stem Cell Reports that further validates rainbow reporter technology and details its usefulness to areas of research beyond the heart.

“This was a deep dive into stem cell biology,” says El-Nachef.  “We know that not all induced pluripotent stem cells in a culture are alike. This study attempts to shed more light on the origin and nature of those differences and to validate approaches for tracking individual cells over time and down through generations.”

In the study, El-Nachef used rainbow reporters to learn more about pluripotent stem cells and three primary differentiated cell types: mesodermal cells, which give rise to bones, muscle, blood, and the heart, endodermal cells, which give rise to the tissue in the gut, and ectodermal cells, which give rise to the skin and brain.

“We were able to watch everything happen over the course of many weeks,” says El-Nachef. “We imaged the cells over and over again. And with the color coding, we saw every day what was happening during stem cell self-renewal and differentiation. That’s the cool factor of bringing together pluripotent stem cells and rainbow technology.”

Left, low magnification image of over 1 cm^2 area containing thousands of neurons. Note the majority of the cells came from 3 clonally dominant progenitors (a red, a purple, and a green progenitor). Middle, zoom in showing neurite (axons/dendrites) outgrowth and connections of neurons from different parental origins. Right, zoom in showing the center of a clonally expanded cluster with neuron progenitors surrounding a ventricular zone.

El-Nachef used three color-coding genes – a red protein extracted from sea anemone, an infrared fluorescent protein from a species of coral, and a protein that enables jellyfish to turn green. The green jellyfish protein was developed largely at the University of Washington’s Friday Harbor marine research laboratories. In different combinations, the three genes can be used to generate up to 18 colors.

Documenting the use of rainbow reporters in human stem cells is an important step forward for the field. Specifically, it addresses two key limitations. First, it validates the use of rainbow reporters in human stem cells – historically they have been limited to simpler model organisms. Second, traditional rainbow technology could not label undifferentiated pluripotent stem cells and track them as they differentiate. This paper details a novel method by which El-Nachef was able to label stem cells before they matured into specific cell types and track the individual cells and their progeny over several weeks.

In another insight, the researchers noted that cells on the outside of the growing colonies had different properties than cells closer to the center. Perhaps most significant, however, was an observation specific to one cell type – the ectodermal neural stem cells associated with brain development.

As El-Nachef watched through a microscope, the color-coded stem cells differentiated into neurons and began producing generations of daughter cells that self-organized into dahlia-shaped colonies.  As these colonies expanded, they formed ventricles surrounded by radially aligned neuron progenitors and neurons. “We could see the morphology of every neuron progenitor and neuron,” says El-Nachef.  “I realized these were basically developing brains in a dish.”

While most self-assembled cortical structures with radially aligned progenitors surrounding a ventricle were comprised of a single clonally dominant progenitor, ~12% were made from multiple parental cells, in this example there were two parental cells that clonally expanded and contributed to this structure (a purple and a green progenitor)

One aspect of the neuron differentiations caught El-Nachef’s attention – an emergence of a patching pattern, like a quilt, where the entire culture of tens of thousands of neurons was made from a dozen highly proliferative parent cells that dominated, while other less productive parent cells died off.  While similar observations have been made in chicks, El-Nachef believes this is the first study to detail this clonal dominance phenomenon in human cells.

The ability to track and potentially regulate neural progenitors has real-world implications for diseases in which the brain is either too big or too small, often the result of progenitor cells expanding too slowly or too quickly, such as microencephaly that results from Zika virus and other genetic diseases. “Outside of tool development the main major biological discovery in the paper was in the context of neuron differentiation,” say El-Nachef.

In the course of the investigation El-Nachef collaborated with several ISCRM faculty members, including Kelly Stevens, PhD, and Chuck Murry, MD, PhD, who guided him on protocols for differentiating the pluripotent stem cells into liver cells and heart cells, respectively. Jessica Young, PhD, an Assistant Professor in Laboratory Medicine and Pathology, was a third partner, specially on neurons.

Young noted the significance of the findings for brain research. “What is really great about this study is that, for the first time, we can study live human neurogenesis at single cell resolution. The identification of unique clonal progenitors that give rise populations of differentiated cells can tell us a lot about the diversity of the human central nervous system and provides a model amenable to functional studies. This model can be further used to provide insights not only into neurological disorders but human neurogenesis in general which is important for future studies on regenerating the brain.”

While his work on rainbow reporter technology is advancing research in multiple ISCRM labs,  El-Nachef himself has transitioned to a new role in the biomedical field. He is now working as a Senior Scientist at Sana Biotechnology iwhere he is working with ISCRM Director Chuck Murry to develop cell therapies.

Acknowledgements

This work was supported by the following funding sources: NIH HL141187 and HL142624 (to J.D.), NSF CMMI-1661730 (to N.J.S.), NIHDP2HL137188 (to K.R.S.), NIH F32HL143851 (to D.E.-N.), Washington Research Foundation postdoctoral fellowship (to M.C.R.), Gree Family Gift (JD/NJS/KRS/CEM), the University of Washington Ellison Foundation (to J.E.Y.). C.E.M. was supported by NIH grants R01HL128362 , U54DK107979 , R01HL128368 , R01HL141570 , and R01HL146868 , and a grant from the Fondation Leducq Transatlantic Network of Excellence .