November 6, 2025 | By Asher Jones
Each day, the bone marrow pumps out more than 200 billion blood cells, including oxygen-carrying red blood cells and an incredible variety of innate and adaptive white blood cells, which form the immune system.
Known as hematopoiesis, this process starts with a single cell type: the hematopoietic stem cell (HSC). Exactly how one cell type differentiates into multitudes—whether as a series of committed steps or a more continual process—has intrigued scientists for decades and led to competing models.
Now, researchers at the University of Pittsburgh and Cincinnati Children’s Hospital Medical Center have used a new experimental framework and developed a novel tool called ChromLinker that unites traditional immunological techniques and modern single-cell genomic analyses to resolve this debate. The new findings, published in Nature Immunology, indicate that cells pass through distinct genomic states during hematopoiesis.
“Until now, the old world of immunology with its emphasis on using surface proteins to isolate cells by flow cytometry and the new world of single-cell sequencing have existed almost in parallel,” said co-senior author Harinder Singh, professor of systems immunology at the Pitt School of Medicine. “By combining high dimensional flow cytometry with single-cell genomics and using the tool ChromLinker, we unified these worlds.”
In doing so, Singh and his team have developed a resource for the immunology community that will help advance fundamental understanding of immune cell development.
The Old and the New
The old world, as Singh calls it, refers to the traditional way that immunologists study blood and immune cells. This approach involves developing antibodies that recognize surface proteins that are unique to different cell types, then using a technique called flow cytometry to characterize and isolate cells, which can be cultured in vitro and transferred back into animals to study their functions in vivo.
According to Singh, these traditional experiments led to a theory of hematopoiesis as discrete steps: HSCs diverge into increasingly distinct cell types like choosing to turn left or right on a forking path.
“Then along came the single-cell ‘omics revolution,” he said. “This ‘new world’ gave us the ability to analyze gene expression and chromatin in individual cells, which has generated an extraordinary amount of information. It revealed more variability in immune cells than previously appreciated, which led to a different view of hematopoiesis as a more continuous process.”
Under this steady-state view of hematopoiesis, HSCs gradually accumulate changes that take them further toward maturity, more like meandering down a winding trail than a path with discrete branches.
Unifying Worlds
Both worlds are important: ‘Omics tools provide highly detailed information on the regulatory networks that control gene expression in immune cells and novel insights into cell states, but traditional approaches are still needed to isolate these cells and experimentally confirm hypotheses.
To bridge these parallel worlds, Singh and his colleagues used single-cell genomics to identify rare multilineage progenitor cells—critical intermediates in blood cell development—from mouse bone marrow. They then overlaid these high dimensional genomic analyses with data on over a hundred cell surface proteins to build an atlas where each distinct genomic state could be identified with a unique pattern of proteins expressed on the surface of the cells, making possible their isolation and characterization.
The researchers validated their atlas by comparing analyses from 13 published datasets that have been widely used to distinguish different types of blood and immune cell progenitors.
Resolving Competing Models of Blood Cell Development
By merging the world of single-cell analyses and genomics with the world of cell surface markers, Singh and his colleagues concluded that blood cells pass through discrete intermediates as they differentiate during hematopoiesis.
“Our view is close to the classical view of hematopoiesis, but we don’t rest that claim on traditional methods,” explained Singh. “By using unbiased approaches to analyze underlying transcriptional networks operating in cells, we argue that such networks lead to discrete states. When cells move from one state to another, they reconfigure these networks.”
A Map for Exploring the Unified World
The new mouse bone marrow atlas is now available as a public resource. According to Singh, the atlas and tools will enable researchers with genomic data to identify surface proteins that define their cells and use antibodies to physically isolate the cells and perform functional studies using traditional methods.
“This is a tremendous resource to understand the different lineages of the immune system, including both innate and adaptive cells,” said Singh. “This atlas will help analyze relationships between lineages, identify putative regulators of differentiation, understand environmental triggers that influence immune cell production, enhance understanding of the origin of immune cell cancers, and eventually develop therapeutic targets for disease.”
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Image: Harinder Singh, professor of systems immunology in the Pitt School of Medicine. [Credit: Asher Jones]