Long before birth, the prenatal liver fosters the stem cells that will power our blood and immune systems for a lifetime. The fetal liver functions like a finely tuned incubator, providing the perfect environment to allow these hematopoietic stem cells (HSCs) to grow while preserving their extraordinary capacity for indefinite regeneration. These cells offer an effective treatment for cancers and other diseases that affect the blood system.
Studying these cells is challenging because true hematopoietic stem cells—those rare cells capable of lifelong blood regeneration—closely resemble short-lived blood precursor cells. Distinguishing between these distinct cell types is critical for identifying the cells with the greatest potential for long-term transplantation success. Further, the specialized fetal liver niche is challenging to study. It exists only transiently during development and is made up of a complex mixture of cell types that are difficult to recreate outside the body.
Thankfully, researchers in the Translational Science & Therapeutics Division at Fred Hutchinson Cancer Center were up to the challenge. Dr. Takashi Ishida and colleagues in the Hadland Lab engineered an ex vivo co-culture system that mimics the fetal liver environment; the latter of which is made up of endothelial and stromal cells. With this novel system, they were able to capture and functionally analyze single fetal liver HSCs in unprecedented detail. The results of their work were recently published in Cell Reports.
The researchers used cells extracted from the fetal mouse liver to create their model system. To mimic the developmental vascular niche of the fetal liver, endothelial cells derived from mouse fetal liver were genetically modified to express an activated AKT protein. This enables endothelial cell propagation in serum-free culture while maintaining their ability to create a supportive feeder layer. Single HSCs (defined by the surface marker expression pattern (CD45+GR1⁻F4/80−SCA1highEPCRhighCD150+) were isolated from mouse livers at embryonic day 15-16 and seeded onto this feeder layer under serum-free conditions with addition of cytokines representative of stroma-supplied growth signals. Remarkably, this approach maintained HSCs capable of serially repopulating all lineages of the hematopoietic system in transplantation experiments. Importantly, cytokine-only cultures without the feeder layer could not replicate this, indicating cell-cell contacts are crucial for the process.
Using this platform, they expanded single HSC clones to create identical cell cultures which they tested in a series of sophisticated experiments: time-lapse imaging, serial transplantation assays, single-cell RNA sequencing and flow cytometry phenotyping. This approach enabled them to link single-cell behavior like cell division kinetics or long-term engraftment capacity with transcriptional state and immunophenotypes of cell progeny.
Among the rare fetal liver HSCs capable of serial engraftment, three features stood out. First, these cells exhibited differentiation latency—a delay in progressing toward specialized cell fates, allowing preservation of stem cell identity. Second, they showed symmetric self-renewal—a bias toward dividing into two stem-like daughters rather than one stem cell and one differentiated cell. Third, they had transcriptional signatures of biosynthetic dormancy—reduced metabolic and ribosomal activity compared with shorter-lived progenitors and higher expression of genes involved in maintaining quiescence, chromatin stability, and self-renewal.
“Our work overturns the traditional view that HSCs are highly active during development. We discovered that the rare HSCs capable of lifelong blood production actually remain in a state of metabolic dormancy and divide symmetrically to expand their numbers without rushing into differentiation,” Hadland explains.
The authors also identified cell surface molecules such as CD63 with elevated expression on self-renewing HSCs with long-term engraftment potential, offering a potential tool to enrich for these rare, long-term regenerative cells. Finally, the team applied a computational algorithm to their single-cell RNA sequencing data to identify a network of fetal liver endothelial ligands—including TGFβ1, ICAM1, COL4A1, LAMB2, SELP, and JAM3 and other ligands not previously linked to HSC maintenance—that interact with HSC receptors to regulate self-renewal, cell anchorage, and dormancy. These signals converge on key stemness genes like MECOM, PRDM16, and ANGPT1, revealing that coordinated, multi-pathway communication between endothelial and hematopoietic cells underlies the unique self-renewing potential of fetal liver HSCs.
“This work provides new insights into strategies to grow transplantable HSCs in the lab, something that has been a major challenge for decades.” Hadland shares.
By identifying dormancy and differentiation latency programs and linking them to niche signals, this work creates a roadmap to expand clinically useful HSCs ex vivo. Moreover, the transcriptional resource generated via single cell RNA-seq from unique HSC clones will likely inform mechanistic studies of how intrinsic programs and niche cues synergize to maintain stemness.
“These findings raise exciting questions: How is this dormant state established during development, and can we harness it to improve HSC production and expansion, such as from human pluripotent stem cells?” Hadland asks. “Looking ahead, we’re also developing machine learning tools to analyze live-cell imaging data from our platform, aiming to predict HSC fate based on subtle behavioral patterns. This could transform how we identify and select the most potent HSCs for clinical use.”
“This research was made possible by the Cancer Consortium’s shared genomics and imaging resources, which allowed us to link HSC behavior with molecular signatures at single-cell resolution. These integrated platforms were critical for uncovering the unique properties of fetal HSCs.” according to Dr. Hadland.
The spotlighted research was funded by the NIH, the American Society of Hematology, the Birth Defects Research Laboratory Takeda Science Foundation, the Nakatomi Foundation, and the Kitasato University School of Medicine Alumni Association.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Brandon Hadland, Irwin Bernstein, Kimberly Aldinger and Cole Trapnell contributed to this research.
Ishida T, Mercoli J, Heck AM, Phelps I, Varnum-Finney B, Dozono S, Nourigat-McKay C, Kraskouskas K, Wellington R, Waltner O, Jackson DL, Delaney C, Rafii S, Bernstein ID, Aldinger KA, Birth Defects Research Laboratory, Trapnell C, Zhao HG, & Hadland B. 2025. Differentiation latency and dormancy signatures define fetal liver hematopoietic stem cells at single-cell resolution. Cell Reports. https://doi.org/10.1016/j.celrep.2025.116289
Science Spotlight writer Kelly Mitchell is a postdoctoral fellow in the Paddison Lab at Fred Hutch Cancer Center. She utilizes live cell reporters and CRISPR screening to study how glioblastoma cancer cells resist chemotherapy and radiation treatment. She obtained her PhD in cellular biology from Albert Einstein College of Medicine.