While many of us have memorized the tidy, organized flowchart describing hematopoietic differentiation, researchers at Cincinnati Children’s Hospital Medical Center recently developed advanced imaging techniques to turn this two-dimensional diagram into a real-life understanding of how hematopoiesis occurs within the bone marrow — under both steady-state and stress conditions.
Qingqing Wu, PhD, and colleagues initially used a large flow cytometry panel (247-markers) to develop staining strategies to define hematopoietic progenitors. They validated their method by sort-purifying populations of interest and confirming function using transplantation and colony-forming assays.
The study authors then performed fluorescence microscopy on whole mounts of bones (e.g., sterna) to visualize stepwise hematopoiesis in mice.
Dr. Wu and her colleagues first examined parent multipotent progenitors/hematopoietic stem cells (HSCs). Imaging revealed they were present in the marrow as single cells (rather than in clusters), with a median distance to their nearest neighboring HSC of 100 microns (more than 10 cell diameters, or approximately the same diameter as a human hair). Curiously, as daughter cells are by necessity immediately adjacent to each other following cell division, these findings suggest either rapid differentiation of one daughter cell, ejection into the circulation, or migration within the bone marrow.
In vivo imaging of the calvarium after microscopy-guided transplantation of HSCs confirmed the latter: HSCs rapidly move away from each other when they find themselves in close proximity in vivo.
The authors next used a “confetti” mouse, i.e., a mouse in which fluorescent proteins of green, yellow, red, or cyan are expressed irreversibly in response to transient Cre activation.1,2 This allows clonal relationships to be tracked in short-lived cells. Colony-forming unit-erythroid (CFU-E) progenitors were visualized in oligoclonal strings, with the more mature erythroblasts seen in monoclonal clusters. Common lymphoid progenitors (CLPs) were found as single cells, far away from the multipotent progenitors, and were selectively enriched near the arterioles and depleted near the sinusoids. As lymphoid cells matured, they moved away from the CLPs but remained associated with each other in loose clusters.
The bone marrow can initiate emergency responses to a variety of stressors, increasing or reducing the output of mature cells into the periphery. To model acute stress, the authors used phlebotomy (mimicking hemorrhage), listeria monocytogenes infection, and granulocyte colony-stimulating factor (G-CSF), which predictably increases granulocyte production. All these situations revealed that the spatial patterns seen at steady state are maintained during an acute stress response. Following G-CSF stimulation, there was an expected increase in granulopoiesis in the long bones; however, the authors were surprised to find a decrease in the sternum, suggesting an anatomical variation in stress granulopoiesis throughout the skeleton, specifically in response to G-CSF.
In Brief
This work provides new insights into the behavior of HSCs and their progeny within the bone marrow, offering more detail of the physiology of this process than previously known. While the authors did not test chemotherapy or repopulation of the marrow after allogeneic or autologous stem cell transplantation, these methods could perhaps be applied in the future toward understanding the determinants of immune recovery and optimal graft function in the context of treatment for hematological malignancy.
Competing Interests
Dr. Markey indicated no relevant conflicts of interest.
